The present invention provides novel polynucleotides encoding MMP-29 polypeptides, fragments and homologues thereof. Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel MMP-29 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

Description

75 parts

›This application is a divisional application of non-provisional…

This application is a divisional application of non-provisional application U.S. Ser. No. 10/133,797, filed Apr. 26, 2002, now U.S. Pat. No. 7,034,134, which claims benefit to provisional application U.S. Ser. No. 60/286,764 filed Apr. 26, 2001. The entire teachings of the referenced applications are incorporated herein by reference.

›FIELD OF THE INVENTION

›BACKGROUND OF THE INVENTION · 1 of 2

Proteases hydrolyze specific peptide bonds in proteins. The residues at the active site are used to classify proteases (Rawlings & Barrett, 1995). Proteases that hydrolyze peptide bonds using metal ions are referred to as metalloproteases (“MP”). The metalloproteinases may be one of the older classes of proteases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures, but many contain a zinc ion. In some cases, zinc may be replaced by another metal such as cobalt or nickel.

The gene and protein of the present invention codes for a human protease belonging to the peptidase M10 family (see Rawlings & Barrett, 1995 for review of protease familial classification). This family contains the sequence . . . HE[ILF]GHXXGLXH . . . (SEQ ID NO:81), which is thought to contain amino acids (histidines and or glutamic acid) which coordinate metal ion binding. Such metal ion coordination facilitates catalysis through the stabilization of a noncovalent, tetrahedral intermediate after the attack of a metal-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group. Metal ion coordination is thought to stabilize the negative charges formed within the active site of the enzyme during catalysis. Such stabilization lowers the transition state energy requirements, and thus results in significant rate enhancements during enzymatic catalysis over non-metal ion coordination conditions (Fersht, A., “Enzyme Structure and Mechanism”, 2 nd edition, W. H. Freeman and Company, New York, 1985).

Another important feature of peptidase M10 family is a highly conserved octapeptide ( . . . PRC[GN]XP[DR][LIVSAPKQ] . . . (SEQ ID NO:82)) that has been shown to be involved in autoinhibition of metalloproteases (Breathnach R. et al, 1988; Navre M. et al. 1991). A cysteine within the octapeptide chelates the active site zinc ion, thus inhibiting the enzyme. Between the autoinhibitive octapeptide and the catalytic peptide resides a pair of conserved basic amino acids [RK][RK] that serves as the putative cleavage site for activation by furin proteinases.

The prototype of this family is a human secreted interstitial collagenase called matrix metalloproteinase 1. Substrate proteins for the matrix metalloproteinase 1 include the interstitial collagen group—types I, II, III and alpha-macroglobulins (Vincenti MP et al, 1996). A metalloproteinase gene (XMMP) transiently expressed in Xenopus laevis early embryo development has been discovered (Yang M, Murray M T, Kurkinen M, 1997). It is undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo, suggesting that XMMP plays a role in Xenopus early development. The human MMP-29 gene described herein represents the human ortholog of the Xenopus XMMP.

Metalloproteinases in Disease

Limited-proteolysis by metalloproteases plays a central regulatory role in many physiological and pathophysiological processes. There are many examples of inhibitors of metalloproteases that are useful medications in the treatment of hypertension, heart failure, various forms of cancer and other diseases.

Metalloproteases play many important biological roles in the nervous system, including the spinal cord. There is a balance between the synthesis and degradation of extracellular matrix proteins in the process of synapse formation during development and regeneration. The timing of MP activation is therefore potentially critical. Some MPs have been shown to be upregulated in the spinal cord either during development or in pathological states such as multiple sclerosis, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis. Since MPs degrade extracellular matrix proteins, they would be toxic to developing neurons that depend upon the matrix proteins for survival, neurite outgrowth, and synapse formation. Degradation of the matrix proteins would also cause the breakdown of the blood brain barrier and infiltration of immune cells into the CNS, which occurs in inflammatory conditions such as MS. Other biological processes that metalloproteinases are involved in include fibrillogenesis, angiogenesis, rheumatoid arthritis, osteoarthritis, enamel formation, atherosclerosis, neural degeneration, diabetic renal lesions and ulceration.

Using the above examples, it is clear the availability of a novel cloned metalloproteinase provides opportunities for adjunct or replacement therapy, and are useful for the identification of metalloproteinase agonists, or stimulators (which might stimulate and/or bias metalloproteinase action), as well as, in the identification of metalloproteinase inhibitors. All of which might be therapeutically useful under different circumstances. The metalloproteinase of the present invention can also be used as a scaffold to tailor-make specific metalloproteinase inhibitors.

The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length MMP-29 gene and its encoded polypeptide. Also provided are polypeptide alignments illustrating the strong conservation of the MMP-29 polypeptide to other known metalloproteinases. Data is also provided illustrating the unique tissue expression profile of the MMP-29 polypeptide in testis tissues, which has not been appreciated heretofore.

The invention also provides methods for designing, evaluating and identifying compounds which bind to all or parts of the aforementioned regions. The methods include three dimensional model building (homology modeling) and methods of computer assisted-drug design which can be used to identify compounds which bind or modulate the forementioned regions of the MMP-29 polypeptide. Such compounds are potential inhibitors of MMP-29 or its homologues. The invention also provides novel classes of compounds, and pharmaceutical compositions thereof, that are useful as inhibitors of MMP-29 or its homologues.

›BACKGROUND OF THE INVENTION · 2 of 2

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of MMP-29 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MMP-29 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.

›BRIEF SUMMARY OF THE INVENTION · 1 of 4

The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the MMP-29 protein having the amino acid sequence shown in FIGS. 1A-B (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone, MMP-29 (also referred to as BGS26).

The invention further provides an isolated MMP-29 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

The invention further relates to a polynucleotide encoding a polypeptide fragment of SEQ ID NO:2, or a polypeptide fragment encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to a polynucleotide encoding a polypeptide domain of SEQ ID NO:2 or a polypeptide domain encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to a polynucleotide encoding a polypeptide epitope of SEQ ID NO:2 or a polypeptide epitope encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to a polynucleotide encoding a polypeptide of SEQ ID NO:2 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1, having biological activity.

The invention further relates to a polynucleotide which is a variant of SEQ ID NO:1.

The invention further relates to a polynucleotide which is an allelic variant of SEQ ID NO:1.

The invention further relates to a polynucleotide which encodes a species homologue of the SEQ ID NO:2.

The invention further relates to a polynucleotide which represents the complimentary sequence (antisense) of SEQ ID NO:1.

The invention further relates to a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified herein, wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:2, wherein the polynucleotide fragment comprises a nucleotide sequence encoding an metalloprotease protein.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:2 or the polypeptide encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to an isolated nucleic acid molecule of of SEQ ID NO:1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:1 or the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:1, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated polypeptide comprising an amino acid sequence that comprises a polypeptide fragment of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide fragment of SEQ ID NO:2 or the encoded sequence included in the deposited clone, having biological activity.

The invention further relates to a polypeptide domain of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide epitope of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a full length protein of SEQ ID NO:2 or the encoded sequence included in the deposited clone.

The invention further relates to a variant of SEQ ID NO:2.

The invention further relates to an allelic variant of SEQ ID NO:2. The invention further relates to a species homologue of SEQ ID NO:2.

The invention further relates to the isolated polypeptide of of SEQ ID NO:2, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated antibody that binds specifically to the isolated polypeptide of SEQ ID NO:2.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of SEQ ID NO:2 or the polynucleotide of SEQ ID NO:1.

The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or absence of a mutation in the polynucleotide of SEQ ID NO:1; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

The invention further relates to a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising the steps of (a) determining the presence or amount of expression of the polypeptide of of SEQ ID NO:2 in a biological sample; and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

›BRIEF SUMMARY OF THE INVENTION · 2 of 4

The invention further relates to a method for identifying a binding partner to the polypeptide of SEQ ID NO:2 comprising the steps of (a) contacting the polypeptide of SEQ ID NO:2 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide.

The invention further relates to a gene corresponding to the cDNA sequence of SEQ ID NO:1.

The invention further relates to a method of identifying an activity in a biological assay, wherein the method comprises the steps of expressing SEQ ID NO:1 in a cell, (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity.

The invention further relates to a process for making polynucleotide sequences encoding gene products having altered SEQ ID NO:2 activity comprising the steps of (a) shuffling a nucleotide sequence of SEQ ID NO:1, (b) expressing the resulting shuffled nucleotide sequences and, (c) selecting for altered activity as compared to the activity of the gene product of said unmodified nucleotide sequence.

The invention further relates to a shuffled polynucleotide sequence produced by a shuffling process, wherein said shuffled DNA molecule encodes a gene product having enhanced tolerance to an inhibitor of SEQ ID NO:2 activity.

The invention further relates to a method for preventing, treating, or ameliorating a medical condition with the polypeptide provided as SEQ ID NO:2, in addition to, its encoding nucleic acid, wherein the medical condition is a female reproductive disorder.

›BRIEF SUMMARY OF THE INVENTION · 3 of 4

The invention further relates to a method of identifying a compound that modulates the biological activity of MMP-29, comprising the steps of, (a) combining a candidate modulator compound with MMP-29 having the sequence set forth in one or more of SEQ ID NO:2; and measuring an effect of the candidate modulator compound on the activity of MMP-29.

The invention further relates to a method of identifying a compound that modulates the biological activity of a metalloproteinase, comprising the steps of, (a) combining a candidate modulator compound with a host cell expressing MMP-29 having the sequence as set forth in SEQ ID NO:2; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed MMP-29.

The invention further relates to a method of identifying a compound that modulates the biological activity of MMP-29, comprising the steps of, (a) combining a candidate modulator compound with a host cell containing a vector described herein, wherein MMP-29 is expressed by the cell; and, (b) measuring an effect of the candidate modulator compound on the activity of the expressed MMP-29.

The invention further relates to a method of screening for a compound that is capable of modulating the biological activity of MMP-29, comprising the steps of: (a) providing a host cell described herein; (b) determining the biological activity of MMP-29 in the absence of a modulator compound; (c) contacting the cell with the modulator compound; and (d)determining the biological activity of MMP-29 in the presence of the modulator compound; wherein a difference between the activity of MMP-29 in the presence of the modulator compound and in the absence of the modulator compound indicates a modulating effect of the compound.

The invention further relates to a compound that modulates the biological activity of human MMP-29 as identified by the methods described herein.

The invention further relates to a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises the structural coordinates of a member of the group consisting of: the MMP-29 propeptide domain model provided in FIG. 7 in accordance with Table IV; the MMP-29 catalytic domain model provided in FIG. 8 in accordance with Table V; and the MMP-29 hemopexin-like domain model provided in FIG. 9 in accordance with Table VI, wherein said computer comprises: (a) A machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the set of structure coordinates of the model; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central-processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into said three-dimensional representation; and (d) a display coupled to said central-processing unit for displaying said three-dimensional representation.

The invention further relates to a method for identifying a MMP-29 mutant with altered biological properties, function, or activity using a model selected from the group consisting of: the MMP-29 propeptide domain model provided in FIG. 7 in accordance with Table IV; the MMP-29 catalytic domain model provided in FIG. 8 in accordance with Table V; and the MMP-29 hemopexin-like domain model provided in FIG. 9 in accordance with Table VI, wherein said method comprises the steps of: (a) using a model of said polypeptide according to the structural coordinates of said model to identify amino acids to mutate; and (b) mutating said amino acids to create a mutant MMP-29 protein with altered biological function or properties wherein the mutant is a member of the group consisting of: (a) a mutant with one or more mutations in the propeptide domain of MMP-29 comprised of amino acids from about A48 to about P120 of SEQ ID NO:2 according to Table IV with altered metalloprotease function or properties; (b) a mutant with one or more mutations in the catalytic domain of MMP-29 comprised of amino acids from about G161 to about E330 of SEQ ID NO:2 according to Table V with altered metalloprotease function or properties; (c) a mutant with one or more mutations in the hemopexin-like domain of MMP-29 comprised of amino acids from about Q346 to about N544 of SEQ ID NO:2 according to Table VI with altered metalloprotease function or properties; (d) a mutant with one or more mutations in the catalytic domain of MMP-29 comprised of amino acids C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and/or Q302 of SEQ ID NO:2 according to Table V with altered metalloprotease function or properties; and (e) a mutant with one or more mutations in the S1 pocket of MMP-29 comprised of amino acids Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2 according to Table V with altered metalloprotease function or properties;

›BRIEF SUMMARY OF THE INVENTION · 4 of 4

The invention further relates to a method for designing or selecting compounds as potential modulators of a member of the group consisting of: the MMP-29 propeptide domain model provided in FIG. 7 in accordance with Table IV; the MMP-29 catalytic domain model provided in FIG. 8 in accordance with Table V; and the MMP-29 hemopexin-like domain model provided in FIG. 9 in accordance with Table VI, wherein said method comprises the steps of: (a) identifying a structural or chemical feature of said member using the structural coordinates of said member; (b) and

rationally designing compounds that bind to said feature.

The present invention also relates to an isolated polynucleotide consisting of a portion of the human MMP-29 gene consisting of at least 8 bases, specifically excluding Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to an isolated polynucleotide consisting of a nucleotide sequence encoding a fragment of the human MMP-29 protein, wherein said fragment displays one or more functional activities specifically excluding Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to the polynucleotide of SEQ ID NO:1 consisting of at least 10 to 50 bases, wherein said at least 10 to 50 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to the polynucleotide of SEQ ID NO:1 consisting of at least 15 to 100 bases, wherein said at least 15 to 100 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to the polynucleotide of SEQ ID NO:1 consisting of at least 100 to 1000 bases, wherein said at least 100 to 1000 bases specifically exclude the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

The present invention also relates to an isolated polypeptide fragment of the human MMP-29 protein, wherein said polypeptide fragment does not consist of the polypeptide encoded by the polynucleotide sequence of Genbank Accession Nos. AW665196, and/or BG234242.

›BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS · 1 of 3

FIGS. 1A-B show the polynucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of the novel human metalloproteinase, MMP-29, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 1707 nucleotides (SEQ ID NO:1), encoding a polypeptide of 569 amino acids (SEQ ID NO:2). An analysis of the MMP-29 polypeptide determined that it comprised the following features: a predicted signal sequence located from about amino acid 1 to amino acid 24 of SEQ ID NO:2 ( FIGS. 1A-B ) represented by double underlining; a predicted propeptide domain located from about amino acid 48 to amino acid 120 of SEQ ID NO:2 ( FIGS. 1A-B ) represented by single underlining; a predicted catalytic domain located from about amino acid 161 to amino acid 330 of SEQ ID NO:2 ( FIGS. 1A-B ) represented by light shading; a predicted hemopexin-like domain located from about amino acid 346 to amino acid 544 of SEQ ID NO:2 ( FIGS. 1A-B ) represented by dotted underlinine; three amino acid residues predicted to coordinate the catalytic zinc ion located at amino acid 283, 287, and 293 of SEQ ID NO:2 ( FIGS. 1A-B ) represented by dark shading; and a predicted “Met-turn” methionine is located at amino acid 301 of SEQ ID NO:2 ( FIGS. 1A-B ) represented in bold.

FIGS. 2A-C show the regions of identity and similarity between the encoded MMP-29 protein (SEQ ID NO:2) to other metalloproteinases, specifically, the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4). The alignment was performed using the CLUSTALW algorithm described elsewhere herein. The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Lines between residues indicate gapped regions for the aligned polypeptides.

FIG. 3 shows an expression profile of the novel human metalloproteinase, MMP-29. The figure illustrates the relative expression level of MMP-29 amongst various mRNA tissue sources. As shown, transcripts corresponding to MMP-29 expressed highly in the testis. The MMP-29 polypeptide was also expressed significantly in small intestine, liver, and to a lesser extent, in lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain. Expression data was obtained by measuring the steady state MMP-29 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:30 and 31 as described herein.

FIG. 4 shows a hydrophobicity plot of MMP-29 according to the BioPlot Hydrophobicity algorithm of Vector NTI (version 5.5). The six hydrophilic peaks of the HGPRBMY29 polypeptide are clearly visible.

FIG. 5 shows a table illustrating the percent identity and percent similarity between the MMP-29 polypeptide of the present invention with other metalloproteinases, specifically, the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4). The percent identity and percent similarity values were determined using the GAP algorithm (GCG suite of programs; and Henikoff, S. and Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89: 10915-10919(1992)) using the following parameters: gap weight=8, and length weight=2.

FIG. 6 shows an expanded expression profile of the novel human metalloproteinase, MMP-29. The figure illustrates the relative expression level of MMP-29 amongst various mRNA tissue sources. As shown, the MMP-29 polypeptide was expressed at relatively low levels in a majority of the tissues tested, though predominately in tissues of the female reproductive system, such as the ovary, and significant expression observed in the brain sub region nucleus accumbens, followed by the choroid-plexus and the testis. Expression data was obtained by measuring the steady state MMP-29 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:68 and 69, and Taqman probe (SEQ ID NO:70) as described in Example 5 herein.

FIG. 7 shows a three-dimensional homology model of the propeptide domain of the MMP-29 polypeptide based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73). The predicted cysteine (“C117”) that is predicted to ligate the catalytic zinc is displayed in space filling representation and labeled, accordingly. The structural coordinates of the propeptide domain of MMP-29 polypeptide are provided in Table IV herein. The homology model of MMP-29 was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

FIG. 8 shows a three-dimensional homology model of the catalytic domain of the MMP-29 polypeptide based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73). The predicted active site amino acids of the catalytic domain of the human MMP-29 polypeptide (“H283”, “H287”, and “H293”) are labeled. The structural coordinates of the propeptide domain of MMP-29 polypeptide are provided in Table V herein. The homology model of MMP-29 was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

›BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS · 2 of 3

FIG. 9 shows a three-dimensional homology model of the hemopexin-like domain of the MMP-29 polypeptide based upon the homologous structure of a portion of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74). The hemopexin-like domain amino acids of the human MMP-29 polypeptide correspond to amino acids Q346 to N544 of SEQ ID NO:2. The structural coordinates of the hemopexin-like domain of MMP-29 polypeptide are provided in Table VI herein. The homology model of MMP-29 was derived from generating a sequence alignment with the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) using the Proceryon suite of software (Proceryon Biosciences, Inc. N.Y., N.Y.), and the overall atomic model including plausible sidechain orientations using the program LOOK (V3.5.2, Molecular Applications Group).

FIG. 10 shows an energy graph for the model of the MMP-29 propeptide domain (see FIG. 7 ) of the present invention (dotted line) and the human fibroblast stromelysin-1 proenzyme template (PDB code 1slmA) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the MMP-29 propeptide domain model and 1slmA template have similar energies over the aligned region, suggesting that the structural model of the MMP-29 propeptide domain represents a “native-like” conformation of the MMP-29 propeptide domain. This graph supports the motif and sequence alignments in confirming that the three-dimensional structure coordinates of MMP-29 propeptide domain are an accurate and useful representation of the structure of the MMP-29 propeptide domain.

FIG. 11 shows an energy graph for the model of the MMP-29 catalytic domain (see FIG. 8 ) of the present invention (dotted line) and the human fibroblast stromelysin-1 proenzyme template (PDB code 1slmA) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the MMP-29 catalytic domain model and 1slmA template have similar energies over the aligned region, suggesting that the structural model of the MMP-29 catalytic domain represents a “native-like” conformation of the MMP-29 catalytic domain. This graph supports the motif and sequence alignments in confirming that the three-dimensional structure coordinates of MMP-29 catalytic domain are an accurate and useful representation of the structure of the MMP-29 catalytic domain.

FIG. 12 shows an energy graph for the model of the MMP-29 hemopexin-like domain (see FIG. 9 ) of the present invention (dotted line) and the pig fibroblast (Interstitial) collagenase Mmp-1 (PDB code 1fblA) (solid line) from which the model was generated. The energy distribution for each protein fold is displayed on the y-axis, while the amino acid residue position of the protein fold is displayed on the x-axis. As shown, the MMP-29 hemopexin-like domain model and 1slmA template have similar energies over the aligned region, suggesting that the structural model of the MMP-29 hemopexin-like domain represents a “native-like” conformation of the MMP-29 hemopexin-like domain. This graph supports the motif and sequence alignments in confirming that the three-dimensional structure coordinates of MMP-29 hemopexin-like domain are an accurate and useful representation of the structure of the MMP-29 hemopexin-like domain.

FIGS. 13A-B shows the regions of identity and similarity between the encoded MMP-29 protein (SEQ ID NO:2) to a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73), and to a portion of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74). Both stromelysin-1 and collagenase Mmp-1 are metalloproteinases in which the x-ray crystal structure has been solved (Bernstein et al., J. Mol. Biol. 112:535-542 (1977); and Li et al., Structure 3: 541-549 (1995); respectively). As described herein, portions of the x-ray structures of the stromelysin-1 proenzyme and collagenase Mmp-1 were used as the basis for building the various homology models of propeptide domain, catalytic domain, and hemopexin-like domain of the MMP-29 polypeptide of the present invention, as represented in Tables IV, V, and VI. Amino acids defining the cysteine switch are highlighted with the (“@”) sign above each intended residue; and the histidines from the catalytic domain are highlighted with asterisks (“*”) above each intended residue.

Table I provides a summary of the novel polypeptides and their encoding polynucleotides of the present invention.

Table II illustrates the preferred hybridization conditions for the polynucleotides of the present invention. Other hybridization conditions may be known in the art or are described elsewhere herein.

Table III provides a summary of various conservative substitutions encompassed by the present invention.

Table IV provides the structural coordinates of the homology model of the propeptide domain of MMP-29 provided in FIG. 7 . A description of the headings are as follows: “Atom No” refers to the atom number within the MMP-29 propeptide domain homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the MMP-29 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

Table V provides the structural coordinates of the homology model of the catalytic domain of MMP-29 provided in FIG. 8 . A description of the headings are as follows: “Atom No” refers to the atom number within the MMP-29 catalytic domain homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the MMP-29 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

›BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS · 3 of 3

Table VI provides the structural coordinates of the homology model of the hemopexin-like domain of MMP-29 provided in FIG. 9 . A description of the headings are as follows: “Atom No” refers to the atom number within the MMP-29 hemopexin-like domain homology model; “Atom Name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid of the MMP-29 polypeptide within which the atom resides, in addition to the amino acid position in which the atom resides; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

›DETAILED DESCRIPTION OF THE INVENTION · 1 of 64

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein.

The invention provides a novel human sequence that encodes a metalloproteinase with substantial homology to the class of metalloproteinases known as matrix metalloproteinases, particularly members of the peptidase M10 class of proteases. Metalloproteinases of this class have been implicated in a number of diseases and disorders which include, for example, fibrillogenesis, angiogenesis, rheumatoid arthritis, osteoarthritis, enamel formation, atherosclerosis, neural degeneration, diabetic renal lesions, and ulceration, for example. In addition, expression analysis indicates the MMP-29 has strong preferential expression in testis; significant expression in small intestine, liver, and to a lesser extent, in lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain. Based on this information, we have provisionally named the gene and protein MMP-29.

In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:1 or the cDNA contained within the clone deposited with the ATCC. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without a signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ ID NO:1 was often generated by overlapping sequences contained in one or more clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:1 was deposited with the American Type Culture Collection (“ATCC”). As shown in Table I, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The deposited clone is inserted in the pSport1 plasmid (Life Technologies) using the NotI and SalI restriction endonuclease cleavage sites.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373, preferably a Model 3700, from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequence in FIGS. 1A-B (SEQ ID NO: 1), a nucleic acid molecule of the present invention encoding the MMP-29 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 1A-B (SEQ ID NO: 1) was discovered in a cDNA library derived from human brain and testis.

›DETAILED DESCRIPTION OF THE INVENTION · 2 of 64

A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:1, the complement thereof, or the cDNA within the clone deposited with the ATCC. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% form amide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:1” refers to a polynucleotide sequence while “SEQ ID NO:2” refers to a polypeptide sequence, both sequences identified by an integer specified in Table I.

“A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention.)

›DETAILED DESCRIPTION OF THE INVENTION · 3 of 64

As used herein the terms “modulate” or “modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein. The definition of “modulate” or “modulates” as used herein is meant to encompass agonists and/or antagonists of a particular activity, DNA, RNA, or protein.

The term “organism” as referred to herein is meant to encompass any organism referenced herein, though preferably to eukaryotic organisms, more preferably to mammals, and most preferably to humans.

The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that described by Ozenberger and Young (Mol Endocrinol., 9(10):1321-9, (1995); and Ann. N.Y. Acad. Sci., 7;766:279-81, (1995)).

The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of this or a related sequence, as probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.

Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.

Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, paralogs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the invention, non-coding regions of the invention, regulatory sequences of the invention, and secreted, mature, pro-, prepro-, forms of the invention (as applicable).

In preferred embodiments, the invention provides methods for identifying the glycosylation sites inherent in the polynucleotides and polypeptides of the invention, and the subsequent alteration, deletion, and/or addition of said sites for a number of desirable characteristics which include, but are not limited to, augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

In further preferred embodiments, methods are provided for evolving the polynucleotides and polypeptides of the present invention using molecular evolution techniques in an effort to create and identify novel variants with desired structural, functional, and/or physical characteristics.

The present invention further provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods (e.g., quantitative PCR), anti-sense methodology, gene knockout experiments, and other procedures that could use sequence information from clones to build a primer or a hybrid partner.

Polynucleotides and Polypeptides of the Invention

Features of the Polypeptide Encoded by Gene No:1

The polypeptide of this gene provided as SEQ ID NO:2 ( FIGS. 1A-B ), encoded by the polynucleotide sequence according to SEQ ID NO:1 ( FIGS. 1A-B ), and/or encoded by the polynucleotide contained within the deposited clone, MMP-29, has significant homology at the nucleotide and amino acid level to a number of metalloproteinases, which include, the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4). An alignment of the MMP-29 polypeptide with these proteins is provided in FIGS. 2A-C . Based upon such strong conservation, the inventors have ascribed the MMP-29 polypeptide as having proteolytic activity, preferably metalloproteinase activity.

The MMP-25 matrix metalloproteinase, also referred to as leukolysin and/or MT6-MMP, is thought to function in the activation of progelatinase A. MMP-25 was found to require calcium and zinc for activity, and is attached to the membrane by a GPI-anchor. MMP-25 was found to be expressed predominantly in leukocytes, lung, spleen, in addition to, colon carcinomas, astrocytomas, and glioblastomas. The precursor of MMP-25 is cleaved by a furin endopeptidase. MMP-25 is thought to be specifically involved in the proteolytic arsenal deployed by leukocytes during inflammatory responses (Cell Res. 9 (4), 291-303 (1999)). More recently, MMP-25 has been shown to be expressed in brain tissues where it facilitates tumor progression through its ability to activate progelatinase A at the membrane of cells from colon carcinomas or brain tumors (Cancer Res. 60 (4), 877-882 (2000)). Addition information related to MMP-25 may be found in reference to the following publication: FEBS Lett. 480 (2-3), 142-146 (2000), which is hereby incorporated herein by reference.

The MMP-17 matrix metalloproteinase, also referred to as MT4-MMP, is also thought to function in the activation of membrane-bound precursors of growth factors or inflammatory mediators, such as tumor necrosis factor-alpha. MMP-17 may also be involved in tumoral processes, although its ability to proteolytically activate progelatinase A is not known. Interestingly, MMP-17 is not able to hydrolyse collagen types I, II, III, IV and V, gelatin, fibronectin, laminin, decorin nor alpha1-antitrypsin. MMP-17 is a member of the peptidase m10 family, also referred to as the matrixin family, the members of which are zinc metalloproteases that degrade various components of the extracellular matrix, such as fibrin.

›DETAILED DESCRIPTION OF THE INVENTION · 4 of 64

MMP-17 requires calcium and zinc for activity, and is capable of cleaving pro-TNF-alpha at the 74-ALA-|-GLN-75 site. Like MMP-25 above, MMP-17 is attached to the membrane by a GPI-anchor. MMP-17 is primarily expressed in brain, leukocytes, colon, ovary, testis and breast cancer, in addition to, many transformed and non-transformed cell types. Additional information relative to MMP-17 may be found in reference to the following publications: Biol. Chem. 380 (9), 1103-1108 (1999); J. Biol. Chem . . . 274 (48), 34260-34266 (1999); J. Biol. Chem . . . 274 (46), 33043-33049 (1999); Cancer Res. 56 (5), 944-949 (1996); and FEBS Lett. 457 (3), 353-356 (1999); which are hereby incorporated herein by reference.

The MMP-14 matrix metalloproteinase, also refered to as MT-MMP, seems to specifically activate pro-gelatinase A (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E. & Seiki, M., Nature (London) 370, 61-65 (1994)), and may thus trigger invasion by tumor cells by activating pro-gelatinase A on the tumor cell surface (Proc. Natl. Acad. Sci. U.S.A. 92 (7), 2730-2734 (1995)). Like other matrix metalloproteinases, MT-MMP requires calcium and zinc for activity. Conversely, MT-MMP is believed to represent a type I membrane protein. MMP-14 is a zinc metalloproteinase and belongs to peptidase family M10A.

The Xenopus XMMP metalloproteinase is believed to play a role in Xenopus early development based upon its transient expression in Xenupus embryos (J. Biol. Chem . . . 272 (21), 13527-13533 (1997)). Specifically, XMMP mRNA was undetected in the blastula stage embryo, induced in gastrula embryo, expressed in neurula embryo, and then down-regulated in pretailbud embryo.

The Dm1-MMP metalloproteinase is believed to be involved in the extracellular matrix remodeling taking place during the development of the central nervous system in Drosophila (J. Biol. Chem . . . 275 (46), 35978-35985 (2000)).

The MMP-29 polypeptide was determined to have 33.2% identity and 41.0% similarity with the human matrix metalloproteinase 25 protein (MMP-25; SWISS-PROT Accession No: Q9NPA2; SEQ ID NO:7); to have 33.3% identity and 42.1% similarity with the human matrix metalloproteinase 17 protein (MMP-17; SWISS-PROT Accession No: Q9ULZ9; SEQ ID NO:5); to have 35.5% identity and 44.5% similarity with the rat matrix metalloproteinase 14 protein (MMP-14; SWISS-PROT Accession No: Q10739; SEQ ID NO:6); to have 61.6% identity and 70.4% similarity with the Xenopus matrix metalloproteinase protein (XMMP; Genbank Accession No: gi|AAC21447; SEQ ID NO:3); and to have 35.8% identity and 41.4% similarity with the Drosophila matrix metalloproteinase 1 protein (Dm1-MMP; Genbank Accession No: gi|AAG33131; SEQ ID NO:4); as shown in FIG. 5 .

The MMP-29 polypeptide was found to contain the conserved sequence HEIGHVLGLPH (SEQ ID NO:8), fitting the consensus sequence pattern of HE[ILF]GHXXGLXH (SEQ ID NO:81) for all metallopoteinases. MMP-29 also contains an octapeptide PRCGVPDM (SEQ ID NO:9) that fits the highly conserved octapeptide pattern of PRC[GN]XP[DR][LIVSAPKQ] (SEQ ID NO:82) that has been shown to be involved in autoinhibition of metalloproteases (Breathnach R. et al, 1988; Navre M. et al. 1991). Between the autoinhibitive octapeptide and the catalytic peptide there are three pair of RR that possibly serve as the putative cleavage site for activation by furin proteinases. Thus, based upon the sequence and structural homology to known metalloproteases, the novel MMP-29 is believed to represent a novel human secreted metalloprotease.

The MMP-29 polypeptide was determined to comprise a signal sequence from about amino acid 1 to about amino acid 24 of SEQ ID NO:2 ( FIGS. 1A-B ) according to the SPScan computer algorithm (Genetics Computer Group suite of programs). Based upon the predicted signal peptide cleavage site, the mature MMP-29 polypeptide is expected to be from about amino acid 25 to about amino acid 569 of SEQ ID NO:2 ( FIGS. 1A-B ). As this determination was based upon the prediction from a computer algorithm, the exact physiological cleavage site may vary, as discussed more particularly herein. In this context, the term “about” should be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 more amino acids in either the N- or C-terminal direction of the above referenced polypeptide. Polynucleotides encoding these polypeptides are also provided.

In addition to the mature polypeptide above, the polynucleotides encoding the mature polypeptide are also encompassed by the present invention. Specifically, from about nucleotide position 73 to about nucleotide position 1707 of SEQ ID NO:1 ( FIGS. 1A-B ).

As discussed more particularly herein, metalloproteinases are a group of structurally diverse, high molecular weight (400 to 500 amino acids) proteins that have a metal ion within their active site, typically zinc. Despite the structural heterogeneity, metalloproteinases share some well defined structural-functional characteristics, particularly in the active site domain (Zhang, X., Gonnella, N C., Koehn, J., Pathak, N., Ganu, V., Melton, R., Parker, D., Hu, SI., Nam, K Y, J. Mol, Biol., 301(2):513-24, (2000)). Non limiting examples of proteins which are known to belong to the metalloproteinase family of proteins are the following: metalloproteinase 1 thru 26 (MMP-1 to MMP-26); membrane-type 1 matrix metalloproteinase (MT1-MMP); Matrilysin-2; Stromelysin-1, Collagenase-1, ADAMs.

More information relating to metalloproteinases can be found elsewhere herein, or in reference to the following publications: Westerk, J., Kahari, V M, FASEB, J., 13(8):781-92, (1999); Ohtani, H, Pathol, Int., 48(1):1-9, (1998); Stack, M S., Ellerbroek, S M., Fishman, D A, Int, J. Oncol., 12(3):569-76, (1998); Tanaka, S., Hamanishi, C., Kikuchi, H., Fukuda, K, Semin, Arthritis, Rheum., 27(6):392-9, (1998); Yu, A E., Hewitt, R E., Connor, E W., Stetler, Stevenson, W G, Drugs, Aging., 11(3):229-44, (1997).

In preferred embodiments, the MMP-29 polypeptide of the present invention is directed to a polypeptide having structural similarity to metalloproteinases.

›DETAILED DESCRIPTION OF THE INVENTION · 5 of 64

Based upon the strong homology to members of the metalloproteinase family, the MMP-29 polypeptide is expected to share at least some biological activity with metalloproteinases, preferably with matrix metalloproteinases, in addition to other metalloproteinases referenced herein and/or otherwise known in the art.

Expression profiling designed to measure the steady state mRNA levels encoding the MMP-29 polypeptide showed predominately high expression levels in expressed highly in the testis. The MMP-29 polypeptide was also expressed significantly in small intestine, liver, and to a lesser extent, in lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain (See FIG. 4 ).

Expanded analysis of MMP-29 expression levels by TaqMan™ quantitative PCR (see FIG. 6 ) confirmed that the MMP-29 polypeptide is expressed at very low levels compared to the results obtained with SYBR green ( FIG. 4 ). MMP-29 mRNA was expression in the majority of the tissues tested, with slightly greater steady state transcript levels observed in tissues of the female reproductive system, with ovary being the highest. Significant expression was also observed in the brain sub region nucleus accumbens, followed by the choroid-plexus, testis, and to a lesser extent in other tissues as shown. These data suggest that modulators of MMP-29 function may have utility in the treatment of various female reproductive disorders including ovarian cancers. Due to MMP-29 expression in the nucleus accumbens, modulators of MMP-29 may also be useful in the treatment of dysphoria, depression, irritability, and anxiety associated with various drug addictions, especially cocaine.

The analysis of MMP-29 expression in various tumor tissue RNA samples indicates that the steady state levels of MMP-29 are 3.4 fold higher in breast tumors than in matched controls. Considerable literature exists on the role of MMPs and their involvement in breast cancer initiation, invasion and metastasis. Consistent with their role in breast cancer progression, high levels of at least two MMPs (MMP-2 and stromelysin-3) have also been found to correlate with poor prognosis in patients with breast cancer (Duffy M J, Maguire T M, Hill A, McDermott E, O'Higgins N. Breast Cancer Res 2000;2(4):252-7). Because MMPs are apparently involved in breast cancer initiation and dissemination, inhibition of MMP-29 may be of value both in preventing breast cancer and in blocking metastasis of established tumors.

The MMP-29 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating cellular adhesion events, cellular proliferation, and inflammation, in various cells, tissues, and organisms, and particularly in mammalian ovary, testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and brain tissue, preferably human tissue. MMP-29 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing reproductive, gastrointestinal, hepatic, pulmonary, cardiovascular, renal, neural, immune, hematopoietic, metabolic, endocrine, and/or proliferative diseases or disorders.

In preferred embodiments, MMP-29 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, ameliorating, and/or preventing the following diseases or disorders: fibrinolysis, susceptibility to infectious diseases (such as, for example, AIDS), emphysema, liver cirrhosis, hepatocellular carcinoma, thrombosis, embolisms, thrombin-mediated vascular injury, microcirculation in severe sepsis, arterial thrombosis, myocardial infarction, unstable angina, stroke, venous thrombosis, pulmonary embolism, angiogenesis, rheumatoid arthritis, osteoarthritis, enamel formation, atherosclerosis, neural degeneration, diabetic renal lesions and ulceration, multiple sclerosis, experimental autoimmune encephalomyelitis, amyotrophic lateral sclerosis, degenerative conditions affecting extracellular matrix proteins, conditions affecting neurite outgrowth, and synapse formation.

The strong homology to human metalloproteinases, combined with the predominate localized expression in testis tissue suggests the MMP-29 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing reproductice diseases and disorders, particularly male reproductice disorders In preferred embodiments, MMP-29 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the testis: spermatogenesis, infertility, Klinefelter's syndrome, XX male, epididymitis, genital warts, germinal cell aplasia, cryptorchidism, varicocele, immotile cilia syndrome, and viral orchitis. The MMP-29 polynucleotides and polypeptides including agonists and fragments thereof, may also have uses related to modulating testicular development, embryogenesis, reproduction, and in ameliorating, treating, and/or preventing testicular proliferative disorders (e.g., cancers, which include, for example, choriocarcinoma, Nonseminoma, seminona, and testicular germ cell tumors).

Likewise, the predominate localized expression in testis tissue also emphasizes the potential utility for MMP-29 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing metabolic diseases and disorders which include the following, not limiting examples: premature puberty, incomplete puberty, Kallman syndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis, congenital adrenal hyperplasia, FSH deficiency, and granulomatous disease, for example.

This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.

›DETAILED DESCRIPTION OF THE INVENTION · 6 of 64

Other metalloproteinases are known to be expressed in testis and are believed to play a role in reproductive processes. Specifically, MMP-23 (Velasco, G., Pendas, A, M., Fueyo, A., Knauper, V., Murphy, G., Lopez, Otin, C, J. Biol, Chem., 274(8):4570-6, (1999)), MT4-MMP (Puente, X, S., Pendas, A, M., Llano, E., Velasco, G., Lopez, Otin, C, Cancer, Res., 56(5):944-9, (1996)), MMP-18 (Cossins, J., Dudgeon, T, J., Catlin, G., Gearing, A, J., Clements, J. M, Biochem, Biophys, Res, Commun., 228(2):494-8, (1996)), (Will, H., Hinzmann, B, Eur, J. Biochem., 231(3):602-8, (1995)), ADAM20 and ADAM21 (Poindexter, K., Nelson, N., DuBose, R, F., Black, R, A., Cerretti, D, P, Gene., 237(1):61-70, (1999)), and ADAM29 and ADAM30 (Cerretti, D, P., DuBose, R, F., Black, R, A., Nelson, N, Biochem, Biophys, Res, Commun., 263(3):810-5, (1999)); which are incorporated by reference in their entirety.

The strong homology to human metalloproteinase proteins, combined with the localized expression in small intestine suggests the MMP-29 polynucleotides and polypeptides may be useful in treating, diagnosing, prognosing, and/or preventing gastrointesinal diseases and/or disorders, which include, but are not limited to, ulcers, irritable bowel syndrome, inflammatory bowel disease, diarrhea, traveler's diarrhea, drug-related diarrhea polyps, absorption disorders, constipation, diverticulitis, vascular disease of the intestines, intestinal obstruction, intestinal infections, ulcerative colitis, Shigellosis, cholera, Crohn's Disease, amebiasis, enteric fever, Whipple's Disease, peritonitis, intrabdominal abcesses, hereditary hemochromatosis, gastroenteritis, viral gastroenteritis, food poisoning, mesenteric ischemia, mesenteric infarction, in addition to, metabolic diseases and/or disorders.

Moreover, polynucleotides and polypeptides, including fragments and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing susceptibility to the following, non-limiting, gastrointestinal infections: Salmonella infection, E. coli infection, E. coli O157:H7 infection, Shiga Toxin-producing E. coli infection, Campylobacter infection (e.g., Campylobacter fetus, Campylobacter upsaliensis, Campylobacter hyointestinalis, Campylobacter lari, Campylobacter jejuni, Campylobacter concisus, Campylobacter mucosalis, Campylobacter sputorum, Campylobacter rectus, Campylobacter curvus, Campylobacter sputorum , etc.), Heliobacter infection (e.g., Heliobacter cinaedi, Heliobacter fennelliae , etc.) Yersinia enterocolitica infection, Vibrio sp. Infection (e.g., Vibrio mimicus, Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio vulnificus, Vibrio alginolyticus, Vibrio metschnikovii, Vibrio damsela, Vibrio cincinnatiensis , etc.) Aeromonas infection (e.g., Aeromonas hydrophila, Aeromonas sobira, Aeromonas caviae , etc.), Plesiomonas shigelliodes infection, Giardia infection (e.g., Giardia lamblia , etc.), Cryptosporidium infection, Listeria infection, Entamoeba histolytica infection, Rotavirus infection, Norwalk virus infection, Clostridium difficile infection, Clostriudium perfringens infection, Staphylococcus infection, Bacillus infection, in addition to any other gastrointestinal disease and/or disorder implicated by the causative agents listed above or elsewhere herein.

Moreover, the tissue distribution in liver indicates the protein product of this clone would be useful for the detection and treatment of liver disorders and cancers. Representative uses are described in the “Hyperproliferative Disorders”, “Infectious Disease”, and “Binding Activity” sections below, and elsewhere herein. Briefly, the protein can be used for the detection, treatment, and/or prevention of hepatoblastoma, jaundice, hepatitis, liver metabolic diseases and conditions that are attributable to the differentiation of hepatocyte progenitor cells, cirrhosis, hepatic cysts, pyrogenic abscess, amebic abcess, hydatid cyst, cystadenocarcinoma, adenoma, focal nodular hyperplasia, hemangioma, hepatocellulae carcinoma, cholangiocarcinoma, angiosarcoma, granulomatous liver disease. In addition the expression in fetus would suggest a useful role for the protein product in developmental abnormalities, fetal deficiencies, pre-natal disorders and various would-healing diseases and/or tissue trauma.

Moreover, polynucleotides and polypeptides, including fragments and/or antagonists thereof, have uses which include, directly or indirectly, treating, preventing, diagnosing, and/or prognosing the following, non-limiting, hepatic infections: liver disease caused by sepsis infection, liver disease caused by bacteremia, liver disease caused by Pneomococcal pneumonia infection, liver disease caused by Toxic shock syndrome, liver disease caused by Listeriosis, liver disease caused by Legionnaries' disease, liver disease caused by Brucellosis infection, liver disease caused by Neisseria gonorrhoeae infection, liver disease caused by Yersinia infection, liver disease caused by Salmonellosis, liver disease caused by Nocardiosis, liver disease caused by Spirochete infection, liver disease caused by Treponema pallidum infection, liver disease caused by Brrelia burgdorferi infection, liver disease caused by Leptospirosis, liver disease caused by Coxiella burnetii infection, liver disease caused by Rickettsia richettsii infection, liver disease caused by Chlamydia trachomatis infection, liver disease caused by Chlamydia psittaci infection, in addition to any other hepatic disease and/or disorder implicated by the causative agents listed above or elsewhere herein.

In addition, antagonists of the MMP-29 polynucleotides and polypeptides may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to hyper metalloproteinase activity, which may include immune and/or proliferative diseases or disorders, particularly thrombosis, embolism, and other blood disorders. Therapeutic and/or pharmaceutical compositions comprising the MMP-29 polypeptides may be formulated to comprise heparin.

›DETAILED DESCRIPTION OF THE INVENTION · 7 of 64

Moreover, MMP-29 polynucleotides and polypeptides, including fragments and agonists thereof, may have uses which include treating, diagnosing, prognosing, and/or preventing hyperproliferative disorders, particularly of the reproductive, gastrointestinal, and hepatic systems. Such disorders may include, for example, cancers, and metastasis.

MMP-29 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have uses which include identification of modulators of MMP-29 function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains (including MMP-29 epitopes provided herein) of the MMP-29 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.

MMP-29 polypeptides and polynucleotides are useful for diagnosing diseases related to over or under expression of MMP-29 proteins by identifying mutations in the MMP-29 gene using MMP-29 probes, or determining MMP-29 protein or mRNA expression levels. MMP-29 polypeptides are also useful for screening for compounds, which affect activity of the protein. Diseases that can be treated with MMP-29 include, the following, non-limiting examples: neuro-regeneration, neuropathic pain, obesity, anorexia, HIV infections, cancers, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, osteoporosis, angina pectoris, myocardial infarction, psychotic, immune, metabolic, cardiovascular, and neurological disorders.

The MMP-29 polynucleotides and polypeptides, including fragments and/or antagonsists thereof, may have used which include identification of modulators of metalloproteinase function including antibodies (for detection or neutralization), naturally-occurring modulators and small molecule modulators. Antibodies to domains of the MMP-29 protein could be used as diagnostic agents of inflammatory conditions in patients, are useful in monitoring the activation and presence of cognate proteases, and can be used as a biomarker for the protease involvement in disease states and in the evaluation of inhibitors of the cognate protease in vivo.

Molecular genetic manipulation of the structure of the active site domain, particularly the metal binding domain, and of other functional domains in the metalloproteinase superfamily enables the production of metalloproteinases with tailor-made activities. Thus, the MMP-29 polypeptides, and fragments thereof, as well as any homologous product resulting from genetic manipulation of the structure, are useful for NMR-based design of modulators of MMP-29 biological activity, and metalloproteinase, in general.

MMP-29 polypeptides and polynucleotides have additional uses which include diagnosing diseases related to the over and/or under expression of MMP-29 by identifying mutations in the MMP-29 gene by using MMP-29 sequences as probes or by determining MMP-29 protein or mRNA expression levels. MMP-29 polypeptides may be useful for screening compounds that affect the activity of the protein. MMP-29 peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with MMP-29 (described elsewhere herein).

Although it is believed the encoded polypeptide may share at least some biological activities with human metalloproteinases (particularly O-sialoglycoprotein endopeptidases), a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the MMP-29 polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from diseased testicular tissue, as compared to, normal tissue might indicate a function in modulating reproductive function, for example. In the case of MMP-29, testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and/or brain tissue should be used to extract RNA to prepare the probe.

In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the MMP-29 gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. In the case of MMP-29, a disease correlation related to MMP-29 may be made by comparing the mRNA expression level of MMP-29 in normal tissue, as compared to diseased tissue (particularly diseased tissue isolated from the following: ovary, testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, and/or brain tissue). Significantly higher or lower levels of MMP-29 expression in the diseased tissue may suggest MMP-29 plays a role in disease progression, and antagonists against MMP-29 polypeptides would be useful therapeutically in treating, preventing, and/or ameliorating the disease. Alternatively, significantly higher or lower levels of MMP-29 expression in the diseased tissue may suggest MMP-29 plays a defensive role against disease progression, and agonists of MMP-29 polypeptides may be useful therapeutically in treating, preventing, and/or ameliorating the disease. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:1 ( FIGS. 1A-B ).

›DETAILED DESCRIPTION OF THE INVENTION · 8 of 64

The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the MMP-29, transforming yeast deficient in metalloproteinase activity, and assessing their ability to grow would provide convincing evidence the MMP-29 polypeptide has metalloproteinase activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.

Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a testis, small intestine, liver, lung, stomach, heart, kidney, spinal cord, lymph node, pancreas, bone marrow, prostate, uterus, thymus, or brain tissue specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.

In the case of MMP-29 transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (reproductive, gastrointestinal, hepatic, pulmonary, cardiovascular, renal, neural, immune, hematopoietic, metabolic, endocrine, cancers etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.

In preferred embodiments, the following N-terminal MMP-29 deletion polypeptides are encompassed by the present invention: M1-M569, L2-M569, A3-M569, A4-M569, S5-M569, I6-M569, F7-M569, R8-M569, P9-M569, T10-M569, L11-M569, L12-M569, L13-M569, C14-M569, W15-M569, L16-M569, A17-M569, A18-M569, P19-M569, W20-M569, P21-M569, T22-M569, Q23-M569, P24-M569, E25-M569, S26-M569, L27-M569, F28-M569, H29-M569, S30-M569, R31-M569, D32-M569, R33-M569, S34-M569, D35-M569, L36-M569, E37-M569, P38-M569, S39-M569, P40-M569, L41-M569, R42-M569, Q43-M569, A44-M569, K45-M569, P46-M569, I47-M569, A48-M569, D49-M569, L50-M569, H51-M569, A52-M569, A53-M569, Q54-M569, R55-M569, F56-M569, L57-M569, S58-M569, R59-M569, Y60-M569, G61-M569, W62-M569, S63-M569, G64-M569, V65-M569, W66-M569, A67-M569, A68-M569, W69-M569, G70-M569, P71-M569, S72-M569, P73-M569, E74-M569, G75-M569, P76-M569, P77-M569, E78-M569, T79-M569, P80-M569, K81-M569, G82-M569, A83-M569, A84-M569, L85-M569, A86-M569, E87-M569, A88-M569, V89-M569, R90-M569, R91-M569, F92-M569, Q93-M569, R94-M569, A95-M569, N96-M569, A97-M569, L98-M569, P99-M569, A10-M569, S101-M569, G102-M569, E103-M569, L104-M569, D105-M569, A106-M569, A107-M569, T108-M569, L109-M569, A10-M569, A111-M569, M112-M569, N113-M569, R114-M569, P115-M569, R116-M569, C117-M569, G118-M569, V119-M569, P120-M569, D121-M569, M122-M569, R123-M569, P124-M569, P125-M569, P126-M569, P127-M569, S128-M569, A129-M569, P130-M569, P131-M569, S132-M569, P133-M569, P134-M569, G135-M569, P136-M569, P137-M569, P138-M569, R139-M569, A140-M569, R141-M569, S142-M569, R143-M569, R144-M569, S145-M569, P146-M569, R147-M569, A148-M569, P149-M569, L150-M569, S151-M569, L152-M569, S153-M569, R154-M569, R155-M569, G156-M569, W157-M569, Q158-M569, P159-M569, R160-M569, G161-M569, Y162-M569, P163-M569, D164-M569, G165-M569, G166-M569, A167-M569, A168-M569, Q169-M569, A170-M569, F171-M569, S172-M569, K173-M569, R174-M569, T175-M569, L176-M569, S177-M569, W178-M569, R179-M569, L180-M569, L181-M569, G182-M569, E183-M569, A184-M569, L185-M569, S186-M569, S187-M569, Q188-M569, L189-M569, S190-M569, V191-M569, A192-M569, D193-M569, Q194-M569, R195-M569, R196-M569, I197-M569, V198-M569, A199-M569, L200-M569, A201-M569, F202-M569, R203-M569, M204-M569, W205-M569, S206-M569, E207-M569, V208-M569, T209-M569, P210-M569, L211-M569, D212-M569, F213-M569, R214-M569, E215-M569, D216-M569, L217-M569, A218-M569, A219-M569, P220-M569, G221-M569, A222-M569, A223-M569, V224-M569, D225-M569, I226-M569, K227-M569, L228-M569, G229-M569, F230-M569, G231-M569, R232-M569, G233-M569, R234-M569, H235-M569, L236-M569, G237-M569, C238-M569, P239-M569, R240-M569, A241-M569, F242-M569, D243-M569, G244-M569, S245-M569, G246-M569, Q247-M569, E248-M569, F249-M569, A250-M569, H251-M569, A252-M569, W253-M569, R254-M569, L255-M569, G256-M569, D257-M569, I258-M569, H259-M569, F260-M569, D261-M569, D262-M569, D263-M569, E264-M569, H265-M569, F266-M569, T267-M569, P268-M569, P269-M569, T270-M569, S271-M569, D272-M569, T273-M569, G274-M569, I275-M569, S276-M569, L277-M569, L278-M569, K279-M569, V280-M569, A281-M569, V282-M569, H283-M569, E284-M569, I285-M569, G286-M569, H287-M569, V288-M569, L289-M569, G290-M569, L291-M569, P292-M569, H293-M569, T294-M569, Y295-M569, R296-M569, T297-M569, G298-M569, S299-M569, I300-M569, M301-M569, Q302-M569, P303-M569, N304-M569, Y305-M569, I306-M569, P307-M569, Q308-M569, E309-M569, P310-M569, A311-M569, F312-M569, E313-M569, L314-M569, D315-M569, W316-M569, S317-M569, D318-M569, R319-M569, K320-M569, A321-M569, 1322-M569, Q323-M569, K324-M569, L325-M569, Y326-M569, G327-M569, S328-M569, C329-M569, E330-M569, G331-M569, S332-M569, F333-M569, D334-M569, T335-M569, A336-M569, F337-M569, D338-M569, W339-M569, 1340-M569, R341-M569, K342-M569, E343-M569, R344-M569, N345-M569, Q346-M569, Y347-M569, G348-M569, E349-M569, V350-M569, M351-M569, V352-M569, R353-M569, F354-M569, S355-M569, T356-M569, Y357-M569, F358-M569, F359-M569, R360-M569, N361-M569, S362-M569, W363-M569, Y364-M569, W365-M569, L366-M569, Y367-M569, E368-M569, N369-M569, R370-M569, N371-M569, N372-M569, R373-M569, T374-M569, R375-M569, Y376-M569, G377-M569, D378-M569, P379-M569, I380-M569, Q381-M569, 1382-M569, L383-M569, T384-M569, G385-M569, W386-M569, P387-M569, G388-M569, 1389-M569, P390-M569, T391-M569, H392-M569, N393-M569, 1394-M569, D395-M569, A396-M569, F397-M569, V398-M569, H399-M569, 1400-M569, W401-M569, T402-M569, W403-M569, K404-M569, R405-M569, D406-M569, E407-M569, R408-M569, Y409-M569, F410-M569, F411-M569, Q412-M569, G413-M569, N414-M569, Q415-M569, Y416-M569, W417-M569, R418-M569, Y419-M569, D420-M569, S421-M569, D422-M569, K423-M569, D424-M569, Q425-M569, A426-M569, L427-M569, T428-M569, E429-M569, D430-M569, E431-M569, Q432-M569, G433-M569, K434-M569, S435-M569, Y436-M569, P437-M569, K438-M569, L439-M569, 1440-M569, S441-M569, E442-M569, G443-M569, F444-M569, P445-M569, G446-M569, 1447-M569, P448-M569, S449-M569, P450-M569, L451-M569, D452-M569, T453-M569, A454-M569, F455-M569, Y456-M569, D457-M569, R458-M569, R459-M569, Q460-M569, K461-M569, L462-M569, 1463-M569, Y464-M569, F465-M569, F466-M569, K467-M569, E468-M569, S469-M569, L470-M569, V471-M569, F472-M569, A473-M569, F474-M569, D475-M569, V476-M569, N477-M569, R478-M569, N479-M569, R480-M569, V481-M569, L482-M569, N483-M569, S484-M569, Y485-M569, P486-M569, K487-M569, R488-M569, 1489-M569, T490-M569, E491-M569, V492-M569, F493-M569, P494-M569, A495-M569, V496-M569, 1497-M569, P498-M569, Q499-M569, N500-M569, H501-M569, P502-M569, F503-M569, R504-M569, N505-M569, 1506-M569, D507-M569, S508-M569, A509-M569, Y510-M569, Y511-M569, S512-M569, Y513-M569, A514-M569, Y515-M569, N516-M569, S517-M569, 1518-M569, F519-M569, F520-M569, F521-M569, K522-M569, G523-M569, N524-M569, A525-M569, Y526-M569, W527-M569, K528-M569, V529-M569, V530-M569, N531-M569, D532-M569, K533-M569, D534-M569, K535-M569, Q536-M569, Q537-M569, N538-M569, S539-M569, W540-M569, L541-M569, P542-M569, A543-M569, N544-M569, G545-M569, L546-M569, F547-M569, P548-M569, K549-M569, K550-M569, F551-M569, I552-M569, S553-M569, E554-M569, K555-M569, W556-M569, F557-M569, D558-M569, V559-M569, C560-M569, D561-M569, V562-M569, and/or H563-M569 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 9 of 64

In preferred embodiments, the following C-terminal MMP-29 deletion polypeptides are encompassed by the present invention: M1-M569, M1-N568, M1-L567, M1-T566, M1-S565, M1-I564, M1-H563, M1-V562, M1-D561, M1-C560, M1-V559, M1-D558, M1-F557, M1-W556, M1-K555, M1-E554, M1-S553, M1-1552, M1-F551, M1-K550, M1-K549, M1-P548, M1-F547, M1-L546, M1-G545, M1-N544, M1-A543, M1-P542, M1-L541, M1-W540, M1-S539, M1-N538, M1-Q537, M1-Q536, M1-K535, M1-D534, M1-K533, M1-D532, M1-N531, M1-V530, M1-V529, M1-K528, M1-W527, M1-Y526, M1-A525, M1-N524, M1-G523, M1-K522, M1-F521, M1-F520, M1-F519, M1-I518, M1-S517, M1-N516, M1-Y515, M1-A514, M1-Y513, M1-S512, M1-Y511, M1-Y510, M1-A509, M1-S508, M1-D507, M1-I506, M1-N505, M1-R504, M1-F503, M1-P502, M1-H501, M1-N500, M1-Q499, M1-P498, M1-I497, M1-V496, M1-A495, M1-P494, M1-F493, M1-V492, M1-E491, M1-T490, M1-I489, M1-R488, M1-K487, M1-P486, M1-Y485, M1-S484, M1-N483, M1-L482, M1-V481, M1-R480, M1-N479, M1-R478, M1-N477, M1-V476, M1-D475, M1-F474, M1-A473, M1-F472, M1-V471, M1-L470, M1-S469, M1-E468, M1-K467, M1-F466, M1-F465, M1-Y464, M1-I463, M1-L462, M1-K461, M1-Q460, M1-R459, M1-R458, M1-D457, M1-Y456, M1-F455, M1-A454, M1-T453, M1-D452, M1-L451, M1-P450, M1-S449, M1-P448, M1-I447, M1-G446, M1-P445, M1-F444, M1-G443, M1-E442, M1-S441, M1-I440, M1-L439, M1-K438, M1-P437, M1-Y436, M1-S435, M1-K434, M1-G433, M1-Q432, M1-E431, M1-D430, M1-E429, M1-T428, M1-L427, M1-A426, M1-Q425, M1-D424, M1-K423, M1-D422, M1-S421, M1-D420, M1-Y419, M1-R418, M1-W417, M1-Y416, M1-Q415, M1-N414, M1-G413, M1-Q412, M1-F411, M1-F410, M1-Y409, M1-R408, M1-E407, M1-D406, M1-R405, M1-K404, M1-W403, M1-T402, M1-W401, M1-I400, M1-H399, M1-V398, M1-F397, M1-A396, M1-D395, M1-I394, M1-N393, M1-H392, M1-T391, M1-P390, M1-I389, M1-G388, M1-P387, M1-W386, M1-G385, M1-T384, M1-L383, M1-I382, M1-Q381, M1-I380, M1-P379, M1-D378, M1-G377, M1-Y376, M1-R375, M1-T374, M1-R373, M1-N372, M1-N371, M1-R370, M1-N369, M1-E368, M1-Y367, M1-L366, M1-W365, M1-Y364, M1-W363, M1-S362, M1-N361, M1-R360, M1-F359, M1-F358, M1-Y357, M1-T356, M1-S355, M1-F354, M1-R353, M1-V352, M1-M351, M1-V350, M1-E349, M1-G348, M1-Y347, M1-Q346, M1-N345, M1-R344, M1-E343, M1-K342, M1-R341, M1-I340, M1-W339, M1-D338, M1-F337, M1-A336, M1-T335, M1-D334, M1-F333, M1-S332, M1-G331, M1-E330, M1-C329, M1-S328, M1-G327, M1-Y326, M1-L325, M1-K324, M1-Q323, M1-I322, M1-A321, M1-K320, M1-R319, M1-D318, M1-S317, M1-W316, M1-D315, M1-L314, M1-E313, M1-F312, M1-A311, M1-P310, M1-E309, M1-Q308, M1-P307, M1-I306, M1-Y305, M1-N304, M1-P303, M1-Q302, M1-M301, M1-I300, M1-S299, M1-G298, M1-T297, M1-R296, M1-Y295, M1-T294, M1-H293, M1-P292, M1-L291, M1-G290, M1-L289, M1-V288, M1-H287, M1-G286, M1-I285, M1-E284, M1-H283, M1-V282, M1-A281, M1-V280, M1-K279, M1-L278, M1-L277, M1-S276, M1-I275, M1-G274, M1-T273, M1-D272, M1-S271, M1-T270, M1-P269, M1-P268, M1-T267, M1-F266, M1-H265, M1-E264, M1-D263, M1-D262, M1-D261, M1-F260, M1-H259, M1-I258, M1-D257, M1-G256, M1-L255, M1-R254, M1-W253, M1-A252, M1-H251, M1-A250, M1-F249, M1-E248, M1-Q247, M1-G246, M1-S245, M1-G244, M1-D243, M1-F242, M1-A241, M1-R240, M1-P239, M1-C238, M1-G237, M1-L236, M1-H235, M1-R234, M1-G233, M1-R232, M1-G231, M1-F230, M1-G229, M1-L228, M1-K227, M1-I226, M1-D225, M1-V224, M1-A223, M1-A222, M1-G221, M1-P220, M1-A219, M1-A218, M1-L217, M1-D216, M1-E215, M1-R214, M1-F213, M1-D212, M1-L211, M1-P210, M1-T209, M1-V208, M1-E207, M1-S206, M1-W205, M1-M204, M1-R203, M1-F202, M1-A201, M1-L200, M1-A199, M1-V198, M1-I197, M1-R196, M1-R195, M1-Q194, M1-D193, M1-A192, M1-V191, M1-S190, M1-L189, M1-Q188, M1-S187, M1-S186, M1-L185, M1-A184, M1-E183, M1-G182, M1-L181, M1-L180, M1-R179, M1-W178, M1-S177, M1-L176, M1-T175, M1-R174, M1-K173, M1-S172, M1-F171, M1-A170, M1-Q169, M1-A168, M1-A167, M1-G166, M1-G165, M1-D164, M1-P163, M1-Y162, M1-G161, M1-R160, M1-P159, M1-Q158, M1-W157, M1-G156, M1-R155, M1-R154, M1-S153, M1-L152, M1-S151, M1-L150, M1-P149, M1-A148, M1-R147, M1-P146, M1-S145, M1-R144, M1-R143, M1-S142, M1-R141, M1-A140, M1-R139, M1-P138, M1-P137, M1-P136, M1-G135, M1-P134, M1-P133, M1-S132, M1-P131, M1-P130, M1-A129, M1-S128, M1-P127, M1-P126, M1-P125, M1-P124, M1-R123, M1-M122, M1-D121, M1-P120, M1-V119, M1-G118, M1-C117, M1-R116, M1-P115, M1-R114, M1-N113, M1-M112, M1-A111, M1-A110, M1-L109, M1-T108, M1-A107, M1-A106, M1-D105, M1-L104, M1-E103, M1-G102, M1-S101, M1-A100, M1-P99, M1-L98, M1-A97, M1-N96, M1-A95, M1-R94, M1-Q93, M1-F92, M1-R91, M1-R90, M1-V89, M1-A88, M1-E87, M1-A86, M1-L85, M1-A84, M1-A83, M1-G82, M1-K81, M1-P80, M1-T79, M1-E78, M1-P77, M1-P76, M1-G75, M1-E74, M1-P73, M1-S72, M1-P71, M1-G70, M1-W69, M1-A68, M1-A67, M1-W66, M1-V65, M1-G64, M1-S63, M1-W62, M1-G61, M1-Y60, M1-R59, M1-S58, M1-L57, M1-F56, M1-R55, M1-Q54, M1-A53, M1-A52, M1-H51, M1-L50, M1-D49, M1-A48, M1-I47, M1-P46, M1-K45, M1-A44, M1-Q43, M1-R42, M1-L41, M1-P40, M1-S39, M1-P38, M1-E37, M1-L36, M1-D35, M1-S34, M1-R33, M1-D32, M1-R31, M1-S30, M1-H29, M1-F28, M1-L27, M1-S26, M1-E25, M1-P24, M1-Q23, M1-T22, M1-P21, M1-W20, M1-P19, M1-A18, M1-A17, M1-L16, M1-W15, M1-C14, M1-L13, M1-L12, M1-L11, M1-T10, M1-P9, M1-R8, and/or M1-F7 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the MMP-29 polypeptide (e.g., any combination of both N- and C-terminal MMP-29 polypeptide deletions) of SEQ ID NO:2. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of MMP-29 (SEQ ID NO:2), and where CX refers to any C-terminal deletion polypeptide amino acid of MMP-29 (SEQ ID NO:2). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 10 of 64

The present invention also encompasses immunogenic and/or antigenic epitopes of the MMP-29 polypeptide.

The MMP-29 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the MMP-29 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the MMP-29 polypeptide to associate with other polypeptides, particularly the serine protease substrate for MMP-29, or its ability to modulate serine protease function.

Specifically, the MMP-29 polypeptide was predicted to comprise one tyrosine phosphorylation site using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y (SEQ ID NO:83), or [RK]-x(3)-[DE]-x(2)-Y (SEQ ID NO:84), where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977(1982); Hunter T., J. Biol. Chem. 257:4843-4848(1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. 259:7835-7841(1984), which are hereby incorporated herein by reference.

In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: QNHPFRNIDSAYYSYAY (SEQ ID NO:22). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these MMP-29 tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The MMP-29 polypeptide was predicted to comprise eleven PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem . . . . 260:12492-12499(1985); which are hereby incorporated by reference herein.

In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: EGPPETPKGAALA (SEQ ID NO:10), PPRARSRRSPRAP (SEQ ID NO:11), ARSRRSPRAPLSL (SEQ ID NO:12), APLSLSRRGWQPR (SEQ ID NO:13), AAQAFSKRTLSWR (SEQ ID NO:14), SKRTLSWRLLGEA (SEQ ID NO:15), LGLPHTYRTGSIM (SEQ ID NO:16), FELDWSDRKAIQK (SEQ ID NO:17), FVHIWTWKRDERY (SEQ ID NO:18), YWRYDSDKDQALT (SEQ ID NO:19), and/or PKKFISEKWFDVC (SEQ ID NO:20). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the MMP-29 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

The MMP-29 polypeptide has been shown to comprise one glycosylation site according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.

In preferred embodiments, the following asparagine glycosylation site polypeptide is encompassed by the present invention: YENRNNRTRYGDPI (SEQ ID NO:21). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In confirmation of the MMP-29 polypeptide representing a member of the metalloproteinase family, the MMP-29 polypeptide has been shown to comprise one neutral zinc metallopeptidase zinc-binding site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The majority of zinc-dependent metallopeptidases (with the notable exception of the carboxypeptidases) share a common pattern of primary structure in the part of their sequence involved in the binding of zinc, and can be grouped together as a superfamily, known as the metzincins, on the basis of this sequence similarity. They can be classified into a number of distinct families which are listed below along with the proteases which are currently known to belong to these families. Family M1: Bacterial aminopeptidase N (EC 3.4.11.2) (gene pepN), Mammalian aminopeptidase N (EC 3.4.11.2), Mammalian glutamyl aminopeptidase (EC 3.4.11.7) (aminopeptidase A)—which may play a role in regulating growth and differentiation of early B-lineage cells, Yeast aminopeptidase yscII (gene APE2), Yeast alanine/arginine aminopeptidase (gene AAP1), Yeast hypothetical protein YIL137c., Leukotriene A-4 hydrolase (EC 3.3.2.6)—which is the enzyme is responsible for the hydrolysis of an epoxide moiety of LTA-4 to form LTB-4 (it has been shown that it binds zinc and is capable of peptidase activity); Family M2: Angiotensin-converting enzyme (EC 3.4.15.1) (dipeptidyl carboxypeptidase I) (ACE) the enzyme responsible for hydrolyzing angiotensin I to angiotensin II—There are two forms of ACE: a testis-specific isozyme and a somatic isozyme which has two active centers; Family M3: Thimet oligopeptidase (EC 3.4.24.15)—a mammalian enzyme involved in the cytoplasmic degradation of small peptides, Neurolysin (EC 3.4.24.16) (also known as mitochondrial oligopeptidase M or microsomal endopeptidase), Mitochondrial intermediate peptidase precursor (EC 3.4.24.59) (MIP)—which is involved the second stage of processing of some proteins imported in the mitochondrion, Yeast saccharolysin (EC 3.4.24.37) (proteinase yscD), Escherichia coli and related bacteria dipeptidyl carboxypeptidase (EC 3.4.15.5) (gene dcp), Escherichia coli and related bacteria oligopeptidase A (EC 3.4.24.70) (gene opdA or prlC), Yeast hypothetical protein YKL134c; Family M4: Thermostable thermolysins (EC 3.4.24.27), and related thermolabile neutral proteases (bacillolysins) (EC 3.4.24.28) from various species of Bacillus , Pseudolysin (EC 3.4.24.26) from Pseudomonas aeruginosa (gene lasB), Extracellular elastase from Staphylococcus epidermidis , Extracellular protease prt1 from Erwinia carotovora, Extracellular minor protease smp from Serratia marcescens , Vibriolysin (EC 3.4.24.25) from various species of Vibrio , Protease prtA from Listeria monocytogenes , Extracellular proteinase proA from Legionella pneumophila ; Family M5: Mycolysin (EC 3.4.24.31) from Streptomyces cacaoi ; Family M6: Immune inhibitor A from Bacillus thuringiensis (gene ina). Ina degrades two classes of insect antibacterial proteins, attacins and cecropins; Family M7: Streptomyces extracellular small neutral proteases; Family M8 : Leishmanolysin . (EC 3.4.24.36) (surface glycoprotein gp63), a cell surface protease from various species of Leishmania ; Family M9: Microbial collagenase (EC 3.4.24.3) from Clostridium perfringens and Vibrio alginolyticus ; Family M10A: Serralysin (EC 3.4.24.40), an extracellular metalloprotease from Serratia , Alkaline metalloproteinase from Pseudomonas aeruginosa (gene aprA), Secreted proteases A, B, C and G from Erwinia chrysanthemi , Yeast hypothetical protein YIL108w; Family M10B: Mammalian extracellular matrix metalloproteinases (known as matrixins), MMP-1 (EC 3.4.24.7) (interstitial collagenase), MMP-2 (EC 3.4.24.24) (72 Kd gelatinase), MMP-9 (EC 3.4.24.35) (92 Kd gelatinase), MMP-7 (EC 3.4.24.23) (matrylisin), MMP-8 (EC 3.4.24.34) (neutrophil collagenase), MMP-3 (EC 3.4.24.17) (stromelysin-1), MMP-10 (EC 3.4.24.22) (stromelysin-2), and MMP-11 (stromelysin-3), MMP-12 (EC 3.4.24.65) (macrophage metalloelastase), Sea urchin hatching enzyme (envelysin) (EC 3.4.24.12)—A protease that allows the embryo to digest the protective envelope derived from the egg extracellular matrix, Soybean metalloendoproteinase 1; Family M11: Chlamydomonas reinhardtii gamete lytic enzyme (GLE);Family M12A: Astacin (EC 3.4.24.21), a crayfish endoprotease, Meprin A (EC 3.4.24.18), a mammalian kidney and intestinal brush border metalloendopeptidase, Bone morphogenic protein 1 (BMP-1)—a protein which induces cartilage and bone formation and which expresses metalloendopeptidase activity ( Drosophila homologue of BMP-1 is the dorsal-ventral patterning protein tolloid), Blastula protease 10 (BP10) from Paracentrotus lividus and the related protein SpAN from Strongylocentrotus purpuratus, Caenorhabditis elegans hypothetical proteins F42A10.8 and R151.5, Choriolysins L and H (EC 3.4.24.67) (also known as embryonic hatching proteins LCE and HCE) from the fish Oryzias lapides —these proteases participates in the breakdown of the egg envelope, which is derived from the egg extracellular matrix, at the time of hatching; Family M12B: Snake venom metalloproteinases—This subfamily mostly groups proteases that act in hemorrhage. Examples are: adamalysin II (EC 3.4.24.46), atrolysin C/D (EC 3.4.24.42), atrolysin E (EC 3.4.24.44), fibrolase (EC 3.4.24.72), trimerelysin I (EC 3.4.25.52) and II (EC 3.4.25.53), Mouse cell surface antigen MS2; Family M13: Mammalian neprilysin (EC 3.4.24.11) (neutral endopeptidase) (NEP), Endothelin-converting enzyme 1 (EC 3.4.24.71) (ECE-1)—which process the precursor of endothelin to release the active peptide, Kell blood group glycoprotein, a major antigenic protein of erythrocytes, The Kell protein is very probably a zinc endopeptidase, Peptidase 0 from Lactococcus lactis (gene pepo); Family M27: Clostridial neurotoxins, including tetanus toxin (TeTx) and the various botulinum toxins (BONT)—these toxins are zinc proteases that block neurotransmitter release by proteolytic cleavage of synaptic proteins such as synaptobrevins, syntaxin and SNAP-25; Family M30: Staphylococcus hyicus neutral metalloprotease; Family M32: Thermostable carboxypeptidase 1 (EC 3.4.17.19) (carboxypeptidase Taq)—an enzyme from Thermus aquaticus which is most active at high temperature; Family M34: Lethal factor (LF) from Bacillus anthracis , one of the three proteins composing the anthrax toxin; Family M35: Deuterolysin (EC 3.4.24.39) from Penicillium citrinum and related proteases from various species of Aspergillus ; and Family M36: Extracellular elastinolytic metalloproteinases from Aspergillus.

›DETAILED DESCRIPTION OF THE INVENTION · 11 of 64

Based on the tertiary structure of thermolysin, the position of the residues acting as zinc ligands and those involved in the catalytic activity are known. Two of the zinc ligands are histidines which are very close together in the sequence; C-terminal to the first histidine is a glutamic acid residue which acts as a nucleophile and promotes the attack of a water molecule on the carbonyl carbon of the substrate. A consensus sequence for neutral zinc metallopeptidases zinc-binding domains is as follows: [GSTALIVN]-x(2)-H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ] (SEQ ID NO:85), wherein the two H's are zinc ligands, E is the active site residue, and X represents any amino acid.

Additional information relative to metalloproteinases and neutral zinc metallopeptidases zinc-binding domains may be found by reference to the following publications: Jongeneel C. V., Bouvier J., Bairoch A., FEBS Lett. 242:211-214(1989); Murphy G. J. P., Murphy G., Reynolds J. J., FEBS Lett. 289:4-7(1991); Bode W., Grams F., Reinemer P., Gomis-Rueth F.-X., Baumann U., McKay D. B., Stoecker W., Zoology 99:237-246(1996); Rawlings N. D., Barrett A. J., Meth. Enzymol. 248:183-228(1995); Woessner J. Jr., FASEB J. 5:2145-2154(1991); Hite L. A., Fox J. W., Bjarnason J.B., Biol. Chem. Hoppe-Seyler 373:381-385(1992); Montecucco C., Schiavo G., Trends Biochem. Sci. 18:324-327(1993); Niemann H., Blasi J., Jahn R., Trends Cell Biol. 4:179-185(1994); which are hereby incorporated herein by reference in their entirety.

In preferred embodiments, the following neutral zinc metallopeptidases zinc-binding domain polypeptide is encompassed by the present invention: ISLLKVAVHEIGHVLGLPHT (SEQ ID NO:23). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this neutral zinc metallopeptidases zinc-binding domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also provides a three-dimensional homology model of several MMP-29 polypeptide domains. Specifically, three-dimensional homology models of the MMP-29 propeptide, catalytic, and hemopexin-like domains are provided (see FIGS. 7 , 8 , and 9 ) representing amino acids from about A48 to about P120, from about G161 to about E330, and from about Q346 to about N544 of SEQ ID NO:2, respectively.

Protein threading and molecular modeling of MMP29 suggests that there are three functional domains for which three dimensional models can be accurately created. Amino acids from about A48 to about P120 of SEQ ID NO:2 comprise the N-terminal domain known as the propeptide region. This region is similar to the propeptide region of human fibroblast Stromelysin-1 proenzyme Protein DataBank (PDB) code (Bernstein et al. 1977) 1slmA. This region contains the cysteine switch. Amino acids from about D121 to about R160 of SEQ ID NO:2 comprise a proline rich linker region. This region is followed by amino acids from about G161 to about E330 of SEQ ID NO:2 which comprise the catalytic domain. Amino acids from about G331 to about N345 of SEQ ID NO:2 make up a linker region between the catalytic domain and the C-terminal hemopexin-like domain. Amino acids from about Q346 to about N544 of SEQ ID NO:2 comprise the hemopexin-like domain which has a short C-terminal region of unknown function composed of amino acids from about G545 to about M569 of SEQ ID NO:2. Based on sequence, structure and known metalloprotease signature sequences, MMP29 is a novel metaloproteinase.

The three dimensional crystallographic and NMR structures of matrix metalloproteinases (MMPs), also referred to as matrixins, allow for the characterization and functional analyses which can be used to understand the role of MMPs in biology and pathology. The detailed structural analysis for MMP29 allows for the characterization of functional domains and development of three dimensional models for the domains of MMP29.

In most cases the matrixins are synthesized as prepro-enzymes and secreted as inactive pro-enzymes. The primary structure and bioinformatics analyses and structural considerations for matrixins and related metalloproteinase families such as the metzincins (astacins, reprolysins, adamalysins, and serralysins) were used to describe the structural and functional organization of the matrixin metalloproteinases (Massova et al., 1998). The matrixins are organized into three distinctive functional domains. The N-terminal domain is a propeptide domain (consisting of about 80-90 amino acids) which has a conserved sequence motif PRCGxPD (SEQ ID NO:86). The cysteine residue within this motif is known as the “cysteine switch”. This cysteine interacts with the catalytic zinc to maintain latency of the pro-enzyme. This domain consists of a three helices connected by loops and turns.

The second domain is the catalytic domain (consisting of about 170 amino acids). The catalytic domain contains a zinc binding motif (HexxHxxGxxH) (SEQ ID NO:87) and a conserved methionine, which forms a unique “Met-turn” structure. The catalytic domain also contains two zinc ions and at least one calcium ion coordinated to various residues. One of the zinc ions is the catalytic zinc and is in the active site. The other zinc ion and (known as the structural zinc) and a calcium ion are located approximately 12 angstroms from the zinc in the active site. The catalytic zinc is essential for proteolytic activity of the matrixins. Three histidine residues coordinate the catalytic zinc and are conserved in all matrixins and related metalloproteinases. This domain consists of a five beta-stranded sheet and three alpha-helices connected by bridging loops (Bode et al., 1993). The structure can be classified as an alpha/beta three-layer sandwich.

The third conserved domain is the hemopexin-like domain (consisting of about 210 amino acids). This domain is a four bladed beta-propeller where each blade consists of four anti-parallel beta-strands and a alpha-helix (Gomis-Ruth et al., 1996) The hemopexin-like domains show sequence and structural similarity to the plasma protein hemopexin. This domain has been shown to play a functional role in substrate binding and interactions with tissue inhibitors of the metalloproteinases.

›DETAILED DESCRIPTION OF THE INVENTION · 12 of 64

A three-dimensional homology model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995). The homology model of the MMP-29 propeptide domain, corresponding to amino acid residues A48 to P120 of SEQ ID NO:2, respectively, was based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and is defined by the set of structural coordinates set forth in Table IV herein.

The homology model of MMP29 propeptide domain was derived from the sequence alignment set forth in FIG. 13 . The sequence identity of the propeptide domain to the structural template is 26%, but there is significant amino acid homology and predicted structural similarity. The three dimensional model for the propeptide domain comprises amino acids A48 through P120 of SEQ ID NO:2. An overall atomic model including plausible side chain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for MMP29 propeptide domain is defined by the set of structure coordinates as set forth in Table IV and is shown in FIG. 7 rendered by backbone secondary structures. The cysteine, C117 is the cysteine that ligates the catalytic zinc is also displayed. The role of the cysteine is to ligate the catalytic zinc to maintain the latency of the pro-enzyme while the propeptide is attached. This cysteine is essential and conserved in all matrixin metalloproteinases.

The homology model of the MMP-29 catalytic domain, corresponding to amino acid residues from about G161 to about E330 of SEQ ID NO:2, respectively, was based upon the homologous structure of a portion of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) and is defined by the set of structural coordinates set forth in Table V herein.

The homology model of MMP29 catalytic domain was derived from the sequence alignment set forth in FIG. 13 . The sequence identity of the catalytic domain to the structural templates is 39%. There is also significant amino acid homology and predicted structural similarity. The three dimensional model for the catalytic domain comprises amino acids G161 through E330 of SEQ ID NO:2. An overall atomic model including plausible side chain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for MMP29 catalytic domain is defined by the set of structure coordinates as set forth in Table V and is shown in FIG. 8 rendered by backbone secondary structures. FIG. 8 also displays the side chains for the three histidines (H283, H287, H293) that are predicted to coordinate the catalytic zinc ion. The histidines are essential and conserved in all matrix metalloproteinases.

The homology model of the MMP-29 hemopexin-like domain, corresponding to amino acid residues from about Q346 to about N544 of SEQ ID NO:2, respectively, was based upon the homologous structure of a portion of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and is defined by the set of structural coordinates set forth in Table VI herein.

The homology model of MMP29 hemopexin-like domain was derived from the sequence alignment set forth in FIG. 13 . The sequence identity of the hemopexin-like domain to the template is 23%, but there is significant amino acid homology and predicted structural similarity. The three dimensional model for the hemopexin-like domain comprises amino acids Q346 through N544 of SEQ ID NO:2. An overall atomic model including plausible side chain orientations was generated using the program LOOK (Levitt, 1992). The three dimensional model for MMP29 hemopexin-like domain is defined by the set of structure coordinates as set forth in Table VI and is shown in FIG. 9 rendered by backbone secondary structures.

A description of the headings in Tables IV, V, and VI are as follows: “Atom No” refers to the atom number within the MMP-29 homology model; “Atom name” refers to the element whose coordinates are measured, the first letter in the column defines the element; “Residue” refers to the amino acid within which the atom resides, and the provided number after the amino acid refers to the amino acid number of the “residue”; “X Coord”, “Y Coord”, and “Z Coord” structurally define the atomic position of the element measured in three dimensions.

The MMP-29 propeptide, catalytic, and hemopexin-like domain homology models of the present invention may provide one basis for designing rational stimulators (agonists) and/or inhibitors (antagonists) of one or more of the biological functions of MMP-29, or of MMP-29 mutants having altered specificity (e.g., molecularly evolved MMP-29 polypeptides, engineered site-specific MMP-29 mutants, MMP-29 allelic variants, etc.).

Homology models are not only useful for designing rational agonists and/or antagonists, but are also useful in predicting the function of a particular polypeptide. The functional predictions from homology models are typically more accurate than the functional attributes derived from traditional polypeptide sequence homology alignments (e.g., CLUSTALW), particularly when the three dimensional structure of a related polypeptide is known (e.g., 1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73; and 1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74). The increased prediction accuracy is based upon the fact that homology models approximate the three-dimensional structure of a protein, while homology based alignments only take into account the one dimension polypeptide sequence. Since the function of a particular polypeptide is determined not only by its primary, secondary, and tertiary structure, functional assignments derived solely upon homology alignments using the one dimensional protein sequence may be less reliable. A 3-dimensional model can be constructed on the basis of the known structure of a homologous protein (Greer et al, 1991, Lesk, et al, 1992, Cardozo, et al, 1995, Yuan, et al, 1995).

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Prior to developing a homology model, those of skill in the art would appreciate that a template of a known protein, or model protein, must first be identified which will be used as a basis for constructing the homology model for the protein of unknown structure (query template). In the case of the MMP-29 polypeptide of the present invention, the model protein templates used in constructing the MMP-29 propeptide, catalytic, and hemopexin-like domain homology models were portions of the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and portions of the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74).

Identifying a template can be accomplished using pairwise alignment of protein sequences using such programs as FASTA (Pearson, et al 1990) and BLAST (Altschul, et al, 1990). In cases where sequence similarity is high (greater than 30%), such pairwise comparison methods may be adequate for identifying an appropriate template. Likewise, multiple sequence alignments or profile-based methods can be used to align a query sequence to an alignment of multiple (structurally and biochemically) related proteins. When the sequence similarity is low, more advanced techniques may be used. Such techniques, include, for example, protein fold recognition (protein threading; Hendlich, et al, 1990), where the compatibility of a particular polypeptide sequence with the 3-dimensional fold of a potential template protein is gauged on the basis of a knowledge-based potential.

A pairwise alignment of the MMP-29 polypeptide of the present invention to the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) is provided in FIG. 13 .

Following the initial sequence alignment, an optional second step would be to optimally align the query template to the model template by manual manipulation and/or by the incorporation of features specific to the polypeptides (e.g., motifs, secondary structure predictions, and allowed conservations). Preferably, the incorporated features are found within both the model and query template.

The next step could be to identify structurally conserved regions that could be used to construct secondary core structure (Sali, et al, 1995). Loops could be added using knowledge-based techniques, and by performing forcefield calculations (Sali, et al, 1995).

In order to recognize errors in a three-dimensional structure, knowledge based mean fields can be used to judge the quality of protein folds (Sippl 1993). The methods can be used to recognize misfolded structures as well as faulty parts of structural models. The technique generates an energy graph where the energy distribution for a given protein fold is displayed on the y-axis and residue position in the protein fold is displayed on the x-axis. The knowledge based mean fields compose a force field derived from a set of globular protein structures taken as a subset from the Protein Data Bank (Bernstein et. al. 1977). To analyze the quality of a model the energy distribution is plotted and compared to the energy distribution of the template from which the model was generated.

FIG. 10 shows the energy graph for the MMP-29 propeptide model (dotted line) and the 1slmA template (solid line) from which the model was generated. It is clear that the model has slightly higher energies in the C-terminal region while the N-terminal region appears to be “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of the MMP-29 propeptide domain are an accurate and useful representation for the polypeptide.

FIG. 11 shows the energy graph for the MMP-29 catalytic model (dotted line) and the 1fblA template (solid line) from which the model was generated. It is clear that the model has slightly higher energies in the C-terminal region while the N-terminal region appears to be “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of the MMP-29 catalytic domain are an accurate and useful representation for the polypeptide.

FIG. 12 shows the energy graph for the MMP-29 hemopexin-like model (dotted line) and the 1slmA template (solid line) from which the model was generated. It is clear that the model has slightly higher energies in the C-terminal region while the N-terminal region appears to be “native-like”. This graph supports the motif and sequence alignments in confirming that the three dimensional structure coordinates of the MMP-29 hemopexin-like domain are an accurate and useful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model.

In this invention, the propeptide domain homology model of residues A48 to about P120 of MMP-29 (SEQ ID NO:2) was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc., N.Y., N.Y.).

In this invention, the catalytic domain homology model of residues G161 through E330 of MMP-29 (SEQ ID NO:2) was derived from generating a sequence alignment with the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74) using the Proceryon suite of software (Proceryon Biosciences, Inc., N.Y., N.Y.).

In this invention, the hemopexin-like domain homology model of residues Q346 through N544 of MMP-29 (SEQ ID NO:2) was derived from generating a sequence alignment with the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) using the Proceryon suite of software (Proceryon Biosciences, Inc., N.Y., N.Y.).

The sequence alignment was then used to guide three dimensional model construction whereby the backbone and side chain conformations were constructed using the LOOK suite of software (Molecular Applications Group) and homology modeling module SEGMOD (Levitt, M., 1992).

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The skilled artisan would appreciate that a set of structure coordinates for a protein represents a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from the generation of similar homology models using different alignment templates (i.e., other than the the human fibroblast stromelysin-1 proenzyme (1slmA; Genbank Accession No. gi|1942848; SEQ ID NO:73) and the pig fibroblast (Interstitial) collagenase Mmp-1 (1fblA; Genbank Accession No. gi|1310872; SEQ ID NO:74), and/or using different methods in generating the homology model, will likely have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Tables IV, V, and VI could be manipulated by fractionalization of the structure coordinates; integer additions, or integer subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

Therefore, various computational analyses are necessary to determine whether a template molecule or a portion thereof is sufficiently similar to all or part of a query template (e.g., propeptide, catalytic, or hemopexin-like domains of MMP-29) in order to be considered the same. Such analyses may be carried out in current software applications, such as INSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as described in the User's Guide, or software applications available in the SYBYL software suite (Tripos Inc., St. Louis, Mo.).

Using the superimposition tool in the program SYBYL, comparisons can be made between different structures and different conformations of the same structure. The procedure used in SYBYL to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. The atom equivalency within SYBYL is defined by user input. For the purpose of this invention, we will define equivalent atoms as protein backbone atoms (N, Cα, X, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by the SYBYL program. For the purpose of the present invention, any homology model of MMP-29 that has a root mean square deviation of conserved residue backbone atoms (N, Cα, X, C, O) of less than 3.0 A when superimposed on the relevant backbone atoms described by structure coordinates listed in Tables IV, V, or VI are considered identical. More preferably, the root mean square deviation for the homology models of the MMP-29 propeptide, catalytic, or hemopexin-like domains is less than about 2.0 Å, less than about 1.5 Å, less than about 1.0 Å, less than about 0.9 Å, less than about 0.8 Å, less than about 0.7 Å, less than about 0.6 Å, less than about 0.5 Å, less than about 0.4 Å, less than about 0.3 Å, less than about 0.2 Å, or less than about 0.1 Å.

The homology models of the present invention are useful for the structure-based design of modulators of MMP-29 biological function, as well as mutants with altered biological function and/or specificity.

There is 39% sequence identity between catalytic domain of MMP29 and the catalytic domain of matrixin metalloproteinases used in the composite alignment of the human fibroblast Stromelysin-1 proenzyme Protein DataBank (PDB) code (Bernstein et al. 1977) 1slmA (Becker et al., 1995) and the Fibroblast (Interstitial) Collagenase Mmp-1, from pig, PDB code 1fbl (L1 et al., 1995) as shown in FIG. 13 .

For MMP29 there are functionally important residues are located in the propeptide domain and the catalytic domain. C117 is part of the cysteine switch. This region is part of a conserved motif (PRCGXPD (SEQ ID NO:86)) that forms a loop on which C117 is presented into the active site of the catalytic domain so that the thiol group of the cysteine can interact with the catalytic zinc rendering the pro-enzyme latent. Upon processing the propeptide domain is removed and the active site of the catalytic domain exposed to solvent for substrate binding. In the homology model of the catalytic domain (Table V) residues proposed to coordinate the catalytic zinc ion include: H283, H287, and H293 of SEQ ID NO:2. These residues are conserved in both of the templates used to model the MMP29 domains and in all other matrixin metalloproteinases (denoted by the asterisks (*) in FIG. 13 ). The conservation of the amino acids that are required for metal coordination emphasizes the key role metal ions play in generating the nucleophile for catalysis. These residues are located in the active site formed at the interface of the propeptide and catalytic domains. The substrate binding cleft is surface exposed adjacent to the catalytic zinc active site and is shallow. The matrixin metalloproteinases also contain a conserved methionine, which forms a unique “Met-turn”, 1-4 beta-turn structure. The conserved methionine of MMP29 that forms the turn that supports the active site is M236 of SEQ ID NO:2. This is part of the local substructure of the active site region and is adjacent to the S1 pocket of the substrate binding site.

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In addition to the residues proposed to coordinate the metal ions, there is an adjacent region in the binding site called the S1 pocket that can best be described as a hydrophobic pocket. The hydrophobic pocket is composed of residues Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2. The shape and character of the S 1 pocket is responsible for substrate specificity for a given matrixin. The more preferred description of the active site includes residues in the hydrophobic pocket, residues that coordinate the metal ion and additional residues that are involved in hydrogen bonding networks and van der Waal's interactions in the region of the active. The binding site residues include but are not limited to: C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2. These active site residues play critical roles in the mechanism of catalysis and substrate specificity and binding.

In a preferred embodiment of the present invention, the molecule comprises the active site region defined by structure coordinates of MMP29 amino acids described above according to Tables IV and V, or a mutant of said molecule. The active site is defined by residues C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2.

More preferred are molecules comprising all or any part of the active site region (C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2) or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP29 amino acids of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

In accordance with the structural coordinates provided in Table IV and the three dimensional homology model of MMP-29, the MMP-29 polypeptide has been shown to comprise a propeptide domain embodied by the following amino acids: at about amino acid A48 to about amino acid P120 of SEQ ID NO:2 ( FIGS. 1A-B ). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced amino acids.

Also more preferred are polypeptides comprising all or any part of the MMP-29 propeptide domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

In preferred embodiments, the following MMP-29 propeptide domain polypeptide is encompassed by the present invention: ADLHAAQRFLSRYGWSGVWAAWGPSPEGPPETPKGAALAEAVRRFQRANA LPASGELDAATLAAMNRPRCGVP (SEQ ID NO:75). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 propeptide domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of the MMP-29 propeptide domain (SEQ ID NO: 75). Such polypeptides may correspond, for example, to the N- and/or C-terminal deletions of the methionine aminopeptidase domain.

In preferred embodiments, the following N-terminal MMP-29 propeptide domain deletion polypeptides are encompassed by the present invention: A1-P73, D2-P73, L3-P73, H4-P73, A5-P73, A6-P73, Q7-P73, R8-P73, F9-P73, L10-P73, S11-P73, R12-P73, Y13-P73, G14-P73, W15-P73, S16-P73, G17-P73, V18-P73, W19-P73, A20-P73, A21-P73, W22-P73, G23-P73, P24-P73, S25-P73, P26-P73, E27-P73, G28-P73, P29-P73, P30-P73, E31-P73, T32-P73, P33-P73, K34-P73, G35-P73, A36-P73, A37-P73, L38-P73, A39-P73, E40-P73, A41-P73, V42-P73, R43-P73, R44-P73, F45-P73, Q46-P73, R47-P73, A48-P73, N49-P73, A50-P73, L51-P73, P52-P73, A53-P73, S54-P73, G55-P73, E56-P73, L57-P73, D58-P73, A59-P73, A60-P73, T61-P73, L62-P73, A63-P73, A64-P73, M65-P73, N66-P73, and/or R67-P73 of SEQ ID NO:75. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 propeptide domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal MMP-29 propeptide domain deletion polypeptides are encompassed by the present invention: A1-P73, A1-V72, A1-G71, A1-C70, A1-R69, A1-P68, A1-R67, A1-N66, A1-M65, A1-A64, A1-A63, A1-L62, A1-T61, A1-A60, A1-A59, A1-D58, A1-L57, A1-E56, A1-G55, A1-S54, A1-A53, A1-P52, A1-L51, A1-A50, A1-N49, A1-A48, A1-R47, A1-Q46, A1-F45, A1-R44, A1-R43, A1-V42, A1-A41, A1-E40, A1-A39, A1-L38, A1-A37, A1-A36, A1-G35, A1-K34, A1-P33, A1-T32, A1-E31, A1-P30, A1-P29, A1-G28, A1-E27, A1-P26, A1-S25, A1-P24, A1-G23, A1-W22, A1-A21, A1-A20, A1-W19, A1-V18, A1-G17, A1-S16, A1-W15, A1-G14, A1-Y13, A1-R12, A1-S11, A1-L10, A1-F9, A1-R8, and/or A1-Q7 of SEQ ID NO:75. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 propeptide domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

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Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the MMP-29 propeptide domain (e.g., any combination of both N- and C-terminal MMP-29 propeptide domain deletions) of SEQ ID NO:75. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the MMP-29 propeptide domain (SEQ ID NO:75), and where CX refers to any C-terminal amino acid position of the MMP-29 propeptide domain (SEQ ID NO:75). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following MMP-29 propeptide domain amino acid substitutions are encompassed by the present invention: wherein A48 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D49 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L50 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein H51 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A52 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A53 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q54 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein R55 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F56 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L57 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S58 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein R59 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y60 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G61 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W62 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S63 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G64 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V65 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein W66 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein A67 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A68 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W69 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein G70 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P71 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein S72 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P73 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E74 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G75 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P76 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein P77 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein E78 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T79 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein P80 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K81 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G82 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A83 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A84 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L85 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A86 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E87 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A88 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V89 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein R90 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein R91 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F92 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q93 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein R94 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein A95 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N96 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y wherein A97 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L98 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P99 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A100 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S101 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G102 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E103 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L104 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D105 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A106 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A107 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T108 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L109 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A110 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A111 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M112 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein N113 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R114 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein P115 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein R116 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein C117 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G118 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V119 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; and/or wherein P120 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y of SEQ ID NO:2, in addition to any combination thereof. The present invention also encompasses the use of these MMP-29 propeptide domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 17 of 64

In preferred embodiments, the following MMP-29 propeptide domain conservative amino acid substitutions are encompassed by the present invention: wherein A48 is substituted with either a G, I, L, M, S, T, or V; wherein D49 is substituted with an E; wherein L50 is substituted with either an A, I, or V; wherein H51 is substituted with either a K, or R; wherein A52 is substituted with either a G, I, L, M, S, T, or V; wherein A53 is substituted with either a G, I, L, M, S, T, or V; wherein Q54 is substituted with a N; wherein R55 is substituted with either a K, or H; wherein F56 is substituted with either a W, or Y; wherein L57 is substituted with either an A, I, or V; wherein S58 is substituted with either an A, G, M, or T; wherein R59 is substituted with either a K, or H; wherein Y60 is either an F, or W; wherein G61 is substituted with either an A, M, S, or T; wherein W62 is either an F, or Y; wherein S63 is substituted with either an A, G, M, or T; wherein G64 is substituted with either an A, M, S, or T; wherein V65 is substituted with either an A, I, or L; wherein W66 is either an F, or Y; wherein A67 is substituted with either a G, I, L, M, S, T, or V; wherein A68 is substituted with either a G, I, L, M, S, T, or V; wherein W69 is either an F, or Y; wherein G70 is substituted with either an A, M, S, or T; wherein P71 is a P; wherein S72 is substituted with either an A, G, M, or T; wherein P73 is a P; wherein E74 is substituted with a D; wherein G75 is substituted with either an A, M, S, or T; wherein P76 is a P; wherein P77 is a P; wherein E78 is substituted with a D; wherein T79 is substituted with either an A, G, M, or S; wherein P80 is a P; wherein K81 is substituted with either a R, or H; wherein G82 is substituted with either an A, M, S, or T; wherein A83 is substituted with either a G, I, L, M, S, T, or V; wherein A84 is substituted with either a G, I, L, M, S, T, or V; wherein L85 is substituted with either an A, I, or V; wherein A86 is substituted with either a G, I, L, M, S, T, or V; wherein E87 is substituted with a D; wherein A88 is substituted with either a G, I, L, M, S, T, or V; wherein V89 is substituted with either an A, I, or L; wherein R90 is substituted with either a K, or H; wherein R91 is substituted with either a K, or H; wherein F92 is substituted with either a W, or Y; wherein Q93 is substituted with a N; wherein R94 is substituted with either a K, or H; wherein A95 is substituted with either a G, I, L, M, S, T, or V; wherein N96 is substituted with a Q; wherein A97 is substituted with either a G, I, L, M, S, T, or V; wherein L98 is substituted with either an A, I, or V; wherein P99 is a P; wherein A100 is substituted with either a G, I, L, M, S, T, or V; wherein S101 is substituted with either an A, G, M, or T; wherein G102 is substituted with either an A, M, S, or T; wherein E103 is substituted with a D; wherein L104 is substituted with either an A, I, or V; wherein D105 is substituted with an E; wherein A106 is substituted with either a G, I, L, M, S, T, or V; wherein A107 is substituted with either a G, I, L, M, S, T, or V; wherein T108 is substituted with either an A, G, M, or S; wherein L109 is substituted with either an A, I, or V; wherein A110 is substituted with either a G, I, L, M, S, T, or V; wherein A111 is substituted with either a G, I, L, M, S, T, or V; wherein M112 is substituted with either an A, G, S, or T; wherein N113 is substituted with a Q; wherein R114 is substituted with either a K, or H; wherein P115 is a P; wherein R116 is substituted with either a K, or H; wherein C117 is a C; wherein G118 is substituted with either an A, M, S, or T; wherein V119 is substituted with either an A, I, or L; and/or wherein P120 is a P of SEQ ID NO:2 in addition to any combination thereof. Other suitable substitutions within the MMP-29 propeptide domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these MMP-29 propeptide domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In accordance with the structural coordinates provided in Table V and the three dimensional homology model of MMP-29, the MMP-29 polypeptide has been shown to comprise a catalytic domain embodied by the following amino acids: at about amino acid G161 to about amino acid E330 of SEQ ID NO:2 ( FIGS. 1A-B ). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced amino acids.

Also more preferred are polypeptides comprising all or any part of the MMP-29 catalytic domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

In preferred embodiments, the following MMP-29 catalytic domain polypeptide is encompassed by the present invention: GYPDGGAAQAFSKRTLSWRLLGEALSSQLSVADQRRIVALAFRMWSEVTPLD FREDLAAPGAAVDIKLGFGRGRHLGCPRAFDGSGQEFAHAWRLGDIHFDDDE HFTPPTSDTGISLLKVAVHEIGHVLGLPHTYRTGSIMQPNYIPQEPAFELDWSD RKAIQKLYGSCE (SEQ ID NO:76). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 catalytic domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 18 of 64

The present invention also encompasses polypeptides comprising at least a portion of the MMP-29 catalytic domain (SEQ ID NO: 76). Such polypeptides may correspond, for example, to the N- and/or C-terminal deletions of the catalytic domain.

In preferred embodiments, the following N-terminal MMP-29 catalytic domain deletion polypeptides are encompassed by the present invention: G1-E170, Y2-E170, P3-E170, D4-E170, G5-E170, G6-E170, A7-E170, A8-E170, Q9-E170, A10-E170, F11-E170, S12-E170, K13-E170, R14-E170, T15-E170, L16-E170, S17-E170, W18-E170, R19-E170, L20-E170, L21-E170, G22-E170, E23-E170, A24-E170, L25-E170, S26-E170, S27-E170, Q28-E170, L29-E170, S30-E170, V31-E170, A32-E170, D33-E170, Q34-E170, R35-E170, R36-E170, 137-E170, V38-E170, A39-E170, L40-E170, A41-E170, F42-E170, R43-E170, M44-E170, W45-E170, S46-E170, E47-E170, V48-E170, T49-E170, P50-E170, L51-E170, D52-E170, F53-E170, R54-E170, E55-E170, D56-E170, L57-E170, A58-E170, A59-E170, P60-E170, G61-E170, A62-E170, A63-E170, V64-E170, D65-E170, 166-E170, K67-E170, L68-E170, G69-E170, F70-E170, G71-E170, R72-E170, G73-E170, R74-E170, H75-E170, L76-E170, G77-E170, C78-E170, P79-E170, R80-E170, A81-E170, F82-E170, D83-E170, G84-E170, S85-E170, G86-E170, Q87-E170, E88-E170, F89-E170, A90-E170, H91-E170, A92-E170, W93-E170, R94-E170, L95-E170, G96-E170, D97-E170, 198-E170, H99-E170, F100-E170, D101-E170, D102-E170, D103-E170, E104-E170, H105-E170, F106-E170, T107-E170, P108-E170, P109-E170, T110-E170, S111-E170, D112-E170, T113-E170, G114-E170, I115-E170, S116-E170, L117-E170, L118-E170, K119-E170, V120-E170, A121-E170, V122-E170, H123-E170, E124-E170, I125-E170, G126-E170, H127-E170, V128-E170, L129-E170, G130-E170, L131-E170, P132-E170, H133-E170, T134-E170, Y135-E170, R136-E170, T137-E170, G138-E170, S139-E170, 1140-E170, M141-E170, Q142-E170, P3 143-E170, N144-E170, Y145-E170, 1146-E170, P147-E170, Q148-E170, E149-E170, P150-E170, A151-E170, F152-E170, E153-E170, L154-E170, D155-E170, W156-E170, S157-E170, D158-E170, R159-E170, K160-E170, A161-E170, 1162-E170, Q163-E170, and/or K164-E170 of SEQ ID NO:76. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 catalytic domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal MMP-29 catalytic domain deletion polypeptides are encompassed by the present invention: G1-E170, G1-C169, G1-S168, G1-G167, G1-Y166, G1-L165, G1-K164, G1-Q163, G1-1162, G1-A161, G1-K160, G1-R159, G1-D158, G1-S157, G1-W156, G1-D155, G1-L154, G1-E153, G1-F152, G1-A151, G1-P150, G1-E149, G1-Q148, G1-P147, G1-1146, G1-Y145, G1-N144, G1-P143, G1-Q142, G1-M141, G1-1140, G1-S139, G1-G138, G1-T137, G1-R136, G1-Y135, G1-T134, G1-H133, G1-P132, G1-L131, G1-G130, G1-L129, G1-V128, G1-H127, G1-G126, G1-1125, G1-E124, G1-H123, G1-V122, G1-A121, G1-V120, G1-K119, G1-L118, G1-L117, G1-S116, G1-1115, G1-G114, G1-T113, G1-D112, G1-S111, G1-T110, G1-P109, G1-P108, G1-T107, G1-F106, G1-H105, G1-E104, G1-D103, G1-D102, G1-D101, G1-F100, G1-H99, G1-198, G1-D97, G1-G96, G1-L95, G1-R94, G1-W93, G1-A92, G1-H91, G1-A90, G1-F89, G1-E88, G1-Q87, G1-G86, G1-S85, G1-G84, G1-D83, G1-F82, G1-A81, G1-R80, G1-P79, G1-C78, G1-G77, G1-L76, G1-H75, G1-R74, G1-G73, G1-R72, G1-G71, G1-F70, G1-G69, G1-L68, G1-K67, G1-166, G1-D65, G1-V64, G1-A63, G1-A62, G1-G61, G1-P60, G1-A59, G1-A58, G1-L57, G1-D56, G1-E55, G1-R54, G1-F53, G1-D52, G1-L51, G1-P50, G1-T49, G1-V48, G1-E47, G1-S46, G1-W45, G1-M44, G1-R43, G1-F42, G1-A41, G1-L40, G1-A39, G1-V38, G1-137, G1-R36, G1-R35, G1-Q34, G1-D33, G1-A32, G1-V31, G1-S30, G1-L29, G1-Q28, G1-S27, G1-S26, G1-L25, G1-A24, G1-E23, G1-G22, G1-L21, G1-L20, G1-R19, G1-W18, G1-S17, G1-L16, G1-T15, G1-R14, G1-K13, G1-S12, G1-F11, G1-A10, G1-Q9, G1-A8, and/or G1-A7 of SEQ ID NO:76. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 catalytic domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the MMP-29 catalytic domain (e.g., any combination of both N- and C-terminal MMP-29 catalytic domain deletions) of SEQ ID NO:76. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the MMP-29 catalytic domain (SEQ ID NO:76), and where CX refers to any C-terminal amino acid position of the MMP-29 catalytic domain (SEQ ID NO:76). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following MMP-29 catalytic domain amino acid substitutions are encompassed by the present invention: wherein G161 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y162 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein P163 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein D164 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G165 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G166 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A167 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A168 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q169 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein A170 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F171 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S172 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein K173 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R174 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T175 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein L176 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S177 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein W178 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein R179 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L180 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L181 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G182 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E183 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A184 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L185 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S186 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein S187 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Q188 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein L189 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein S190 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein V191 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein A192 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D193 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q194 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein R195 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein R196 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein 1197 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V198 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein A199 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L200 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A201 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F202 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R203 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein M204 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein W205 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S206 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E207 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, k, S, T, V, W, or Y; wherein V208 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein T209 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein P210 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein L211 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D212 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F213 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R214 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein E215 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D216 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L217 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein A218 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A219 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P220 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G221 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A222 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A223 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V224 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein D225 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I226 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K227 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L228 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G229 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F230 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G231 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R232 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein G233 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R234 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein H235 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L236 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G237 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein C238 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P239 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein R240 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein A241 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F242 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D243 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G244 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S245 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein G246 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q247 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein E248 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F249 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A250 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H251 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A252 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W253 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein R254 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein L255 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G256 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D257 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I258 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H259 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F260 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D261 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D262 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D263 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E264 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H265 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F266 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T267 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein P268 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein P269 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein T270 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein S271 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D272 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T273 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G274 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I275 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S276 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L277 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein L278 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein K279 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V280 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein A281 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V282 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein H283 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E284 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I285 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G286 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein H287 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V288 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L289 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein G290 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L291 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P292 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein H293 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T294 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y295 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein R296 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T297 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G298 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S299 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein I300 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein M301 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein Q302 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein P303 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein N 3 O 4 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein Y305 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein I306 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P307 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein Q308 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein E309 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P310 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A311 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F312 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E313 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L314 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D315 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W316 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein S317 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D318 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R319 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein K320 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A321 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I322 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q323 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein K324 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L325 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Y326 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G327 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S328 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein C329 is substituted with either an A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein E330 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y of SEQ ID NO:2, in addition to any combination thereof. The present invention also encompasses the use of these MMP-29 catalytic domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 19 of 64

In preferred embodiments, the following MMP-29 catalytic domain conservative amino acid substitutions are encompassed by the present invention: wherein G161 is substituted with either an A, M, S, or T; wherein Y162 is either an F, or W; wherein P163 is a P; wherein D164 is substituted with an E; wherein G165 is substituted with either an A, M, S, or T; wherein G166 is substituted with either an A, M, S, or T; wherein A167 is substituted with either a G, I, L, M, S, T, or V; wherein A168 is substituted with either a G, I, L, M, S, T, or V; wherein Q169 is substituted with a N; wherein A170 is substituted with either a G, I, L, M, S, T, or V; wherein F171 is substituted with either a W, or Y; wherein S172 is substituted with either an A, G, M, or T; wherein K173 is substituted with either a R, or H; wherein R174 is substituted with either a K, or H; wherein T175 is substituted with either an A, G, M, or S; wherein L176 is substituted with either an A, I, or V; wherein S177 is substituted with either an A, G, M, or T; wherein W178 is either an F, or Y; wherein R179 is substituted with either a K, or H; wherein L180 is substituted with either an A, I, or V; wherein L181 is substituted with either an A, I, or V; wherein G182 is substituted with either an A, M, S, or T; wherein E183 is substituted with a D; wherein A184 is substituted with either a G, I, L, M, S, T, or V; wherein L185 is substituted with either an A, I, or V; wherein S186 is substituted with either an A, G, M, or T; wherein S187 is substituted with either an A, G, M, or T; wherein Q188 is substituted with a N; wherein L189 is substituted with either an A, I, or V; wherein S190 is substituted with either an A, G, M, or T; wherein V191 is substituted with either an A, I, or L; wherein A192 is substituted with either a G, I, L, M, S, T, or V; wherein D193 is substituted with an E; wherein Q194 is substituted with a N; wherein R195 is substituted with either a K, or H; wherein R196 is substituted with either a K, or H; wherein I197 is substituted with either an A, V, or L; wherein V198 is substituted with either an A, I, or L; wherein A199 is substituted with either a G, I, L, M, S, T, or V; wherein L200 is substituted with either an A, I, or V; wherein A201 is substituted with either a G, I, L, M, S, T, or V; wherein F202 is substituted with either a W, or Y; wherein R203 is substituted with either a K, or H; wherein M204 is substituted with either an A, G, S, or T; wherein W205 is either an F, or Y; wherein S206 is substituted with either an A, G, M, or T; wherein E207 is substituted with a D; wherein V208 is substituted with either an A, I, or L; wherein T209 is substituted with either an A, G, M, or S; wherein P210 is a P; wherein L211 is substituted with either an A, I, or V; wherein D212 is substituted with an E; wherein F213 is substituted with either a W, or Y; wherein R214 is substituted with either a K, or H; wherein E215 is substituted with a D; wherein D216 is substituted with an E; wherein L217 is substituted with either an A, I, or V; wherein A218 is substituted with either a G, I, L, M, S, T, or V; wherein A219 is substituted with either a G, I, L, M, S, T, or V; wherein P220 is a P; wherein G221 is substituted with either an A, M, S, or T; wherein A222 is substituted with either a G, I, L, M, S, T, or V; wherein A223 is substituted with either a G, I, L, M, S, T, or V; wherein V224 is substituted with either an A, I, or L; wherein D225 is substituted with an E; wherein 1226 is substituted with either an A, V, or L; wherein K227 is substituted with either a R, or H; wherein L228 is substituted with either an A, I, or V; wherein G229 is substituted with either an A, M, S, or T; wherein F230 is substituted with either a W, or Y; wherein G231 is substituted with either an A, M, S, or T; wherein R232 is substituted with either a K, or H; wherein G233 is substituted with either an A, M, S, or T; wherein R234 is substituted with either a K, or H; wherein H235 is substituted with either a K, or R; wherein L236 is substituted with either an A, I, or V; wherein G237 is substituted with either an A, M, S, or T; wherein C238 is a C; wherein P239 is a P; wherein R240 is substituted with either a K, or H; wherein A241 is substituted with either a G, I, L, M, S, T, or V; wherein F242 is substituted with either a W, or Y; wherein D243 is substituted with an E; wherein G244 is substituted with either an A, M, S, or T; wherein S245 is substituted with either an A, G, M, or T; wherein G246 is substituted with either an A, M, S, or T; wherein Q247 is substituted with a N; wherein E248 is substituted with a D; wherein F249 is substituted with either a W, or Y; wherein A250 is substituted with either a G, I, L, M, S, T, or V; wherein H251 is substituted with either a K, or R; wherein A252 is substituted with either a G, I, L, M, S, T, or V; wherein W253 is either an F, or Y; wherein R254 is substituted with either a K, or H; wherein L255 is substituted with either an A, I, or V; wherein G256 is substituted with either an A, M, S, or T; wherein D257 is substituted with an E; wherein I258 is substituted with either an A, V, or L; wherein H259 is substituted with either a K, or R; wherein F260 is substituted with either a W, or Y; wherein D261 is substituted with an E; wherein D262 is substituted with an E; wherein D263 is substituted with an E; wherein E264 is substituted with a D; wherein H265 is substituted with either a K, or R; wherein F266 is substituted with either a W, or Y; wherein T267 is substituted with either an A, G, M, or S; wherein P268 is a P; wherein P269 is a P; wherein T270 is substituted with either an A, G, M, or S; wherein S271 is substituted with either an A, G, M, or T; wherein D272 is substituted with an E; wherein T273 is substituted with either an A, G, M, or S; wherein G274 is substituted with either an A, M, S, or T; wherein I275 is substituted with either an A, V, or L; wherein S276 is substituted with either an A, G, M, or T; wherein L277 is substituted with either an A, I, or V; wherein L278 is substituted with either an A, I, or V; wherein K279 is substituted with either a R, or H; wherein V280 is substituted with either an A, I, or L; wherein A281 is substituted with either a G, I, L, M, S, T, or V; wherein V282 is substituted with either an A, I, or L; wherein H283 is substituted with either a K, or R; wherein E284 is substituted with a D; wherein I285 is substituted with either an A, V, or L; wherein G286 is substituted with either an A, M, S, or T; wherein H287 is substituted with either a K, or R; wherein V288 is substituted with either an A, I, or L; wherein L289 is substituted with either an A, I, or V; wherein G290 is substituted with either an A, M, S, or T; wherein L291 is substituted with either an A, I, or V; wherein P292 is a P; wherein H293 is substituted with either a K, or R; wherein T294 is substituted with either an A, G, M, or S; wherein Y295 is either an F, or W; wherein R296 is substituted with either a K, or H; wherein T297 is substituted with either an A, G, M, or S; wherein G298 is substituted with either an A, M, S, or T; wherein S299 is substituted with either an A, G, M, or T; wherein I300 is substituted with either an A, V, or L; wherein M301 is substituted with either an A, G, S, or T; wherein Q302 is substituted with a N; wherein P303 is a P; wherein N304 is substituted with a Q; wherein Y305 is either an F, or W; wherein I306 is substituted with either an A, V, or L; wherein P307 is a P; wherein Q308 is substituted with a N; wherein E309 is substituted with a D; wherein P310 is a P; wherein A311 is substituted with either a G, I, L, M, S, T, or V; wherein F312 is substituted with either a W, or Y; wherein E313 is substituted with a D; wherein L314 is substituted with either an A, I, or V; wherein D315 is substituted with an E; wherein W316 is either an F, or Y; wherein S317 is substituted with either an A, G, M, or T; wherein D318 is substituted with an E; wherein R319 is substituted with either a K, or H; wherein K320 is substituted with either a R, or H; wherein A321 is substituted with either a G, I, L, M, S, T, or V; wherein I322 is substituted with either an A, V, or L; wherein Q323 is substituted with a N; wherein K324 is substituted with either a R, or H; wherein L325 is substituted with either an A, I, or V; wherein Y326 is either an F, or W; wherein G327 is substituted with either an A, M, S, or T; wherein S328 is substituted with either an A, G, M, or T; wherein C329 is a C; and/or wherein E330 is substituted with a D of SEQ ID NO:2 in addition to any combination thereof. Other suitable substitutions within the MMP-29 catalytic domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these MMP-29 catalytic domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 20 of 64

In accordance with the structural coordinates provided in Table VI and the three dimensional homology model of MMP-29, the MMP-29 polypeptide has been shown to comprise a hemopexin-like domain embodied by the following amino acids: at about amino acid Q346 to about amino acid N544 of SEQ ID NO:2 ( FIGS. 1A-B ). In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids more in either the N- or C-terminal direction of the above referenced amino acids.

Also more preferred are polypeptides comprising all or any part of the MMP-29 hemopexin-like domain, or a mutant or homologue of said polypeptide or molecular complex. By mutant or homologue of the molecule is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

In preferred embodiments, the following MMP-29 hemopexin-like domain polypeptide is encompassed by the present invention: QYGEVMVRFSTYFFRNSWYWLYENRNNRTRYGDPIQILTGWPGIPTHNIDAF VHIWTWKRDERYFFQGNQYWRYDSDKDQALTEDEQGKSYPKLISEGFPGIPS PLDTAFYDRRQKLIYFFKESLVFAFDVNRNRVLNSYPKRITEVFPAVIPQNHPF RNIDSAYYSYAYNSIFFFKGNAYWKVVNDKDKQQNSWLPAN (SEQ ID NO:77). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of the MMP-29 hemopexin-like domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses polypeptides comprising at least a portion of the MMP-29 hemopexin-like domain (SEQ ID NO:77). Such polypeptides may correspond, for example, to the N- and/or C-terminal deletions of the hemopexin-like domain.

In preferred embodiments, the following N-terminal MMP-29 hemopexin-like domain deletion polypeptides are encompassed by the present invention: Q1-N199, Y2-N199, G3-N199, E4-N199, V5-N199, M6-N199, V7-N199, R8-N199, F9-N199, S10-N199, T11-N199, Y12-N199, F13-N199, F14-N199, R15-N199, N16-N199, S17-N199, W18-N199, Y19-N199, W20-N199, L21-N199, Y22-N199, E23-N199, N24-N199, R25-N199, N26-N199, N27-N199, R28-N199, T29-N199, R30-N199, Y31-N199, G32-N199, D33-N199, P34-N199, 135-N199, Q36-N199, 137-N199, L38-N199, T39-N199, G40-N199, W41-N199, P42-N199, G43-N199, 144-N199, P45-N199, T46-N199, H47-N199, N48-N199, 149-N199, D50-N199, A51-N199, F52-N199, V53-N199, H54-N199, 155-N199, W56-N199, T57-N199, W58-N199, K59-N199, R60-N199, D61-N199, E62-N199, R63-N199, Y64-N199, F65-N199, F66-N199, Q67-N199, G68-N199, N69-N199, Q70-N199, Y71-N199, W72-N199, R73-N199, Y74-N199, D75-N199, S76-N199, D77-N199, K78-N199, D79-N199, Q80-N199, A81-N199, L82-N199, T83-N199, E84-N199, D85-N199, E86-N199, Q87-N199, G88-N199, K89-N199, S90-N199, Y91-N199, P92-N199, K93-N199, L94-N199, I95-N199, S96-N199, E97-N199, G98-N199, F99-N199, P100-N199, G101-N199, 1102-N199, P103-N199, S104-N199, P105-N199, L106-N199, D107-N199, T108-N199, A109-N199, F110-N199, Y111-N199, D112-N199, R113-N199, R114-N199, Q115-N199, K116-N199, L117-N199, I118-N199, Y119-N199, F120-N199, F121-N199, K122-N199, E123-N199, S124-N199, L125-N199, V126-N199, F127-N199, A128-N199, F129-N199, D130-N199, V131-N199, N132-N199, R133-N199, N134-N199, R135-N199, V136-N199, L137-N199, N138-N199, S139-N199, Y140-N199, P141-N199, K142-N199, R143-N199, 1144-N199, T145-N199, E146-N199, V147-N199, F148-N199, P149-N199, A150-N199, V151-N199, 1152-N199, P153-N199, Q154-N199, N155-N199, H156-N199, P157-N199, F158-N199, R159-N199, N160-N199, I161-N199, D162-N199, S163-N199, A164-N199, Y165-N199, Y166-N199, S167-N199, Y168-N199, A169-N199, Y170-N199, N171-N199, S172-N199, I173-N199, F174-N199, F175-N199, F176-N199, K177-N199, G178-N199, N179-N199, A180-N199, Y181-N199, W182-N199, K183-N199, V184-N199, V185-N199, N186-N199, D187-N199, K188-N199, D189-N199, K190-N199, Q191-N199, Q192-N199, and/or N193-N199 of SEQ ID NO:77. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal MMP-29 hemopexin-like domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal MMP-29 hemopexin-like domain deletion polypeptides are encompassed by the present invention: Q1-N199, Q1-A198, Q1-P197, Q1-L196, Q1-W195, Q1-S194, Q1-N193, Q1-Q192, Q1-Q191, Q1-K190, Q1-D189, Q1-K188, Q1-D187, Q1-N186, Q1-V185, Q1-V184, Q1-K183, Q1-W182, Q1-Y181, Q1-A180, Q1-N179, Q1-G178, Q1-K177, Q1-F176, Q1-F175, Q1-F174, Q1-I173, Q1-S172, Q1-N171, Q1-Y170, Q1-A169, Q1-Y168, Q1-S167, Q1-Y166, Q1-Y165, Q1-A164, Q1-S163, Q1-D162, Q1-I161, Q1-N160, Q1-R159, Q1-F158, Q1-P157, Q1-H156, Q1-N155, Q1-Q154, Q1-P153, Q1-I152, Q1-V151, Q1-A150, Q1-P149, Q1-F148, Q1-V147, Q1-E146, Q1-T145, Q1-I144, Q1-R143, Q1-K142, Q1-P141, Q1-Y140, Q1-S139, Q1-N138, Q1-L137, Q1-V136, Q1-R135, Q1-N134, Q1-R133, Q1-N132, Q1-V131, Q1-D130, Q1-F129, Q1-A128, Q1-F127, Q1-V126, Q1-L125, Q1-S124, Q1-E123, Q1-K122, Q1-F121, Q1-F120, Q1-Y119, Q1-I118, Q1-L117, Q1-K116, Q1-Q115, Q1-R114, Q1-R113, Q1-D112, Q1-Y111, Q1-F110, Q1-A109, Q1-T108, Q1-D107, Q1-L106, Q1-P105, Q1-S104, Q1-P103, Q1-I102, Q1-G101, Q1-P100, Q1-F99, Q1-G98, Q1-E97, Q1-S96, Q1-195, Q1-L94, Q1-K93, Q1-P92, Q1-Y91, Q1-S90, Q1-K89, Q1-G88, Q1-Q87, Q1-E86, Q1-D85, Q1-E84, Q1-T83, Q1-L82, Q1-A81, Q1-Q80, Q1-D79, Q1-K78, Q1-D77, Q1-S76, Q1-D75, Q1-Y74, Q1-R73, Q1-W72, Q1-Y71, Q1-Q70, Q1-N69, Q1-G68, Q1-Q67, Q1-F66, Q1-F65, Q1-Y64, Q1-R63, Q1-E62, Q1-D61, Q1-R60, Q1-K59, Q1-W58, Q1-T57, Q1-W56, Q1-155, Q1-H54, Q1-V53, Q1-F52, Q1-A51, Q1-D50, Q1-149, Q1-N48, Q1-H47, Q1-T46, Q1-P45, Q1-144, Q1-G43, Q1-P42, Q1-W41, Q1-G40, Q1-T39, Q1-L38, Q1-137, Q1-Q36, Q1-135, Q1-P34, Q1-D33, Q1-G32, Q1-Y31, Q1-R30, Q1-T29, Q1-R28, Q1-N27, Q1-N26, Q1-R25, Q1-N24, Q1-E23, Q1-Y22, Q1-L21, Q1-W20, Q1-Y19, Q1-W18, Q1-S17, Q1-N16, Q1-R15, Q1-F14, Q1-F13, Q1-Y12, Q1-T11, Q1-S10, Q1-F9, Q1-R8, and/or Q1-V7 of SEQ ID NO:77. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal MMP-29 hemopexin-like domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 21 of 64

Alternatively, such polypeptides may comprise polypeptide sequences corresponding, for example, to internal regions of the MMP-29 hemopexin-like domain (e.g., any combination of both N- and C-terminal MMP-29 hemopexin-like domain deletions) of SEQ ID NO:77. For example, internal regions could be defined by the equation NX to CX, where NX refers to any N-terminal amino acid position of the MMP-29 hemopexin-like domain (SEQ ID NO:77), and where CX refers to any C-terminal amino acid position of the MMP-29 hemopexin-like domain (SEQ ID NO:77). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.

In preferred embodiments, the following MMP-29 hemopexin-like domain amino acid substitutions are encompassed by the present invention: wherein Q346 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein Y347 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G348 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E349 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V350 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein M351 is substituted with either an A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; wherein V352 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein R353 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein F354 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S355 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein T356 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein Y357 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F358 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F359 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R360 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N361 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S362 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein W363 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein Y364 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein W365 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein L366 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein Y367 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein E368 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N369 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R370 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N371 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein N372 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R373 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein T374 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein R375 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y376 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein G377 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D378 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P379 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein I380 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q381 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein I382 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L383 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T384 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein G385 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W386 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein P387 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G388 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I389 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P390 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein T391 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein H392 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N393 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein I394 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D395 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A396 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F397 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V398 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein H399 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I400 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein W401 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein T402 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein W403 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K404 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R405 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein D406 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E407 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R408 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y409 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F410 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F411 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q412 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G413 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N414 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein Q415 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein Y416 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein W417 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein R418 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Y419 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein D420 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S421 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein D422 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K423 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D424 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q425 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein A426 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L27 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein T428 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E429 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D430 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E431 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q432 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein G433 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K434 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S435 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y436 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein P437 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K438 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L439 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I440 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S441 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein E442 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G443 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F444 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P445 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein G446 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein I447 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P448 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein S449 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein P450 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein L451 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein D452 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T453 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein A454 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F455 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y456 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein D457 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R458 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein R459 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein Q460 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein K461 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein L462 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein I463 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y464 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein F465 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F466 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K467 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein E468 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S469 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein L470 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein V471 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F472 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein A473 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F474 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D475 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V476 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N477 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R478 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N479 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein R480 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein V481 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein L482 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein N483 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S484 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y485 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein P486 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein K487 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R488 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein I489 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein T490 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; wherein E491 is substituted with either an A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V492 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein F493 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P494 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A495 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V496 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein I497 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P498 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein Q499 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein N500 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein H501 is substituted with either an A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein P502 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein F503 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein R504 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y; wherein N505 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein I506 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D507 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein S508 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein A509 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y510 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein Y511 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein S512 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein Y513 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein A514 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y515 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein N516 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S517 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein I518 is substituted with either an A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F519 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F520 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein F521 is substituted with either an A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K522 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein G523 is substituted with either an A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein N524 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein A525 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Y526 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; wherein W527 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein K528 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein V529 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein V530 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; wherein N531 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein D532 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K533 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein D534 is substituted with either an A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; wherein K535 is substituted with either an A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; wherein Q536 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein Q537 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; wherein N538 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; wherein S539 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; wherein W540 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; wherein L541 is substituted with either an A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; wherein P542 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; wherein A543 is substituted with either a C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and/or wherein N544 is substituted with either an A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y of SEQ ID NO:2, in addition to any combination thereof. The present invention also encompasses the use of these MMP-29 hemopexin-like domain amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 22 of 64

In preferred embodiments, the following MMP-29 hemopexin-like domain conservative amino acid substitutions are encompassed by the present invention: wherein Q346 is substituted with a N; wherein Y347 is either an F, or W; wherein G348 is substituted with either an A, M, S, or T; wherein E349 is substituted with a D; wherein V350 is substituted with either an A, I, or L; wherein M351 is substituted with either an A, G, S, or T; wherein V352 is substituted with either an A, I, or L; wherein R353 is substituted with either a K, or H; wherein F354 is substituted with either a W, or Y; wherein S355 is substituted with either an A, G, M, or T; wherein T356 is substituted with either an A, G, M, or S; wherein Y357 is either an F, or W; wherein F358 is substituted with either a W, or Y; wherein F359 is substituted with either a W, or Y; wherein R360 is substituted with either a K, or H; wherein N361 is substituted with a Q; wherein S362 is substituted with either an A, G, M, or T; wherein W363 is either an F, or Y; wherein Y364 is either an F, or W; wherein W365 is either an F, or Y; wherein L366 is substituted with either an A, I, or V; wherein Y367 is either an F, or W; wherein E368 is substituted with a D; wherein N369 is substituted with a Q; wherein R370 is substituted with either a K, or H; wherein N371 is substituted with a Q; wherein N372 is substituted with a Q; wherein R373 is substituted with either a K, or H; wherein T374 is substituted with either an A, G, M, or S; wherein R375 is substituted with either a K, or H; wherein Y376 is either an F, or W; wherein G377 is substituted with either an A, M, S, or T; wherein D378 is substituted with an E; wherein P379 is a P; wherein I380 is substituted with either an A, V, or L; wherein Q381 is substituted with a N; wherein I382 is substituted with either an A, V, or L; wherein L383 is substituted with either an A, I, or V; wherein T384 is substituted with either an A, G, M, or S; wherein G385 is substituted with either an A, M, S, or T; wherein W386 is either an F, or Y; wherein P387 is a P; wherein G388 is substituted with either an A, M, S, or T; wherein I389 is substituted with either an A, V, or L; wherein P390 is a P; wherein T391 is substituted with either an A, G, M, or S; wherein H392 is substituted with either a K, or R; wherein N393 is substituted with a Q; wherein I394 is substituted with either an A, V, or L; wherein D395 is substituted with an E; wherein A396 is substituted with either a G, I, L, M, S, T, or V; wherein F397 is substituted with either a W, or Y; wherein V398 is substituted with either an A, I, or L; wherein H399 is substituted with either a K, or R; wherein I400 is substituted with either an A, V, or L; wherein W401 is either an F, or Y; wherein T402 is substituted with either an A, G, M, or S; wherein W403 is either an F, or Y; wherein K404 is substituted with either a R, or H; wherein R405 is substituted with either a K, or H; wherein D406 is substituted with an E; wherein E407 is substituted with a D; wherein R408 is substituted with either a K, or H; wherein Y409 is either an F, or W; wherein F410 is substituted with either a W, or Y; wherein F411 is substituted with either a W, or Y; wherein Q412 is substituted with a N; wherein G413 is substituted with either an A, M, S, or T; wherein N414 is substituted with a Q; wherein Q415 is substituted with a N; wherein Y416 is either an F, or W; wherein W417 is either an F, or Y; wherein R418 is substituted with either a K, or H; wherein Y419 is either an F, or W; wherein D420 is substituted with an E; wherein S421 is substituted with either an A, G, M, or T; wherein D422 is substituted with an E; wherein K423 is substituted with either a R, or H; wherein D424 is substituted with an E; wherein Q425 is substituted with a N; wherein A426 is substituted with either a G, I, L, M, S, T, or V; wherein L427 is substituted with either an A, I, or V; wherein T428 is substituted with either an A, G, M, or S; wherein E429 is substituted with a D; wherein D430 is substituted with an E; wherein E431 is substituted with a D; wherein Q432 is substituted with a N; wherein G433 is substituted with either an A, M, S, or T; wherein K434 is substituted with either a R, or H; wherein S435 is substituted with either an A, G, M, or T; wherein Y436 is either an F, or W; wherein P437 is a P; wherein K438 is substituted with either a R, or H; wherein L439 is substituted with either an A, I, or V; wherein I440 is substituted with either an A, V, or L; wherein S441 is substituted with either an A, G, M, or T; wherein E442 is substituted with a D; wherein G443 is substituted with either an A, M, S, or T; wherein F444 is substituted with either a W, or Y; wherein P445 is a P; wherein G446 is substituted with either an A, M, S, or T; wherein 1447 is substituted with either an A, V, or L; wherein P448 is a P; wherein S449 is substituted with either an A, G, M, or T; wherein P450 is a P; wherein L451 is substituted with either an A, I, or V; wherein D452 is substituted with an E; wherein T453 is substituted with either an A, G, M, or S; wherein A454 is substituted with either a G, I, L, M, S, T, or V; wherein F455 is substituted with either a W, or Y; wherein Y456 is either an F, or W; wherein D457 is substituted with an E; wherein R458 is substituted with either a K, or H; wherein R459 is substituted with either a K, or H; wherein Q460 is substituted with a N; wherein K461 is substituted with either a R, or H; wherein L462 is substituted with either an A, I, or V; wherein 1463 is substituted with either an A, V, or L; wherein Y464 is either an F, or W; wherein F465 is substituted with either a W, or Y; wherein F466 is substituted with either a W, or Y; wherein K467 is substituted with either a R, or H; wherein E468 is substituted with a D; wherein S469 is substituted with either an A, G, M, or T; wherein L470 is substituted with either an A, I, or V; wherein V471 is substituted with either an A, I, or L; wherein F472 is substituted with either a W, or Y; wherein A473 is substituted with either a G, I, L, M, S, T, or V; wherein F474 is substituted with either a W, or Y; wherein D475 is substituted with an E; wherein V476 is substituted with either an A, I, or L; wherein N477 is substituted with a Q; wherein R478 is substituted with either a K, or H; wherein N479 is substituted with a Q; wherein R480 is substituted with either a K, or H; wherein V481 is substituted with either an A, I, or L; wherein L482 is substituted with either an A, I, or V; wherein N483 is substituted with a Q; wherein S484 is substituted with either an A, G, M, or T; wherein Y485 is either an F, or W; wherein P486 is a P; wherein K487 is substituted with either a R, or H; wherein R488 is substituted with either a K, or H; wherein I489 is substituted with either an A, V, or L; wherein T490 is substituted with either an A, G, M, or S; wherein E491 is substituted with a D; wherein V492 is substituted with either an A, I, or L; wherein F493 is substituted with either a W, or Y; wherein P494 is a P; wherein A495 is substituted with either a G, I, L, M, S, T, or V; wherein V496 is substituted with either an A, I, or L; wherein I497 is substituted with either an A, V, or L; wherein P498 is a P; wherein Q499 is substituted with a N; wherein N500 is substituted with a Q; wherein H501 is substituted with either a K, or R; wherein P502 is a P; wherein F503 is substituted with either a W, or Y; wherein R504 is substituted with either a K, or H; wherein N505 is substituted with a Q; wherein I506 is substituted with either an A, V, or L; wherein D507 is substituted with an E; wherein S508 is substituted with either an A, G, M, or T; wherein A509 is substituted with either a G, I, L, M, S, T, or V; wherein Y510 is either an F, or W; wherein Y511 is either an F, or W; wherein S512 is substituted with either an A, G, M, or T; wherein Y513 is either an F, or W; wherein A514 is substituted with either a G, I, L, M, S, T, or V; wherein Y515 is either an F, or W; wherein N516 is substituted with a Q; wherein S517 is substituted with either an A, G, M, or T; wherein I518 is substituted with either an A, V, or L; wherein F519 is substituted with either a W, or Y; wherein F520 is substituted with either a W, or Y; wherein F521 is substituted with either a W, or Y; wherein K522 is substituted with either a R, or H; wherein G523 is substituted with either an A, M, S, or T; wherein N524 is substituted with a Q; wherein A525 is substituted with either a G, I, L, M, S, T, or V; wherein Y526 is either an F, or W; wherein W527 is either an F, or Y; wherein K528 is substituted with either a R, or H; wherein V529 is substituted with either an A, I, or L; wherein V530 is substituted with either an A, I, or L; wherein N531 is substituted with a Q; wherein D532 is substituted with an E; wherein K533 is substituted with either a R, or H; wherein D534 is substituted with an E; wherein K535 is substituted with either a R, or H; wherein Q536 is substituted with a N; wherein Q537 is substituted with a N; wherein N538 is substituted with a Q; wherein S539 is substituted with either an A, G, M, or T; wherein W540 is either an F, or Y; wherein L541 is substituted with either an A, I, or V; wherein P542 is a P; wherein A543 is substituted with either a G, I, L, M, S, T, or V; and/or wherein N544 is substituted with a Q of SEQ ID NO:2 in addition to any combination thereof. Other suitable substitutions within the MMP-29 hemopexin-like domain are encompassed by the present invention and are referenced elsewhere herein. The present invention also encompasses the use of these MMP-29 hemopexin-like domain conservative amino acid substituted polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.

›DETAILED DESCRIPTION OF THE INVENTION · 23 of 64

For purposes of the present invention, by “at least a portion of” is meant all or any part of the MMP-29 propeptide, catalytic, or hemopexin-like domain defined by the structure coordinates according to Tables IV, V, or VI (e.g., fragments thereof). More preferred are molecules comprising all or any parts of the MMP-29 propeptide, catalytic, or hemopexin-like domain, according to Tables IV, V, or VI, or a mutant or homologue of said molecule or molecular complex. By mutant or homologue of the molecule it is meant a molecule that has a root mean square deviation from the backbone atoms of said MMP-29 amino acids of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a term that expresses the deviation or variation from a trend or object. For the purposes of the present invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the AR portion of the complex as defined by the structure coordinates described herein.

A preferred embodiment is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structure coordinates of all of the amino acids in Tables IV, V, or VI +/− a root mean square deviation from the backbone atoms of those amino acids of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms.

The structure coordinates of a MMP-29 propeptide, catalytic, or hemopexin-like domain homology models, including portions thereof, is stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.

Accordingly, in one embodiment of this invention is provided a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Tables IV, V, or VI.

One embodiment utilizes System 10 as disclosed in WO 98/11134, the disclosure of which is incorporated herein by reference in its entirety. Briefly, one version of these embodiments comprises a computer comprising a central processing unit (“CPU”), a working memory which may be, e.g, RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or more keyboards, one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may comprise CD-ROM drives or disk drives. In conjunction with a display terminal, keyboard may also be used as an input device.

Output hardware, coupled to the computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include a CRT display terminal for displaying a graphical representation of a region or domain of the present invention using a program such as QUANTA as described herein. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.

In operation, the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage, and accesses to and from the working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following description of the data storage medium.

For the purpose of the present invention, any magnetic data storage medium which can be encoded with machine-readable data would be sufficient for carrying out the storage requirements of the system. The medium could be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, containing magnetic domains whose polarity or orientation could be altered magnetically, for example. The medium may also have an opening for receiving the spindle of a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as the system described herein.

Another example of a suitable storage medium which could also be encoded with such machine-readable data, or set of instructions, which could be carried out by a system such as the system described herein, could be an optically-readable data storage medium. The medium could be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable. The medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.

›DETAILED DESCRIPTION OF THE INVENTION · 24 of 64

In the case of a CD-ROM, as is well known, the coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of the coating. A protective coating, which preferably is substantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from the coating. The arrangement of the domains encodes the data as described above.

Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of the MMP-29 propeptide, catalytic, or hemopexin-like domain homology models, or portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure. Such data may be used for a variety of purposes, such as drug discovery.

For the first time, the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities that are capable of modulating the biological function of MMP-29.

Accordingly, the present invention is also directed to the design of small molecules which imitates the structure of the MMP-29 propeptide, catalytic, and/or hemopexin-like domain (SEQ ID NO:75, 76, and/or 77), or a portion thereof, in accordance with the structure coordinates provided in Tables IV, V, and/or VI. Alternatively, the present invention is directed to the design of small molecules which may bind to at least part of the MMP-29 propeptide, catalytic, and/or hemopexin-like domain (SEQ ID NO:75, 76, and/or 77), or some portion thereof. For purposes of this invention, by MMP-29 propeptide, catalytic, and/or hemopexin-like domain, it is also meant to include mutants or homologues thereof. In a preferred embodiment, the mutants or homologues have at least 25% identity, more preferably 50% identity, more preferably 75% identity, and most preferably 90% identity to SEQ ID NO:75, 76, and/or 77. In this context, the term “small molecule” may be construed to mean any molecule described known in the art or described elsewhere herein, though may include, for example, peptides, chemicals, carbohydrates, nucleic acids, PNAs, and any derivatives thereof.

The three-dimensional model structure of the MMP-29 will also provide methods for identifying modulators of biological function. Various methods or combination thereof can be used to identify these compounds.

For example, test compounds can be modeled that fit spatially into the catalytic domain in MMP-29 embodied by the sequence at amino C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2, in accordance with the structural coordinates of Table V.

Structure coordinates of the catalytic domain in MMP-29 defined by the amino acids at amino acid C117, at amino acid H283, at amino acid H287, at amino acid H293, at amino acid Q247, at amino acid E248, at amino acid F249, at amino acid A250, at amino acid I258, at amino acid Y273, at amino acid G274, and/or at amino acid Q302 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, C117, H283, H287, H293, Q247, E248, F249, A250, I258, Y273, G274, and/or Q302 of SEQ ID NO:2, or amino acids G161 through E330 in accordance with the structure coordinates of Table V.

Structure coordinates of the propeptide domain in MMP-29 defined by the amino acids at amino acid A48 through P120 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, A48 through P120 of SEQ ID NO:2 in accordance with the structure coordinates of Table IV.

›DETAILED DESCRIPTION OF THE INVENTION · 25 of 64

Structure coordinates of the hemopexin-like domain in MMP-29 defined by the amino acids at amino acid Q346 through N544 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, Q346 through N544 of SEQ ID NO:2 in accordance with the structure coordinates of Table VI.

For example, test compounds can be modeled that fit spatially into the S1 pocket in MMP-29 embodied by the sequence at amino Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2, in accordance with the structural coordinates of Table V.

Structure coordinates of the SI pocket in MMP-29 defined by the amino acids at amino acid Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2, can also be used to identify structural and chemical features. Identified structural or chemical features can then be employed to design or select compounds as potential MMP-29 modulators. By structural and chemical features it is meant to include, but is not limited to, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Alternatively, or in conjunction with, the three-dimensional structural model can be employed to design or select compounds as potential MMP-29 modulators. Compounds identified as potential MMP-29 modulators can then be synthesized and screened in an assay characterized by binding of a test compound to the MMP-29, or in characterizing the ability of MMP-29 to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential MMP-29 modulators include, but are not limited to, screening in silico, in vitro assays and high throughput assays. Finally, these methods may also involve modifying or replacing one or more amino acids at amino acid positions, Q247, E248, F249, A250, I258, Y273, G274, and Q302 of SEQ ID NO:2 in accordance with the structure coordinates of Table V.

However, as will be understood by those of skill in the art upon this disclosure, other structure based design methods can be used. Various computational structure based design methods have been disclosed in the art.

For example, a number of computer modeling systems are available in which the sequence of the MMP-29 and the MMP-29 structure (i.e., atomic coordinates of MMP-29 and/or the atomic coordinates of the active site domain as provided in Table IV) can be input. This computer system then generates the structural details of one or more these regions in which a potential MMP-29 modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with MMP-29. In addition, the compound must be able to assume a conformation that allows it to associate with MMP-29. Some modeling systems estimate the potential inhibitory or binding effect of a potential MMP-29 modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in the active site domain of MMP-29. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic forcefields such as MMFF, CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, their relationship to each other and MMP-29 can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Associates, St. Louis Mo.).

In addition, MMP-29 is overall well suited to modern methods including combinatorial chemistry.

Programs such as DOCK (Kuntz et. al. 1982) can be used with the atomic coordinates from the MMP-29 homology models to identify potential ligands from databases or virtual databases which potentially bind the in the active site region, and which may therefore be suitable candidates for synthesis and testing. The computer programs may utilize a combination of the following steps: (a) selection of fragments or chemical entities from a database and then positioning the chemical entity in one or more orientations within the MMP29 active site defined by residues C117, H283, H287, H293, Q247, E248, F249, A250, 1258, Y273, G274, and Q302; (b) characterization of the structural and chemical features of the chemical entity and active site including van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interactions; (c) search databases for molecular fragments which can be joined to or replace the docked chemical entity and spatially fit into regions defined by the said MMP29 active site; and/or (d) evaluate the docked chemical entity and fragments using a combination of scoring schemes which account for van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic interactions

›DETAILED DESCRIPTION OF THE INVENTION · 26 of 64

Examples of databases that may be used include ACD (Molecular Designs Limited), Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute), Maybridge(Maybridge Chemical Company Ltd), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), in addition to others known in the art.

Additionally, the three-dimensional homology model of MMP-29 will aid in the design of mutants with altered biological activity.

The following are encompassed by the present invention: a machine-readable data storage medium, comprising a data storage material encoded with machine readable data, wherein the data is defined by the structure coordinates of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models according to Tables IV, V, and/or VI or a homologue of said model, wherein said homologue comprises backbone atoms that have a root mean square deviation from the backbone atoms of the complex of not more than about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms; and a machine-readable data storage medium, wherein said molecule is defined by the set of structure coordinates of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models according to Tables IV, V, and/or VI, or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more about 3.5 Angstroms, not more than about 3.0 Angstroms, not more than about 2.5 Angstroms, not more than about 2.0 Angstroms, not more than about 1.5 Angstroms, not more than about 1.0 Angstroms, not more than about 0.9 Angstroms, not more than about 0.8 Angstroms, not more than about 0.7 Angstroms, not more than about 0.6 Angstroms, not more than about 0.5 Angstroms, not more than about 0.4 Angstroms, not more than about 0.3 Angstroms, not more than about 0.2 Angstroms, or not more than about 0.1 Angstroms; a model comprising all or any part of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models defined by structure coordinates according to Tables IV, V, and/or VI, or a mutant or homologue of said molecule or molecular complex.

In a further embodiment, the following are encompassed by the present invention: a method for identifying a mutant of MMP-29 with altered biological properties, function, or reactivity, the method comprising any combination of steps of: use of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models or a homologue of said models according to Tables IV, V, and/or VI, for the design of protein mutants with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; and use of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain models or a homologue of said models, for the design of a protein with mutations in the propeptide domain comprised of the amino acids at amino acid A48 thru P120 according to Table IV with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; or for the design of a protein with mutations in the catalytic domain comprised of the amino acids at amino acid G161 through E330 according to Table V with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein; or for the design of a protein with mutations in the catalytic domain comprised of the amino acids at amino acid Q346 through N544 according to Table VI with altered biological function or properties which exhibit any combination of therapeutic effects provided elsewhere herein

In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of MMP-29 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the catalytic domain defined by all or any portion of residues at amino acid G161 through E330, or preferably as defined by residues C117, H283, H287, H293, Q247, E248, F249, A250, 1258, Y273, G274, and Q302 and of the three-dimensional structural model according to Table V, or using a homologue or portion thereof.

In further preferred embodiments, the following are encompassed by the present invention: a method for identifying modulators of MMP-29 biological properties, function, or reactivity, the method comprising any combination of steps of: modeling test compounds that overlay spatially into the S1 pocket defined by all or any portion of residues at amino acid Q247, E248, F249, A250, I258, Y273, G274, and Q302 and of the three-dimensional structural model according to Table V, or using a homologue or portion thereof.

The present invention encompasses using the structure coordinates as set forth herein to identify structural and chemical features of the MMP-29 polypeptide; employing identified structural or chemical features to design or select compounds as potential MMP-29 modulators; employing the three-dimensional structural models described herien to design or select compounds as potential MMP-29 modulators; synthesizing the potential MMP-29 modulators; screening the potential MMP-29 modulators in an assay characterized by binding of a protein to the MMP-29; selecting the potential MMP-29 modulator from a database; designing the MMP-29 modulator de novo; and/or designing said MMP-29 modulator from a known modulator activity.

›DETAILED DESCRIPTION OF THE INVENTION · 27 of 64

Thus, one embodiment of the invention relaties to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the MMP-29 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. Accordingly, the cDNA sequence for MMP-29 depicted in FIGS. 1A-B as well as vectors and host cells expressing the MMP-29 protein or peptides thereof are useful in methods of identifying agents which alter or inhibit MMP-29 activities through modulation of the catalytic region(s). High-throughput screening assays such as proximity-based assays can also be developed using radiolabeled or fluorescent-labeled molecules.

The invention also relates to in silico screening methods including in silico docking and methods of structure based drug design which utilize the three dimensional structural coordinates of the MMP-29 propeptide domain, catalytic domain, and/or hemopexin-like domain (Tables IV, V, and/or VI). Also provided are methods of identifying modulators of MMP-29 that include modulator building or searching utilizing computer programs and algorithms. In an embodiment of the invention a method is provided for designing potential modulators of MMP-29 comprising any combination of steps which utilize said three dimensional structure to design or select potential modulators.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO: 1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1693 of SEQ ID NO:1, b is an integer between 15 to 1707, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14.

In one embodiment, a MMP-29 polypeptide comprises a portion of the amino sequence depicted in FIGS. 1A-B . In another embodiment, a MMP-29 polypeptide comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of the amino sequence depicted in FIGS. 1A-B . In further embodiments, the following MMP-29 polypeptide fragments are specifically excluded from the present invention: DTAFYDRRQKLIYFFKESLV (SEQ ID NO:78); AFDVNRNRVLNSYPKRITEVFPAVIPQNHPFRNIDSAYYSYAYNSIFFFKGNAY WKVVNDKDKQQNSWLPANGLFPKKFISEKWFDVCDVHISTLNM (SEQ ID NO:79); or LLKVAVHEIGH (SEQ ID NO:80).

Table I summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table I and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO: 1.

The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC Deposit No:Z and Date.” “Vector” refers to the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides in the clone contig identified by “Gene No.” The deposited clone may contain all or most of the sequence of SEQ ID NO:1. The nucleotide position of SEQ ID NO:1 of the putative start codon (methionine) is identified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:2,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ ID NO:2 is identified as “Total AA of ORF”.

SEQ ID NO:1 (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:2 (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further herein. For instance, SEQ ID NO:1 is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:1 or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:2 may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the proteins encoded by the cDNA clones identified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:1 and the predicted translated amino acid sequence identified as SEQ ID NO:2, but also a sample of plasmid DNA containing a cDNA of the invention deposited with the ATCC, as set forth in Table I. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited cDNA, collecting the protein, and determining its sequence.

›DETAILED DESCRIPTION OF THE INVENTION · 28 of 64

The present invention also relates to the genes corresponding to SEQ ID NO:1, or the deposited clone. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologues of genes corresponding to SEQ ID NO:1, or a deposited clone, relying on the sequence from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologues may be isolated and identified by making suitable probes or primers which correspond to the 5′, 3′, or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the full-length form of the protein.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, and/or a cDNA provided in ATCC Deposit No. Z. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:2, and/or a polypeptide encoded by the cDNA provided in ATCC Deposit NO:Z. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:2, and/or a polypeptide sequence encoded by the cDNA contained in ATCC Deposit No:Z.

Preferably, the present invention is directed to a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, and/or a cDNA provided in ATCC Deposit No.: that is less than, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.

The present invention encompasses polynucleotides with sequences complementary to those of the polynucleotides of the present invention disclosed herein. Such sequences may be complementary to the sequence disclosed as SEQ ID NO:1, the sequence contained in a deposit, and/or the nucleic acid sequence encoding the sequence disclosed as SEQ ID NO:2.

The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in Table II below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.

Additional examples of stringency conditions for polynucleotide hybridization are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art, and discussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention, the clone deposited with the ATCC, and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds., Innis et al., Academic Press, New York, (1990).

›DETAILED DESCRIPTION OF THE INVENTION · 29 of 64

Signal Sequences

The present invention also encompasses mature forms of the polypeptide comprising, or alternatively consisting of, the polypeptide sequence of SEQ ID NO:2, the polypeptide encoded by the polynucleotide described as SEQ ID NO:1, and/or the polypeptide sequence encoded by a cDNA in the deposited clone. The present invention also encompasses polynucleotides encoding mature forms of the present invention, such as, for example the polynucleotide sequence of SEQ ID NO:1, and/or the polynucleotide sequence provided in a cDNA of the deposited clone.

According to the signal hypothesis, proteins secreted by eukaryotic cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most eukaryotic cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

The established method for identifying the location of signal sequences, in addition, to their cleavage sites has been the SignalP program (v1.1) developed by Henrik Nielsen et al., Protein Engineering 10:1-6 (1997). The program relies upon the algorithm developed by von Heinje, though provides additional parameters to increase the prediction accuracy.

More recently, a hidden Markov model has been developed (H. Neilson, et al., Ismb 1998;6:122-30), which has been incorporated into the more recent SignalP (v2.0). This new method increases the ability to identify the cleavage site by discriminating between signal peptides and uncleaved signal anchors. The present invention encompasses the application of the method disclosed therein to the prediction of the signal peptide location, including the cleavage site, to any of the polypeptide sequences of the present invention.

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the polypeptide of the present invention may contain a signal sequence. Polypeptides of the invention which comprise a signal sequence have an N-terminus beginning within 5 residues (i.e., + or −5 residues, or preferably at the −5, −4, −3, −2, −1, +1, +2, +3, +4, or +5 residue) of the predicted cleavage point. Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:1 and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO:1, the complementary strand thereto, and/or the cDNA sequence contained in the deposited clone.

The present invention also encompasses variants of the polypeptide sequence, and/or fragments therein, disclosed in SEQ ID NO:2, a polypeptide encoded by the polynucleotide sequence in SEQ ID NO:1, and/or a polypeptide encoded by a cDNA in the deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (b) a nucleotide sequence encoding a mature MMP-29 related polypeptide having the amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (c) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence shown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (d) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence sown in the sequence listing and described in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (e) a nucleotide sequence encoding a MMP-29 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (f) a nucleotide sequence encoding a mature MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (g) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z;

›DETAILED DESCRIPTION OF THE INVENTION · 30 of 64

(h) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 or the cDNA contained in ATCC deposit No:Z; (I) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

Another aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively, consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (b) a nucleotide sequence encoding a mature MMP-29 related polypeptide having the amino acid sequence as shown in the sequence listing and descried in Table I; (c) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (d) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence as shown in the sequence listing and descried in Table I; (e) a nucleotide sequence encoding a MMP-29 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (f) a nucleotide sequence encoding a mature MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I: (g) a nucleotide sequence encoding a biologically active fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC Deposit and described in Table I; (h) a nucleotide sequence encoding an antigenic fragment of a MMP-29 related polypeptide having an amino acid sequence encoded by a human cDNA in a cDNA plasmid contained in the ATCC deposit and described in Table I; (i) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules which comprise, or alternatively, consist of, a nucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO:2, the polypeptide sequence encoded by a cDNA provided in the deposited clone, and/or polypeptide fragments of any of the polypeptides provided herein. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.

The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, the polypeptide sequence shown in SEQ ID NO:2, a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:1, a polypeptide sequence encoded by the cDNA in cDNA plasmid:Z, and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these polypeptides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompasses by the present invention, as are the polypeptides encoded by these polynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referenced in Table I, the ORF (open reading frame), or any fragment specified as described herein.

›DETAILED DESCRIPTION OF THE INVENTION · 31 of 64

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for instance, an amino acid sequence referenced in Table 1 (SEQ ID NO:2) or to the amino acid sequence encoded by cDNA contained in a deposited clone, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).

›DETAILED DESCRIPTION OF THE INVENTION · 32 of 64

The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of N- or C-terminal deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polypeptide alignment. Percent identity calculations based upon global polypeptide alignments are often preferred since they reflect the percent identity between the polypeptide molecules as a whole (i.e., including any polypeptide overhangs, not just overlapping regions), as opposed to, only local matching polypeptides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for N- and C-terminal truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what may be used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the CLUSTALW alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the CLUSTALW alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.

In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modifed CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.

The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the mRNA to those preferred by a bacterial host such as E. coli ).

Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

›DETAILED DESCRIPTION OF THE INVENTION · 33 of 64

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem . . . 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the protein will likely be retained when less than the majority of the residues of the protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptide of the present invention may, in fact, result in a significant increase in one or more of the biological activities of the polypeptide(s). For example, biological activity of many polypeptides are governed by the presence of regulatory domains at either one or both termini. Such regulatory domains effectively inhibit the biological activity of such polypeptides in lieu of an activation event (e.g., binding to a cognate ligand or receptor, phosphorylation, proteolytic processing, etc.). Thus, by eliminating the regulatory domain of a polypeptide, the polypeptide may effectively be rendered biologically active in the absence of an activation event.

Thus, the invention further includes polypeptide variants that show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.

The invention encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptide of the present invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics (e.g., chemical properties). According to Cunningham et al above, such conservative substitutions are likely to be phenotypically silent. Additional guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservative substitutions provided in Table IV below.

Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.

›DETAILED DESCRIPTION OF THE INVENTION · 34 of 64

Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple genes with related function and assessing the relative penalty of each substitution to proper gene function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants created through the application of molecular evolution (“DNA Shuffling”) methodology to the polynucleotide disclosed as SEQ ID NO:1, the sequence of the clone submitted in a deposit, and/or the cDNA encoding the polypeptide disclosed as SEQ ID NO:2. Such DNA Shuffling technology is known in the art and more particularly described elsewhere herein (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples provided herein).

A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5,5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:1 or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:1. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

›DETAILED DESCRIPTION OF THE INVENTION · 35 of 64

Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:1, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:2 or encoded by the cDNA contained in a deposited clone. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:2 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to bind to at least one of the same antibodies which bind to the full-length protein, the fragments ability to interact with at lease one of the same proteins which bind to the full-length, the fragments ability to elicit at least one of the same immune responses as the full-length protein (i.e., to cause the immune system to create antibodies specific to the same epitope, etc.), the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan, some of which are described elsewhere herein.

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:1 or contained in ATCC deposit No. Z under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.

›DETAILED DESCRIPTION OF THE INVENTION · 36 of 64

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length, or longer. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

›DETAILED DESCRIPTION OF THE INVENTION · 37 of 64

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Moreover, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules, as well as, antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation of the animal or plant, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med . . . . 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

›DETAILED DESCRIPTION OF THE INVENTION · 38 of 64

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homologue of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologues of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6 M, 5×10-7 M, 107 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111 (Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).

›DETAILED DESCRIPTION OF THE INVENTION · 39 of 64

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionucleotides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitable method known in the art.

The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2 nd ed. (1988); and Current Protocols, Chapter 2; which are hereby incorporated herein by reference in its entirety). In a preferred method, a preparation of the MMP-29 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. The administration of the polypeptides of the present invention may entail one or more injections of an immunizing agent and, if desired, an adjuvant. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum . Such adjuvants are also well known in the art. For the purposes of the invention, “immunizing agent” may be defined as a polypeptide of the invention, including fragments, variants, and/or derivatives thereof, in addition to fusions with heterologous polypeptides and other forms of the polypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections, though they may also be given intramuscularly, and/or through IV). The immunizing agent may include polypeptides of the present invention or a fusion protein or variants thereof. Depending upon the nature of the polypeptides (i.e., percent hydrophobicity, percent hydrophilicity, stability, net charge, isoelectric point etc.), it may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Such conjugation includes either chemical conjugation by derivitizing active chemical functional groups to both the polypeptide of the present invention and the immunogenic protein such that a covalent bond is formed, or through fusion-protein based methodology, or other methods known to the skilled artisan. Examples of such immunogenic proteins include, but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum . Additional examples of adjuvants which may be employed includes the MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

The antibodies of the present invention may comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2 nd ed. (1988), by Hammerling, et al., Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y., pp. 563-681 (1981); Köhler et al., Eur. J. Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), or other methods known to the artisan. Other examples of methods which may be employed for producing monoclonal antibodies includes, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.

›DETAILED DESCRIPTION OF THE INVENTION · 40 of 64

In a hybridoma method, a mouse, a humanized mouse, a mouse with a human immune system, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include polypeptides of the present invention or a fusion protein thereof. Preferably, the immunizing agent consists of an MMP-29 polypeptide or, more preferably, with a MMP-29 polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 degrees C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. More preferred are the parent myeloma cell line (SP20) as provided by the ATCC. As inferred throughout the specification, human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptides of the present invention. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA). Such techniques are known in the art and within the skill of the artisan. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollart, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra, and/or according to Wands et al. (Gastroenterology 80:225-232 (1981)). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The skilled artisan would acknowledge that a variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In this context, the term “monoclonal antibody” refers to an antibody derived from a single eukaryotic, phage, or prokaryotic clone. The DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). The hydridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transformed into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

›DETAILED DESCRIPTION OF THE INVENTION · 41 of 64

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples described herein. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Imunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

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For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabilly et al., Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. The techniques of cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

›DETAILED DESCRIPTION OF THE INVENTION · 43 of 64

Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and creation of an antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in the following scientific publications: Marks et al., Biotechnol., 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93 (1995).

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

Such anti-idiotypic antibodies capable of binding to the MMP-29 polypeptide can be produced in a two-step procedure. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones that produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein-specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies.

The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, Preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards a polypeptide of the present invention, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/20373; and EP03089). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y.

›DETAILED DESCRIPTION OF THE INVENTION · 44 of 64

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).

More preferably, a clone encoding an antibody of the present invention may be obtained according to the method described in the Example section herein.

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

›DETAILED DESCRIPTION OF THE INVENTION · 45 of 64

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis ) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia ) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli , and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

›DETAILED DESCRIPTION OF THE INVENTION · 46 of 64

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

›DETAILED DESCRIPTION OF THE INVENTION · 47 of 64

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (said references incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

›DETAILED DESCRIPTION OF THE INVENTION · 48 of 64

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

The present invention also encompasses the creation of synthetic antibodies directed against the polypeptides of the present invention. One example of synthetic antibodies is described in Radrizzani, M., et al., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class of synthetic antibodies has been described and are referred to as molecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies, peptides, and enzymes are often used as molecular recognition elements in chemical and biological sensors. However, their lack of stability and signal transduction mechanisms limits their use as sensing devices. Molecularly imprinted polymers (MIPs) are capable of mimicking the function of biological receptors but with less stability constraints. Such polymers provide high sensitivity and selectivity while maintaining excellent thermal and mechanical stability. MIPs have the ability to bind to small molecules and to target molecules such as organics and proteins' with equal or greater potency than that of natural antibodies. These “super” MIPs have higher affinities for their target and thus require lower concentrations for efficacious binding.

During synthesis, the MIPs are imprinted so as to have complementary size, shape, charge and functional groups of the selected target by using the target molecule itself (such as a polypeptide, antibody, etc.), or a substance having a very similar structure, as its “print” or “template.” MIPs can be derivatized with the same reagents afforded to antibodies. For example, fluorescent ‘super’ MIPs can be coated onto beads or wells for use in highly sensitive separations or assays, or for use in high throughput screening of proteins.

Moreover, MIPs based upon the structure of the polypeptide(s) of the present invention may be useful in screening for compounds that bind to the polypeptide(s) of the invention. Such a MIP would serve the role of a synthetic “receptor” by minimicking the native architecture of the polypeptide. In fact, the ability of a MIP to serve the role of a synthetic receptor has already been demonstrated for the estrogen receptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001); Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71, (2001)). A synthetic receptor may either be mimicked in its entirety (e.g., as the entire protein), or mimicked as a series of short peptides corresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys, Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may be employed in any one or more of the screening methods described elsewhere herein.

MIPs have also been shown to be useful in “sensing” the presence of its mimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron., 16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst., 126(6):798-802, (2001)). For example, a MIP designed using a polypeptide of the present invention may be used in assays designed to identify, and potentially quantitate, the level of said polypeptide in a sample. Such a MIP may be used as a substitute for any component described in the assays, or kits, provided herein (e.g., ELISA, etc.).

›DETAILED DESCRIPTION OF THE INVENTION · 49 of 64

A number of methods may be employed to create MIPs to a specific receptor, ligand, polypeptide, peptide, organic molecule. Several preferred methods are described by Esteban et al in J. Anal, Chem., 370(7):795-802, (2001), which is hereby incorporated herein by reference in its entirety in addition to any references cited therein. Additional methods are known in the art and are encompassed by the present invention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem, Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J., De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54, (2001); which are hereby incorporated by reference in their entirety herein.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. For example, such antibodies may be used in diagnostic assays to detect the presence or quantification of the polypeptides of the invention in a sample. Such a diagnostic assay may be comprised of at least two steps. The first, subjecting a sample with the antibody, wherein the sample is a tissue (e.g., human, animal, etc.), biological fluid (e.g., blood, urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract (e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g., See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or a chromatography column, etc. And a second step involving the quantification of antibody bound to the substrate. Alternatively, the method may additionally involve a first step of attaching the antibody, either covalently, electrostatically, or reversibly, to a solid support, and a second step of subjecting the bound antibody to the sample, as defined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., (1987), pp 147-158). The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, green fluorescent protein, or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); and Nygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present invention are useful for the affinity purification of such polypeptides from recombinant cell culture or natural sources. In this process, the antibodies against a particular polypeptide are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the polypeptides to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except for the desired polypeptides, which are bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. The translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

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Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses Of Antibodies

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

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The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M, 10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention are useful for inhibiting allergic reactions in animals. For example, by administering a therapeutically acceptable dose of an antibody, or antibodies, of the present invention, or a cocktail of the present antibodies, or in combination with other antibodies of varying sources, the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibody directed against a polypeptide of the present invention, said polypeptide having the potential to elicit an allergic and/or immune response in an organism, and transforming the organism with said antibody gene such that it is expressed (e.g., constitutively, inducibly, etc.) in the organism. Thus, the organism would effectively become resistant to an allergic response resulting from the ingestion or presence of such an immune/allergic reactive polypeptide. Moreover, such a use of the antibodies of the present invention may have particular utility in preventing and/or ameliorating autoimmune diseases and/or disorders, as such conditions are typically a result of antibodies being directed against endogenous proteins. For example, in the instance where the polypeptide of the present invention is responsible for modulating the immune response to auto-antigens, transforming the organism and/or individual with a construct comprising any of the promoters disclosed herein or otherwise known in the art, in addition, to a polynucleotide encoding the antibody directed against the polypeptide of the present invention could effective inhibit the organisms immune system from eliciting an immune response to the auto-antigen(s). Detailed descriptions of therapeutic and/or gene therapy applications of the present invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced in a plant (e.g., cloning the gene of the antibody directed against a polypeptide of the present invention, and transforming a plant with a suitable vector comprising said gene for constitutive expression of the antibody within the plant), and the plant subsequently ingested by an animal, thereby conferring temporary immunity to the animal for the specific antigen the antibody is directed towards (See, for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferably polyclonal antibodies, more preferably monoclonal antibodies, and most preferably single-chain antibodies, can be used as a means of inhibiting gene expression of a particular gene, or genes, in a human, mammal, and/or other organism. See, for example, International Publication Number WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. The application of such methods for the antibodies of the present invention are known in the art, and are more particularly described elsewhere herein.

In yet another embodiment, antibodies of the present invention may be useful for multimerizing the polypeptides of the present invention. For example, certain proteins may confer enhanced biological activity when present in a multimeric state (i.e., such enhanced activity may be due to the increased effective concentration of such proteins whereby more protein is available in a localized location).

Antibody-based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

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In a preferred aspect, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

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In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

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In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

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The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging With Antibodies

Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

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It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Kits

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, Mo.).

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The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because certain proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Similarly, peptide cleavage sites can be introduced in-between such peptide moieties, which could additionally be subjected to protease activity to remove said peptide(s) from the protein of the present invention. The addition of peptide moieties, including peptide cleavage sites, to facilitate handling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused to marker sequences (also referred to as “tags”). Due to the availability of antibodies specific to such “tags”, purification of the fused polypeptide of the invention, and/or its identification is significantly facilitated since antibodies specific to the polypeptides of the invention are not required. Such purification may be in the form of an affinity purification whereby an anti-tag antibody or another type of affinity matrix (e.g., anti-tag antibody attached to the matrix of a flow-thru column) that binds to the epitope tag is present. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984)).

The skilled artisan would acknowledge the existence of other “tags” which could be readily substituted for the tags referred to supra for purification and/or identification of polypeptides of the present invention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). For example, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology 5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553 (1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ ID NO:28), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitope peptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFP epitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

›DETAILED DESCRIPTION OF THE INVENTION · 58 of 64

The present invention also encompasses the attachment of up to nine codons encoding a repeating series of up to nine arginine amino acids to the coding region of a polynucleotide of the present invention. The invention also encompasses chemically derivitizing a polypeptide of the present invention with a repeating series of up to nine arginine amino acids. Such a tag, when attached to a polypeptide, has recently been shown to serve as a universal pass, allowing compounds access to the interior of cells without additional derivitization or manipulation (Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention, including fragments and/or variants thereof, can be used for the following, non-limiting examples, subcellular localization of proteins, determination of protein-protein interactions via immunoprecipitation, purification of proteins via affinity chromatography, functional and/or structural characterization of protein. The present invention also encompasses the application of hapten specific antibodies for any of the uses referenced above for epitope fusion proteins. For example, the polypeptides of the present invention could be chemically derivatized to attach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to the availability of monoclonal antibodies specific to such haptens, the protein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/or variants thereof, in addition to, antibodies directed against such polypeptides, fragments, and/or variants, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999;886:233-5), or HC toxin (Tonukari N J, et al., Plant Cell. 2000 February; 12(2):237-248), for example. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed against polypeptides of the present invention, including variants and fragments thereof, to said toxins for delivering the toxin to specific locations in a cell, to specific tissues, and/or to specific species. Such bifunctional antibodies are known in the art, though a review describing additional advantageous fusions, including citations for methods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm. 11:548-557, (1999); this publication, in addition to the references cited therein, are hereby incorporated by reference in their entirety herein. In this context, the term “toxin” may be expanded to include any heterologous protein, a small molecule, radionucleotides, cytotoxic drugs, liposomes, adhesion molecules, glycoproteins, ligands, cell or tissue-specific ligands, enzymes, of bioactive agents, biological response modifiers, anti-fungal agents, hormones, steroids, vitamins, peptides, peptide analogs, anti-allergenic agents, anti-tubercular agents, anti-viral agents, antibiotics, anti-protozoan agents, chelates, radioactive particles, radioactive ions, X-ray contrast agents, monoclonal antibodies, polyclonal antibodies and genetic material. In view of the present disclosure, one skilled in the art could determine whether any particular “toxin” could be used in the compounds of the present invention. Examples of suitable “toxins” listed above are exemplary only and are not intended to limit the “toxins” that may be used in the present invention.

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

›DETAILED DESCRIPTION OF THE INVENTION · 59 of 64

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris . See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “ Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG, as required.

In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

›DETAILED DESCRIPTION OF THE INVENTION · 60 of 64

In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein, the addition of epitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin, maltose binding protein, etc.), attachment of affinity tags such as biotin and/or streptavidin, the covalent attachment of chemical moieties to the amino acid backbone, N- or C-terminal processing of the polypeptides ends (e.g., proteolytic processing), deletion of the N-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The invention further encompasses chemical derivitization of the polypeptides of the present invention, preferably where the chemical is a hydrophilic polymer residue. Exemplary hydrophilic polymers, including derivatives, may be those that include polymers in which the repeating units contain one or more hydroxy groups (polyhydroxy polymers), including, for example, poly(vinyl alcohol); polymers in which the repeating units contain one or more amino groups (polyamine polymers), including, for example, peptides, polypeptides, proteins and lipoproteins, such as albumin and natural lipoproteins; polymers in which the repeating units contain one or more carboxy groups (polycarboxy polymers), including, for example, carboxymethylcellulose, alginic acid and salts thereof, such as sodium and calcium alginate, glycosaminoglycans and salts thereof, including salts of hyaluronic acid, phosphorylated and sulfonated derivatives of carbohydrates, genetic material, such as interleukin-2 and interferon, and phosphorothioate oligomers; and polymers in which the repeating units contain one or more saccharide moieties (polysaccharide polymers), including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and is generally about 50 to about 5,000,000, with polymers having a molecular weight of about 100 to about 50,000 being preferred. The polymers may be branched or unbranched. More preferred polymers have a molecular weight of about 150 to about 10,000, with molecular weights of 200 to about 8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatize polypeptides of the invention, include, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymers being particularly preferred. Preferred among the PEG polymers are PEG polymers having a molecular weight of from about 100 to about 10,000. More preferably, the PEG polymers have a molecular weight of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of 2,000, 5,000 and 8,000, respectively, being even more preferred. Other suitable hydrophilic polymers, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, the polymers used may include polymers that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

›DETAILED DESCRIPTION OF THE INVENTION · 61 of 64

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated that the polymeric residues may contain functional groups in addition, for example, to those typically involved in linking the polymeric residues to the polypeptides of the present invention. Such functionalities include, for example, carboxyl, amine, hydroxy and thiol groups. These functional groups on the polymeric residues can be further reacted, if desired, with materials that are generally reactive with such functional groups and which can assist in targeting specific tissues in the body including, for example, diseased tissue. Exemplary materials which can be reacted with the additional functional groups include, for example, proteins, including antibodies, carbohydrates, peptides, glycopeptides, glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used to derivatize the polypeptides of the present invention can be a saccharide residue. Exemplary saccharides which can be derived include, for example, monosaccharides or sugar alcohols, such as erythrose, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol and sedoheptulose, with preferred monosaccharides being fructose, mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides, such as lactose, sucrose, maltose and cellobiose. Other saccharides include, for example, inositol and ganglioside head groups. Other suitable saccharides, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, saccharides which may be used for derivitization include saccharides that can be attached to the polypeptides of the invention via alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of the polypeptides of the present invention, for example, with lipids (including cationic, anionic, polymerized, charged, synthetic, saturated, unsaturated, and any combination of the above, etc.) stabilizing agents.

The invention encompasses derivitization of the polypeptides of the present invention, for example, with compounds that may serve a stabilizing function (e.g., to increase the polypeptides half-life in solution, to make the polypeptides more water soluble, to increase the polypeptides hydrophilic or hydrophobic character, etc.). Polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof. Accordingly, suitable polymers include, for example, proteins, such as albumin, polyalginates, and polylactide-coglycolide polymers. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol (including for example, the class of compounds referred to as Pluronics.RTM., commercially available from BASF, Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof. Methods for the preparation of derivatized polypeptides of the invention which employ polymers as stabilizing compounds will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.

›DETAILED DESCRIPTION OF THE INVENTION · 62 of 64

Moreover, the invention encompasses additional modifications of the polypeptides of the present invention. Such additional modifications are known in the art, and are specifically provided, in addition to methods of derivitization, etc., in U.S. Pat. No. 6,028,066, which is hereby incorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:2 or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.

In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

›DETAILED DESCRIPTION OF THE INVENTION · 63 of 64

Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.

In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could be operatively linked to “artificial” or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in mammals. Further, the trans-acting transcription factors of such “artificial” promoters could also be “artificial” or chimeric in design themselves and could act as activators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:1 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).

›DETAILED DESCRIPTION OF THE INVENTION · 64 of 64

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are known in the art. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected organisms can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence o

›Tables in the description — 11

TABLE 1

	CDNA	ATCC Deposit		NT SEQ	Total NT	5′ NT of Start	3′ NT	AA Seq	Total AA
Gene No.	Clone ID	No. Z and Date	Vector	ID. No. X	Seq of Clone	Codon of ORF	of ORF	ID No. Y	of ORF
1.	MMP-29	N/A	PSport 1	1	1707	1	1707	2	569
	(BSG-26)

TABLE II — Hybridization ‡The “hybrid length” is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucletotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc). †SSPE (1xSSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide. *Tb − Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log 10 [Na+]) + 0.41 (% G + C) − (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165 M). ±The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified polynucleotide. Such modified polynucleotides are known in the art and are more particularly described elsewhere herein.

Stringency	Polynucleotide	Hybrid Length	Temperature	Wash Temperature
Condition	Hybrid±	(bp)‡	and Buffer†	and Buffer†
A	DNA:DNA	> or equal to 50	65° C.; 1xSSC -	65° C.;
			or- 42° C.;	0.3xSSC
			1xSSC, 50%
			formamide
B	DNA:DNA	<50	Tb*; 1xSSC	Tb*; 1xSSC
C	DNA:RNA	> or equal to 50	67° C.; 1xSSC -	67° C.;
			or- 45° C.;	0.3xSSC
			1xSSC, 50%
			formamide
D	DNA:RNA	<50	Td*; 1xSSC	Td*; 1xSSC
E	RNA:RNA	> or equal to 50	70° C.; 1xSSC -	70° C.;
			or- 50° C.;	0.3xSSC
			1xSSC, 50%
			formamide
F	RNA:RNA	<50	Tf*; 1xSSC	Tf*; 1xSSC
G	DNA:DNA	> or equal to 50	65° C.; 4xSSC -	65° C.; 1xSSC
			or- 45° C.;
			4xSSC, 50%
			formamide
H	DNA:DNA	<50	Th*; 4xSSC	Th*; 4xSSC
I	DNA:RNA	> or equal to 50	67° C.; 4xSSC -	67° C.; 1xSSC
			or- 45° C.;
			4xSSC, 50%
			formamide
J	DNA:RNA	<50	Tj*; 4xSSC	Tj*; 4xSSC
K	RNA:RNA	> or equal to 50	70° C.; 4xSSC -	67° C.; 1xSSC
			or- 40° C.;
			6xSSC, 50%
			formamide
L	RNA:RNA	<50	Tl*; 2xSSC	Tl*; 2xSSC
M	DNA:DNA	> or equal to 50	50° C.; 4xSSC -	50° C.; 2xSSC
			or- 40° C.
			6xSSC, 50%
			formamide
N	DNA:DNA	<50	Tn*; 6xSSC	Tn*; 6xSSC
O	DNA:RNA	> or equal to 50	55° C.; 4xSSC -	55° C.; 2xSSC
			or- 42° C.;
			6xSSC, 50%
			formamide
P	DNA:RNA	<50	Tp*; 6xSSC	Tp*; 6xSSC
Q	RNA:RNA	> or equal to 50	60° C.; 4xSSC -	60° C.; 2xSSC
			or- 45° C.;
			6xSSC, 50%
			formamide
R	RNA:RNA	<50	Tr*; 4xSSC	Tr*; 4xSSC

TABLE IV — For Amino

Acid	Code	Replace with any of:
Alanine	A	D-Ala, Gly, beta-Ala, L-Cys, D-Cys
Arginine	R	D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met,
		Ile, D-Met, D-Ile, Orn, D-Orn
Asparagine	N	D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln
Aspartic	D	D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln
Acid
Cysteine	C	D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr
Glutamine	Q	D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp
Glutamic	E	D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln
Acid
Glycine	G	Ala, D-Ala, Pro, D-Pro, β-Ala, Acp
Isoleucine	I	D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met
Leucine	L	D-Leu, Val, D-Val, Met, D-Met
Lysine	K	D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met,
		D-Met, Ile, D-Ile, Orn, D-Orn
Methionine	M	D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-
		Val
Phenyl-	F	D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-
alanine		Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-
		phenylproline
Proline	P	D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-
		1-oxazolidine-4-carboxylic acid
Serine	S	D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O),
		D-Met(O), L-Cys, D-Cys
Threonine	T	D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O),
		D-Met(O), Val, D-Val
Tyrosine	Y	D-Tyr, Phe, D-Phe, L-Dopa, His, D-His
Valine	V	D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

5′ Primer	5′- GCAGCA GCGGCCGC GAGAGTCTCTTCCACAGCCGGGACC -3′	(SEQ ID NO:29)
	NotI
3′ Primer	5′- GCAGCA GTCGAC CATGTTCAGTGTGGAGATATGGACG -3′	(SEQ ID NO:30)
	SalI

5′ Primer	5′- GCAGCA GCGGCCGC ATGCTCGCCGCCTCCATCTTCCGTC -3′	(SEQ ID NO:31)
	NotI
3′ Primer	5′- GCAGCA GTCGAC ATTTTGTGGTATTACTGCTGG -3′	(SEQ ID NO:32)
	SalI

20-25	cycles:	45 sec, 93 degrees
		2 min, 50 degrees
		2 min, 72 degrees
1	cycle:	10 min, 72 degrees

	Segment	Cycles	Temperature	Time
1	1	95° C.	30	seconds
2	12-18	95° C.	30	seconds
		55° C.	1	minute
		68° C.	2	minutes/kb of plasmid length

SEQ ID

Primer name	Primer Sequence	NO:
Primer Sequences Used to Amplify VH domains.
Hu VH1 - 5′	CAGGTGCAGCTGGTGCAGTCTGG	32
Hu VH2 - 5′	CAGGTCAACTTAAGGGAGTCTGG	33
Hu VH3 - 5′	GAGGTGCAGCTGGTGGAGTCTGG	34
Hu VR4 - 5′	CAGGTGCAGCTGCAGGAGTCGGG	35
Hu VH5 - 5′	GAGGTGCAGCTGTTGCAGTCTGC	36
Hu VH6 - 5′	CAGGTACAGCTGCAGCAGTCAGG	37
Hu JH1 - 5′	TGAGGAGACGGTGACCAGGGTGCC	38
Hu JH3 - 5′	TGAAGAGACGGTGACCATTGTCCC	39
Hu JH4 - 5′	TGAGGAGACGGTGACCAGGGTTCC	40
Hu JH6 - 5′	TGAGGAGACGGTGACCGTGGTCCC	41
Primer Sequences Used to Amplify VL domains
Hu Vkappa1 - 5′	GACATCCAGATGACCCAGTCTCC	42
Hu Vkappa2a - 5′	GATGTTGTGATGACTCAGTCTCC	43
Hu Vkappa2b - 5′	GATATTGTGATGACTCAGTCTCC	44
Hu Vkappa3 - 5′	GAAATTGTGTTGACGCAGTCTCC	45
Hu Vkappa4 - 5′	GACATCGTGATGACCCAGTCTCC	46
Hu Vkappa5 - 5′	GAAACGACACTCACGCAGTCTCC	47
Hu Vkappa6 - 5′	GAAATTGTGCTGACTCAGTCTCC	48
Hu Vlambda1 - 5′	CAGTCTGTGTTGACGCAGCCGCC	49
Hu Vlambda2 - 5′	CAGTCTGCCCTGACTCAGCCTGC	50
Hu Vlambda3 - 5′	TCCTATGTGCTGACTCAGCCACC	51
Hu Vlambda3b - 5′	TCTTCTGAGCTGACTCAGGACCC	52
Hu Vlambda4 - 5′	CACGTTATACTGACTCAACCGCC	53
Hu Vlambda5 - 5′	CAGGCTGTGCTCACTCAGCCGTC	54
Hu Vlambda6 - 5′	AATTTTATGCTGACTCAGCCCCA	55
Hu Jkappa1 - 3′	ACGTTTGATTTCCACCTTGGTCCC	56
Hu Jkappa2 - 3′	ACGTTTGATCTCCAGCTTGGTCCC	57
Hu Jkappa3 - 3′	ACGTTTGATATCCACTTTGGTCCC	58
Hu Jkappa4 - 3′	ACGTTTGATCTCCACCTTGGTCCC	59
Hu Jkappa5 - 3′	ACGTTTAATCTCCAGTCGTGTCCC	60
Hu Vlambda1 - 3′	CAGTCTGTGTTGACGCAGCCGCC	61
Hu Vlambda2 - 3′	CAGTCTGCCCTGACTCAGCCTGC	62
Hu Vlambda3 - 3′	TCCTATGTGCTGACTCAGCCACC	63
Hu Vlambda3b - 3′	TCTTTCTGAGCTGACTCAGGACCC	64
Hu Vlambda4 - 3′	CACGTTATACTGACTCAACCGCC	65
Hu Vlambda5 - 3′	CAGGCTGTGCTCACTCAGCCGTC	66
Hu Vlambda6 - 3′	AATTTTATGCTGACTCAGCCCCA	67

TABLE IV

Atom	Atom		Residue
No	Name	Residue	No	X coord	Y coord	Z coord
1	N	ALA	48	−1.453	84.982	97.717
2	CA	ALA	48	−0.751	85.431	96.497
3	C	ALA	48	0.353	86.484	96.681
4	O	ALA	48	1.441	86.276	96.133
5	CB	ALA	48	−1.791	85.970	95.523
6	N	ASP	49	0.210	87.396	97.635
7	CA	ASP	49	1.260	88.406	97.872
8	C	ASP	49	2.544	87.771	98.411
9	O	ASP	49	3.632	87.980	97.859
10	CB	ASP	49	0.787	89.416	98.922
11	CG	ASP	49	−0.421	90.241	98.480
12	OD1	ASP	49	−0.667	90.318	97.285
13	OD2	ASP	49	−1.080	90.788	99.358
14	N	LEU	50	2.373	86.809	99.300
15	CA	LEU	50	3.515	86.184	99.960
16	C	LEU	50	3.930	84.870	99.307
17	O	LEU	50	5.013	84.357	99.600
18	CB	LEU	50	3.192	86.002	101.432
19	CG	LEU	50	1.771	85.516	101.660
20	CD1	LEU	50	1.776	84.149	102.317
21	CD2	LEU	50	0.978	86.508	102.503
22	N	HIS	51	3.151	84.405	98.346
23	CA	HIS	51	3.596	83.298	97.505
24	C	HIS	51	4.442	83.879	96.382
25	O	HIS	51	5.485	83.306	96.044
26	CB	HIS	51	2.384	82.561	96.950
27	CG	HIS	51	2.724	81.325	96.139
28	ND1	HIS	51	3.814	80.542	96.266
29	CD2	HIS	51	1.962	80.784	95.131
30	CE1	HIS	51	3.756	79.544	95.363
31	NE2	HIS	51	2.611	79.695	94.660
32	N	ALA	52	4.168	85.135	96.064
33	CA	ALA	52	5.037	85.890	95.168
34	C	ALA	52	6.353	86.182	95.873
35	O	ALA	52	7.404	85.825	95.332
36	CB	ALA	52	4.352	87.203	94.808
37	N	ALA	53	6.276	86.505	97.156
38	CA	ALA	53	7.488	86.725	97.960
39	C	ALA	53	8.274	85.440	98.229
40	O	ALA	53	9.510	85.462	98.200
41	CB	ALA	53	7.088	87.363	99.285
42	N	GLN	54	7.582	84.315	98.241
43	CA	GLN	54	8.243	83.021	98.378
44	C	GLN	54	8.965	82.635	97.098
45	O	GLN	54	10.146	82.278	97.167
46	CB	GLN	54	7.187	81.975	98.675
47	CG	GLN	54	7.768	80.572	98.607
48	CD	GLN	54	6.631	79.591	98.817
49	OE1	GLN	54	6.334	78.750	97.962
50	NE2	GLN	54	5.923	79.805	99.908
51	N	ARG	55	8.389	82.975	95.957
52	CA	ARG	55	9.070	82.714	94.694
53	C	ARG	55	10.219	83.688	94.477
54	O	ARG	55	11.299	83.203	94.116
55	CB	ARG	55	8.062	82.772	93.557
56	CG	ARG	55	7.290	81.459	93.501
57	CD	ARG	55	6.170	81.490	92.472
58	NE	ARG	55	5.036	82.281	92.964
59	CZ	ARG	55	3.928	82.478	92.249
60	NH1	ARG	55	3.884	82.087	90.973
61	NH2	ARG	55	2.909	83.160	92.774
62	N	PHE	56	10.135	84.849	95.113
63	CA	PHE	56	11.225	85.837	95.070
64	C	PHE	56	12.451	85.381	95.864
65	O	PHE	56	13.561	85.856	95.601
66	CB	PHE	56	10.786	87.175	95.676
67	CG	PHE	56	9.602	87.917	95.052
68	CD1	PHE	56	8.917	88.864	95.800
69	CD2	PHE	56	9.212	87.668	93.743
70	CE1	PHE	56	7.837	89.537	95.256
71	CE2	PHE	56	8.125	88.342	93.196
72	CZ	PHE	56	7.436	89.278	93.956
73	N	LEU	57	12.294	84.405	96.741
74	CA	LEU	57	13.438	83.962	97.528
75	C	LEU	57	13.823	82.502	97.253
76	O	LEU	57	15.014	82.177	97.282
77	CB	LEU	57	13.063	84.167	98.989
78	CG	LEU	57	12.568	85.595	99.229
79	CD1	LEU	57	11.828	85.745	100.551
80	CD2	LEU	57	13.683	86.626	99.098
81	N	SER	58	12.862	81.677	96.867
82	CA	SER	58	13.143	80.249	96.646
83	C	SER	58	13.276	79.859	95.175
84	O	SER	58	13.801	78.782	94.870
85	CB	SER	58	12.034	79.412	97.278
86	OG	SER	58	10.836	79.615	96.538
87	N	ARG	59	12.746	80.674	94.279
88	CA	ARG	59	12.960	80.434	92.852
89	C	ARG	59	13.977	81.425	92.330
90	O	ARG	59	14.620	81.201	91.299
91	CB	ARG	59	11.668	80.621	92.069
92	CG	ARG	59	10.734	79.424	92.161
93	CD	ARG	59	9.597	79.604	91.164
94	NE	ARG	59	10.155	80.003	89.861
95	CZ	ARG	59	9.541	80.844	89.027
96	NH1	ARG	59	8.293	81.239	89.280
97	NH2	ARG	59	10.141	81.208	87.891
98	N	TYR	60	14.048	82.543	93.030
99	CA	TYR	60	15.033	83.575	92.729
100	C	TYR	60	16.179	83.408	93.723
101	O	TYR	60	17.070	82.565	93.561
102	CB	TYR	60	14.460	84.983	92.929
103	CG	TYR	60	13.156	85.479	92.272
104	CD1	TYR	60	12.181	84.624	91.775
105	CD2	TYR	60	12.962	86.854	92.197
106	CE1	TYR	60	10.999	85.130	91.255
107	CE2	TYR	60	11.790	87.367	91.658
108	CZ	TYR	60	10.804	86.501	91.205
109	OH	TYR	60	9.599	87.006	90.768
110	N	GLY	61	16.132	84.213	94.767
111	CA	GLY	61	17.113	84.076	95.840
112	C	GLY	61	18.186	85.152	95.829
113	O	GLY	61	17.934	86.297	96.219
114	N	TRP	62	19.409	84.727	95.559
115	CA	TRP	62	20.533	85.666	95.613
116	C	TRP	62	21.557	85.438	94.501
117	O	TRP	62	22.089	86.409	93.946
118	CB	TRP	62	21.196	85.600	96.990
119	CG	TRP	62	21.984	84.346	97.344
120	CD1	TRP	62	21.526	83.050	97.430
121	CD2	TRP	62	23.387	84.305	97.672
122	NE1	TRP	62	22.566	82.246	97.759
123	CE2	TRP	62	23.698	82.956	97.913
124	CE3	TRP	62	24.373	85.278	97.763
125	CZ2	TRP	62	25.000	82.600	98.240
126	CZ3	TRP	62	25.672	84.910	98.090
127	CH2	TRP	62	25.984	83.576	98.327
128	N	SER	63	21.754	84.187	94.118
129	CA	SER	63	22.770	83.852	93.117
130	C	SER	63	22.898	82.351	92.955
131	O	SER	63	22.183	81.560	93.583
132	CB	SER	63	24.128	84.350	93.593
133	OG	SER	63	24.408	83.650	94.796
134	N	GLY	64	23.834	81.984	92.098
135	CA	GLY	64	24.219	80.583	91.951
136	C	GLY	64	25.021	80.153	93.177
137	O	GLY	64	24.419	79.809	94.201
138	N	VAL	65	26.330	80.367	93.086
139	CA	VAL	65	27.407	79.980	94.044
140	C	VAL	65	27.067	79.241	95.347
141	O	VAL	65	26.155	78.411	95.384
142	CB	VAL	65	28.202	81.234	94.392
143	CG1	VAL	65	29.120	81.632	93.244
144	CG2	VAL	65	27.299	82.390	94.812
145	N	TRP	66	28.057	79.300	96.232
146	CA	TRP	66	28.101	78.707	97.593
147	C	TRP	66	27.875	77.190	97.672
148	O	TRP	66	27.196	76.583	96.833
149	CB	TRP	66	27.194	79.478	98.559
150	CG	TRP	66	25.726	79.102	98.652
151	CD1	TRP	66	24.745	79.281	97.705
152	CD2	TRP	66	25.080	78.508	99.797
153	NE1	TRP	66	23.575	78.814	98.201
154	CE2	TRP	66	23.727	78.350	99.453
155	CE3	TRP	66	25.534	78.120	101.049
156	CZ2	TRP	66	22.841	77.803	100.369
157	CZ3	TRP	66	24.641	77.574	101.961
158	CH2	TRP	66	23.301	77.415	101.622
159	N	ALA	67	28.592	76.598	98.622
160	CA	ALA	67	28.546	75.152	98.917
161	C	ALA	67	29.112	74.298	97.784
162	O	ALA	67	28.542	74.257	96.691
163	CB	ALA	67	27.114	74.727	99.232
164	N	ALA	68	30.272	73.704	98.025
165	CA	ALA	68	30.899	72.810	97.040
166	C	ALA	68	30.854	71.336	97.455
167	O	ALA	68	31.385	70.472	96.749
168	CB	ALA	68	32.349	73.234	96.835
169	N	TRP	69	30.228	71.063	98.589
170	CA	TRP	69	30.251	69.713	99.175
171	C	TRP	69	28.931	68.950	99.044
172	O	TRP	69	28.827	67.808	99.507
173	CB	TRP	69	30.622	69.831	100.649
174	CG	TRP	69	32.028	70.344	100.891
175	CD1	TRP	69	32.391	71.587	101.364
176	CD2	TRP	69	33.255	69.613	100.675
177	NE1	TRP	69	33.746	71.641	101.434
178	CE2	TRP	69	34.301	70.482	101.031
179	CE3	TRP	69	33.532	68.333	100.220
180	CZ2	TRP	69	35.617	70.055	100.924
181	CZ3	TRP	69	34.853	67.914	100.115
182	CH2	TRP	69	35.890	68.771	100.466
183	N	GLY	70	27.939	69.576	98.439
184	CA	GLY	70	26.621	68.949	98.250
185	C	GLY	70	26.120	69.309	96.856
186	O	GLY	70	26.280	68.535	95.906
187	N	PRO	71	25.472	70.459	96.756
188	CA	PRO	71	25.644	71.270	95.554
189	C	PRO	71	27.115	71.651	95.442
190	O	PRO	71	27.831	71.677	96.455
191	CB	PRO	71	24.784	72.477	95.766
192	CG	PRO	71	24.328	72.502	97.218
193	CD	PRO	71	24.910	71.250	97.857
194	N	SER	72	27.585	71.833	94.223
195	CA	SER	72	28.979	72.242	94.041
196	C	SER	72	29.222	73.082	92.786
197	O	SER	72	29.575	72.521	91.742
198	CB	SER	72	29.839	70.987	93.955
199	OG	SER	72	31.195	71.405	93.870
200	N	PRO	73	29.043	74.394	92.862
201	CA	PRO	73	28.155	75.062	93.815
202	C	PRO	73	26.721	75.068	93.286
203	O	PRO	73	26.273	74.084	92.680
204	CB	PRO	73	28.687	76.457	93.849
205	CG	PRO	73	29.434	76.702	92.544
206	CD	PRO	73	29.478	75.351	91.846
207	N	GLU	74	26.002	76.113	93.672
208	CA	GLU	74	24.716	76.571	93.102
209	C	GLU	74	23.547	76.238	94.024
210	O	GLU	74	23.189	75.072	94.242
211	CB	GLU	74	24.471	76.157	91.637
212	CG	GLU	74	23.762	74.820	91.404
213	CD	GLU	74	23.683	74.531	89.909
214	OE1	GLU	74	22.924	75.226	89.247
215	OE2	GLU	74	24.470	73.724	89.435
216	N	GLY	75	23.015	77.287	94.624
217	CA	GLY	75	21.805	77.152	95.435
218	C	GLY	75	21.038	78.465	95.592
219	O	GLY	75	21.235	79.192	96.575
220	N	PRO	76	20.081	78.693	94.706
221	CA	PRO	76	18.984	79.605	95.038
222	C	PRO	76	18.225	79.017	96.220
223	O	PRO	76	17.925	77.818	96.191
224	CB	PRO	76	18.120	79.629	93.816
225	CG	PRO	76	18.566	78.524	92.871
226	CD	PRO	76	19.796	77.900	93.507
227	N	PRO	77	18.010	79.819	97.256
228	CA	PRO	77	17.607	79.320	98.584
229	C	PRO	77	16.356	78.446	98.550
230	O	PRO	77	15.217	78.914	98.455
231	CB	PRO	77	17.423	80.543	99.427
232	CG	PRO	77	17.944	81.751	98.666
233	CD	PRO	77	18.371	81.235	97.302
234	N	GLU	78	16.610	77.162	98.717
235	CA	GLU	78	15.591	76.130	98.544
236	C	GLU	78	14.723	76.006	99.783
237	O	GLU	78	15.224	76.127	100.905
238	CB	GLU	78	16.283	74.798	98.249
239	CG	GLU	78	17.116	74.839	96.966
240	CD	GLU	78	18.620	74.833	97.261
241	OE1	GLU	78	19.072	75.728	97.966
242	OE2	GLU	78	19.268	73.877	96.860
243	N	THR	79	13.447	75.736	99.537
244	CA	THR	79	12.384	75.546	100.552
245	C	THR	79	12.246	76.710	101.538
246	O	THR	79	13.203	77.129	102.199
247	CB	THR	79	12.558	74.220	101.299
248	OG1	THR	79	13.660	74.285	102.194
249	CG2	THR	79	12.756	73.045	100.347
250	N	PRO	80	11.016	77.186	101.652
251	CA	PRO	80	10.706	78.342	102.501
252	C	PRO	80	11.125	78.127	103.955
253	O	PRO	80	10.796	77.112	104.579
254	CB	PRO	80	9.225	78.540	102.376
255	CG	PRO	80	8.662	77.519	101.400
256	CD	PRO	80	9.841	76.680	100.939
257	N	GLY	82	11.864	79.097	104.466
258	CA	GLY	82	12.371	79.040	105.841
259	C	GLY	82	13.088	80.333	106.220
260	O	GLY	82	12.892	81.376	105.585
261	N	ALA	83	14.052	80.213	107.121
262	CA	ALA	83	14.754	81.394	107.657
263	C	ALA	83	15.807	81.992	106.718
264	O	ALA	83	16.106	83.190	106.816
265	CB	ALA	83	15.409	81.012	108.979
266	N	ALA	84	16.142	81.264	105.663
267	CA	ALA	84	17.074	81.774	104.651
268	C	ALA	84	16.392	82.738	103.679
269	O	ALA	84	17.069	83.573	103.067
270	CB	ALA	84	17.651	80.594	103.877
271	N	LEU	85	15.073	82.819	103.774
272	CA	LEU	85	14.294	83.709	102.916
273	C	LEU	85	14.422	85.158	103.386
274	O	LEU	85	14.589	86.050	102.545
275	CB	LEU	85	12.829	83.270	102.976
276	CG	LEU	85	12.387	82.353	101.829
277	CD1	LEU	85	13.232	81.093	101.654
278	CD2	LEU	85	10.912	81.996	101.968
279	N	ALA	86	14.701	85.333	104.670
280	CA	ALA	86	14.904	86.682	105.207
281	C	ALA	86	16.311	87.185	104.900
282	O	ALA	86	16.482	88.367	104.579
283	CB	ALA	86	14.690	86.646	106.716
284	N	GLU	87	17.217	86.248	104.667
285	CA	GLU	87	18.590	86.610	104.314
286	C	GLU	87	18.691	86.919	102.825
287	O	GLU	87	19.450	87.813	102.433
288	CB	GLU	87	19.515	85.452	104.669
289	CG	GLU	87	19.423	85.092	106.149
290	CD	GLU	87	19.829	86.276	107.026
291	OE1	GLU	87	18.932	86.897	107.580
292	OE2	GLU	87	21.021	86.435	107.246
293	N	ALA	88	17.763	86.376	102.055
294	CA	ALA	88	17.698	86.718	100.638
295	C	ALA	88	17.014	88.069	100.440
296	O	ALA	88	17.472	88.851	99.598
297	CB	ALA	88	16.958	85.613	99.895
298	N	VAL	89	16.159	88.455	101.378
299	CA	VAL	89	15.590	89.809	101.342
300	C	VAL	89	16.631	90.833	101.788
301	O	VAL	89	16.763	91.883	101.145
302	CB	VAL	89	14.380	89.901	102.266
303	CG1	VAL	89	13.820	91.317	102.270
304	CG2	VAL	89	13.291	88.918	101.864
305	N	ARG	90	17.516	90.412	102.681
306	CA	ARG	90	18.656	91.237	103.095
307	C	ARG	90	19.509	91.650	101.901
308	O	ARG	90	19.543	92.846	101.566
309	CB	ARG	90	19.512	90.439	104.073
310	CG	ARG	90	18.842	90.287	105.432
311	CD	ARG	90	18.838	91.604	106.200
312	NE	ARG	90	20.209	92.011	106.550
313	CZ	ARG	90	20.804	93.099	106.058
314	NH1	ARG	90	20.177	93.848	105.151
315	NH2	ARG	90	22.050	93.404	106.428
316	N	ARG	91	19.878	90.662	101.102
317	CA	ARG	91	20.731	90.897	99.933
318	C	ARG	91	19.997	91.558	98.760
319	O	ARG	91	20.613	92.366	98.054
320	CB	ARG	91	21.280	89.538	99.513
321	CG	ARG	91	22.086	88.931	100.657
322	CD	ARG	91	22.598	87.530	100.350
323	NE	ARG	91	21.505	86.546	100.336
324	CZ	ARG	91	21.621	85.331	100.877
325	NH1	ARG	91	20.629	84.445	100.758
326	NH2	ARG	91	22.762	84.975	101.472
327	N	PHE	92	18.678	91.443	98.732
328	CA	PHE	92	17.865	92.056	97.674
329	C	PHE	92	17.611	93.546	97.932
330	O	PHE	92	17.504	94.333	96.979
331	CB	PHE	92	16.537	91.302	97.662
332	CG	PHE	92	15.630	91.534	96.457
333	CD1	PHE	92	15.575	90.578	95.451
334	CD2	PHE	92	14.839	92.674	96.375
335	CE1	PHE	92	14.743	90.769	94.357
336	CE2	PHE	92	14.011	92.867	95.278
337	CZ	PHE	92	13.962	91.914	94.270
338	N	GLN	93	17.690	93.952	99.189
339	CA	GLN	93	17.550	95.372	99.516
340	C	GLN	93	18.848	96.111	99.247
341	O	GLN	93	18.839	97.118	98.528
342	CB	GLN	93	17.225	95.505	100.991
343	CG	GLN	93	15.866	94.923	101.340
344	CD	GLN	93	15.922	94.603	102.821
345	OE1	GLN	93	14.940	94.710	103.567
346	NE2	GLN	93	17.118	94.219	103.233
347	N	ARG	94	19.955	95.439	99.524
348	CA	ARG	94	21.265	96.052	99.283
349	C	ARG	94	21.615	96.029	97.793
350	O	ARG	94	22.449	96.812	97.324
351	CB	ARG	94	22.311	95.291	100.086
352	CG	ARG	94	21.836	95.082	101.519
353	CD	ARG	94	22.958	94.566	102.411
354	NE	ARG	94	23.691	93.464	101.772
355	CZ	ARG	94	23.723	92.217	102.246
356	NH1	ARG	94	22.935	91.867	103.264
357	NH2	ARG	94	24.470	91.297	101.636
358	N	ALA	95	20.873	95.224	97.052
359	CA	ALA	95	20.914	95.218	95.595
360	C	ALA	95	20.370	96.501	94.984
361	O	ALA	95	21.121	97.418	94.626
362	CB	ALA	95	19.993	94.100	95.142
363	N	ASN	96	19.052	96.603	94.985
364	CA	ASN	96	18.361	97.636	94.195
365	C	ASN	96	18.121	98.955	94.930
366	O	ASN	96	17.040	99.540	94.794
367	CB	ASN	96	17.028	97.058	93.737
368	CG	ASN	96	17.268	95.754	92.982
369	OD1	ASN	96	18.153	95.666	92.124
370	ND2	ASN	96	16.528	94.731	93.371
371	N	ALA	97	19.141	99.433	95.632
372	CA	ALA	97	19.077	100.677	96.417
373	C	ALA	97	17.845	100.729	97.312
374	O	ALA	97	16.945	101.555	97.119
375	CB	ALA	97	19.091	101.870	95.466
376	N	LEU	98	17.762	99.760	98.205
377	CA	LEU	98	16.652	99.670	99.151
378	C	LEU	98	17.221	99.602	100.560
379	O	LEU	98	18.285	99.008	100.778
380	CB	LEU	98	15.861	98.398	98.858
381	CG	LEU	98	15.354	98.342	97.423
382	CD1	LEU	98	14.924	96.934	97.037
383	CD2	LEU	98	14.240	99.353	97.183
384	N	PRO	99	16.516	100.198	101.507
385	CA	PRO	99	16.987	100.216	102.894
386	C	PRO	99	17.169	98.804	103.445
387	O	PRO	99	16.319	97.929	103.244
388	CB	PRO	99	15.946	100.976	103.655
389	CG	PRO	99	14.841	101.407	102.703
390	CD	PRO	99	15.244	100.901	101.328
391	N	ALA	100	18.385	98.546	103.895
392	CA	ALA	100	18.701	97.277	104.567
393	C	ALA	100	17.928	97.062	105.875
394	O	ALA	100	18.386	97.469	106.948
395	CB	ALA	100	20.199	97.279	104.862
396	N	SER	101	16.783	96.403	105.769
397	CA	SER	101	15.981	96.027	106.937
398	C	SER	101	15.980	94.513	107.140
399	O	SER	101	16.552	93.997	108.107
400	CB	SER	101	14.552	96.504	106.700
401	OG	SER	101	13.747	96.061	107.785
402	N	GLY	102	15.369	93.816	106.198
403	CA	GLY	102	15.267	92.357	106.246
404	C	GLY	102	13.848	91.898	105.926
405	O	GLY	102	13.537	90.703	106.006
406	N	GLU	103	12.989	92.851	105.600
407	CA	GLU	103	11.577	92.532	105.343
408	C	GLU	103	11.087	93.142	104.034
409	O	GLU	103	11.851	93.787	103.305
410	CB	GLU	103	10.712	93.024	106.500
411	CG	GLU	103	11.044	92.293	107.798
412	CD	GLU	103	10.019	92.630	108.873
413	OE1	GLU	103	8.981	91.979	108.880
414	OE2	GLU	103	10.231	93.606	109.577
415	N	LEU	104	9.814	92.917	103.745
416	CA	LEU	104	9.184	93.433	102.517
417	C	LEU	104	8.858	94.921	102.629
418	O	LEU	104	7.718	95.315	102.895
419	CB	LEU	104	7.901	92.649	102.264
420	CG	LEU	104	8.183	91.172	102.007
421	CD1	LEU	104	6.888	90.372	101.920
422	CD2	LEU	104	9.018	90.984	100.744
423	N	ASP	105	9.878	95.733	102.407
424	CA	ASP	105	9.754	97.188	102.503
425	C	ASP	105	8.923	97.720	101.336
426	O	ASP	105	8.912	97.117	100.257
427	CB	ASP	105	11.160	97.789	102.503
428	CG	ASP	105	12.002	97.245	103.666
429	OD1	ASP	105	11.426	96.968	104.711
430	OD2	ASP	105	13.216	97.306	103.547
431	N	ALA	106	8.326	98.888	101.509
432	CA	ALA	106	7.396	99.425	100.497
433	C	ALA	106	8.046	99.742	99.149
434	O	ALA	106	7.539	99.293	98.114
435	CB	ALA	106	6.756	100.690	101.056
436	N	ALA	107	9.287	100.206	99.183
437	CA	ALA	107	10.012	100.478	97.936
438	C	ALA	107	10.553	99.200	97.294
439	O	ALA	107	10.609	99.109	96.062
440	CB	ALA	107	11.164	101.428	98.242
441	N	THR	108	10.622	98.145	98.091
442	CA	THR	108	11.093	96.854	97.604
443	C	THR	108	9.949	96.159	96.878
444	O	THR	108	10.142	95.684	95.755
445	CB	THR	108	11.523	96.021	98.808
446	OG1	THR	108	12.493	96.755	99.544
447	CG2	THR	108	12.134	94.687	98.401
448	N	LEU	109	8.740	96.416	97.358
449	CA	LEU	109	7.530	95.869	96.736
450	C	LEU	109	7.124	96.651	95.487
451	O	LEU	109	6.572	96.054	94.555
452	CB	LEU	109	6.400	95.932	97.757
453	CG	LEU	109	6.703	95.078	98.982
454	CD1	LEU	109	5.714	95.357	100.107
455	CD2	LEU	109	6.724	93.594	98.631
456	N	ALA	110	7.589	97.887	95.374
457	CA	ALA	110	7.357	98.678	94.158
458	C	ALA	110	8.371	98.334	93.062
459	O	ALA	110	8.119	98.543	91.870
460	CB	ALA	110	7.446	100.157	94.512
461	N	ALA	111	9.448	97.678	93.466
462	CA	ALA	111	10.412	97.084	92.532
463	C	ALA	111	10.062	95.626	92.217
464	O	ALA	111	10.862	94.903	91.609
465	CB	ALA	111	11.806	97.172	93.145
466	N	MET	112	8.890	95.207	92.666
467	CA	MET	112	8.390	93.844	92.483
468	C	MET	112	7.006	93.871	91.842
469	O	MET	112	6.099	93.172	92.310
470	CB	MET	112	8.263	93.182	93.850
471	CG	MET	112	9.599	92.864	94.513
472	SD	MET	112	9.446	92.431	96.262
473	CE	MET	112	11.063	91.687	96.562
474	N	ASN	113	6.830	94.681	90.810
475	CA	ASN	113	5.507	94.816	90.179
476	C	ASN	113	5.495	94.487	88.671
477	O	ASN	113	4.510	93.924	88.181
478	CB	ASN	113	4.977	96.238	90.395
479	CG	ASN	113	4.620	96.525	91.858
480	OD1	ASN	113	4.865	97.628	92.360
481	ND2	ASN	113	4.024	95.546	92.518
482	N	ARG	114	6.630	94.648	88.005
483	CA	ARG	114	6.721	94.570	86.526
484	C	ARG	114	7.717	93.474	86.041
485	O	ARG	114	8.041	92.638	86.876
486	CB	ARG	114	7.086	96.003	86.142
487	CG	ARG	114	7.697	96.695	87.362
488	CD	ARG	114	8.115	98.140	87.116
489	NE	ARG	114	8.837	98.648	88.295
490	CZ	ARG	114	10.122	99.007	88.272
491	NH1	ARG	114	10.807	98.967	87.125
492	NH2	ARG	114	10.719	99.425	89.389
493	N	PRO	115	7.951	93.232	84.757
494	CA	PRO	115	8.798	92.052	84.357
495	C	PRO	115	10.323	92.178	84.586
496	O	PRO	115	10.832	93.271	84.405
497	CB	PRO	115	8.522	91.906	82.900
498	CG	PRO	115	7.627	93.037	82.415
499	CD	PRO	115	7.316	93.897	83.619
500	N	ARG	116	11.083	91.107	84.816
501	CA	ARG	116	12.551	91.286	85.082
502	C	ARG	116	13.596	90.574	84.158
503	O	ARG	116	13.908	91.144	83.108
504	CB	ARG	116	12.831	90.983	86.550
505	CG	ARG	116	14.181	91.501	87.058
506	CD	ARG	116	14.352	91.299	88.560
507	NE	ARG	116	13.879	92.466	89.318
508	CZ	ARG	116	14.379	92.814	90.506
509	NH1	ARG	116	15.319	92.066	91.075
510	NH2	ARG	116	13.930	93.908	91.124
511	N	CYS	117	14.164	89.422	84.548
512	CA	CYS	117	15.464	88.957	83.959
513	C	CYS	117	15.915	87.456	84.062
514	O	CYS	117	15.647	86.864	85.095
515	CB	CYS	117	16.506	89.743	84.720
516	SG	CYS	117	16.666	91.532	84.541
517	N	GLY	118	16.962	87.102	83.310
518	CA	GLY	118	17.597	85.743	83.076
519	C	GLY	118	17.889	84.616	84.118
520	O	GLY	118	16.937	84.196	84.773
521	N	VAL	119	19.157	84.163	84.242
522	CA	VAL	119	19.564	82.851	84.891
523	C	VAL	119	20.171	82.707	86.357
524	O	VAL	119	19.621	81.819	87.021
525	CB	VAL	119	20.381	82.067	83.849
526	CG1	VAL	119	21.399	82.913	83.095
527	CG2	VAL	119	21.010	80.794	84.408
528	N	PRO	120	21.295	83.269	86.843
529	CA	PRO	120	21.590	83.253	88.321
530	C	PRO	120	21.287	84.522	89.217
531	O	PRO	120	22.151	85.397	89.288
532	CB	PRO	120	23.072	83.064	88.306
533	CG	PRO	120	23.609	83.655	87.003
534	CD	PRO	120	22.385	83.940	86.142

TABLE V

Atom	Atom		Residue
No	Name	Residue	No	X coord	Y coord	Z coord
1	N	GLY	161	30.189	93.412	91.740
2	CA	GLY	161	29.826	93.284	90.328
3	C	GLY	161	30.977	92.572	89.640
4	O	GLY	161	31.105	91.343	89.681
5	N	TYR	162	31.837	93.390	89.063
6	CA	TYR	162	33.177	92.966	88.660
7	C	TYR	162	33.868	92.360	89.888
8	O	TYR	162	33.608	92.838	90.998
9	CB	TYR	162	33.908	94.222	88.162
10	CG	TYR	162	33.675	95.479	89.009
11	CD1	TYR	162	34.400	95.682	90.179
12	CD2	TYR	162	32.750	96.435	88.598
13	CE1	TYR	162	34.168	96.806	90.959
14	CE2	TYR	162	32.513	97.559	89.379
15	CZ	TYR	162	33.215	97.736	90.562
16	OH	TYR	162	32.888	98.775	91.403
17	N	PRO	163	34.742	91.374	89.733
18	CA	PRO	163	35.388	91.003	88.459
19	C	PRO	163	34.563	90.120	87.512
20	O	PRO	163	33.892	90.625	86.608
21	CB	PRO	163	36.634	90.276	88.868
22	CG	PRO	163	36.611	90.016	90.365
23	CD	PRO	163	35.350	90.687	90.876
24	N	ASP	164	34.791	88.817	87.608
25	CA	ASP	164	34.209	87.847	86.664
26	C	ASP	164	33.087	87.037	87.306
27	O	ASP	164	32.958	87.000	88.535
28	CB	ASP	164	35.309	86.898	86.202
29	CG	ASP	164	36.490	87.679	85.634
30	OD1	ASP	164	37.586	87.461	86.127
31	OD2	ASP	164	36.247	88.610	84.878
32	N	GLY	165	32.290	86.392	86.467
33	CA	GLY	165	31.139	85.615	86.946
34	C	GLY	165	31.536	84.305	87.615
35	O	GLY	165	32.715	84.070	87.916
36	N	GLY	166	30.530	83.476	87.855
37	CA	GLY	166	30.719	82.161	88.491
38	C	GLY	166	31.669	81.282	87.687
39	O	GLY	166	31.394	80.946	86.529
40	N	ALA	167	32.845	81.077	88.268
41	CA	ALA	167	33.960	80.339	87.649
42	C	ALA	167	34.371	80.897	86.283
43	O	ALA	167	34.852	80.135	85.437
44	CB	ALA	167	33.576	78.869	87.513
45	N	ALA	168	34.296	82.218	86.153
46	CA	ALA	168	34.545	82.966	84.906
47	C	ALA	168	34.344	82.172	83.616
48	O	ALA	168	35.332	81.827	82.955
49	CB	ALA	168	35.973	83.498	84.944
50	N	GLN	169	33.098	81.920	83.242
51	CA	GLN	169	32.845	81.192	81.987
52	C	GLN	169	32.920	82.109	80.767
53	O	GLN	169	31.909	82.654	80.304
54	CB	GLN	169	31.479	80.517	82.012
55	CG	GLN	169	31.296	79.674	80.751
56	CD	GLN	169	29.891	79.089	80.662
57	OE1	GLN	169	29.401	78.463	81.611
58	NE2	GLN	169	29.267	79.301	79.517
59	N	ALA	170	34.138	82.313	80.305
60	CA	ALA	170	34.385	83.041	79.064
61	C	ALA	170	34.375	82.069	77.894
62	O	ALA	170	34.285	80.847	78.077
63	CB	ALA	170	35.746	83.722	79.160
64	N	PHE	171	34.360	82.620	76.698
65	CA	PHE	171	34.476	81.787	75.502
66	C	PHE	171	35.924	81.768	75.042
67	O	PHE	171	36.713	82.642	75.411
68	CB	PHE	171	33.556	82.294	74.400
69	CG	PHE	171	32.221	81.552	74.335
70	CD1	PHE	171	31.839	80.937	73.150
71	CD2	PHE	171	31.402	81.467	75.454
72	CE1	PHE	171	30.634	80.249	73.079
73	CE2	PHE	171	30.199	80.778	75.385
74	CZ	PHE	171	29.813	80.171	74.197
75	N	SER	172	36.280	80.719	74.322
76	CA	SER	172	37.648	80.592	73.806
77	C	SER	172	37.782	81.176	72.401
78	O	SER	172	38.892	81.304	71.875
79	CB	SER	172	38.021	79.117	73.782
80	OG	SER	172	37.930	78.630	75.115
81	N	LYS	173	36.655	81.519	71.799
82	CA	LYS	173	36.682	82.134	70.473
83	C	LYS	173	36.385	83.624	70.549
84	O	LYS	173	35.400	84.052	71.161
85	CB	LYS	173	35.652	81.449	69.580
86	CG	LYS	173	36.005	79.986	69.344
87	CD	LYS	173	37.352	79.852	68.639
88	CE	LYS	173	37.736	78.390	68.448
89	NZ	LYS	173	39.045	78.275	67.784
90	N	ARG	174	37.190	84.385	69.826
91	CA	ARG	174	36.998	85.841	69.715
92	C	ARG	174	36.092	86.204	68.539
93	O	ARG	174	35.944	87.385	68.205
94	CB	ARG	174	38.352	86.514	69.499
95	CG	ARG	174	39.323	86.236	70.639
96	CD	ARG	174	38.800	86.761	71.974
97	NE	ARG	174	39.722	86.414	73.066
98	CZ	ARG	174	39.620	85.293	73.786
99	NH1	ARG	174	38.609	84.450	73.570
100	NH2	ARG	174	40.500	85.042	74.757
101	N	THR	175	35.586	85.190	67.857
102	CA	THR	175	34.728	85.384	66.689
103	C	THR	175	33.599	84.356	66.712
104	O	THR	175	33.847	83.154	66.557
105	CB	THR	175	35.606	85.205	65.456
106	OG1	THR	175	36.574	86.247	65.464
107	CG2	THR	175	34.824	85.312	64.155
108	N	LEU	176	32.386	84.820	66.963
109	CA	LEU	176	31.251	83.894	67.120
110	C	LEU	176	30.172	84.091	66.056
111	O	LEU	176	29.608	85.181	65.904
112	CB	LEU	176	30.639	84.069	68.505
113	CG	LEU	176	31.588	83.635	69.617
114	CD1	LEU	176	30.992	83.940	70.988
115	CD2	LEU	176	31.927	82.153	69.500
116	N	SER	177	29.814	82.987	65.424
117	CA	SER	177	28.822	83.002	64.343
118	C	SER	177	27.381	83.050	64.842
119	O	SER	177	26.949	82.222	65.660
120	CB	SER	177	29.015	81.760	63.483
121	OG	SER	177	27.995	81.765	62.491
122	N	TRP	178	26.642	83.978	64.255
123	CA	TRP	178	25.217	84.198	64.524
124	C	TRP	178	24.381	83.452	63.475
125	O	TRP	178	24.921	83.051	62.438
126	CB	TRP	178	24.990	85.718	64.453
127	CG	TRP	178	23.634	86.292	64.860
128	CD1	TRP	178	22.660	85.687	65.621
129	CD2	TRP	178	23.124	87.603	64.532
130	NE1	TRP	178	21.610	86.534	65.748
131	CE2	TRP	178	21.845	87.691	65.109
132	CE3	TRP	178	23.641	88.671	63.813
133	CZ2	TRP	178	21.089	88.841	64.932
134	CZ3	TRP	178	22.879	89.822	63.650
135	CH2	TRP	178	21.607	89.905	64.207
136	N	ARG	179	23.203	83.021	63.885
137	CA	ARG	179	22.162	82.516	62.986
138	C	ARG	179	20.915	83.356	63.231
139	O	ARG	179	20.161	83.109	64.184
140	CB	ARG	179	21.832	81.062	63.283
141	CG	ARG	179	20.694	80.629	62.365
142	CD	ARG	179	20.108	79.273	62.721
143	NE	ARG	179	18.982	78.987	61.819
144	CZ	ARG	179	18.500	77.766	61.585
145	NH1	ARG	179	19.002	76.714	62.231
146	NH2	ARG	179	17.489	77.603	60.729
147	N	LEU	180	20.683	84.316	62.354
148	CA	LEU	180	19.671	85.335	62.649
149	C	LEU	180	18.229	84.890	62.341
150	O	LEU	180	17.638	84.131	63.123
151	CB	LEU	180	20.050	86.732	62.087
152	CG	LEU	180	20.252	86.984	60.577
153	CD1	LEU	180	20.153	88.475	60.270
154	CD2	LEU	180	21.534	86.419	59.987
155	N	LEU	181	17.737	85.207	61.156
156	CA	LEU	181	16.307	85.112	60.840
157	C	LEU	181	15.840	83.722	60.425
158	O	LEU	181	14.656	83.541	60.118
159	CB	LEU	181	15.975	86.118	59.742
160	CG	LEU	181	15.364	87.408	60.293
161	CD1	LEU	181	14.109	87.099	61.095
162	CD2	LEU	181	16.333	88.247	61.125
163	N	GLY	182	16.732	82.747	60.479
164	CA	GLY	182	16.349	81.366	60.213
165	C	GLY	182	15.541	80.798	61.376
166	O	GLY	182	14.644	79.978	61.154
167	N	GLU	183	15.816	81.255	62.590
168	CA	GLU	183	15.088	80.701	63.731
169	C	GLU	183	13.827	81.492	64.083
170	O	GLU	183	12.783	80.863	64.299
171	CB	GLU	183	16.011	80.645	64.939
172	CG	GLU	183	17.222	79.742	64.766
173	CD	GLU	183	16.853	78.260	64.834
174	OE1	GLU	183	16.058	77.822	64.020
175	OE2	GLU	183	17.529	77.568	65.581
176	N	ALA	184	13.891	82.816	64.126
177	CA	ALA	184	12.679	83.568	64.497
178	C	ALA	184	12.677	85.078	64.249
179	O	ALA	184	13.693	85.783	64.322
180	CB	ALA	184	12.391	83.339	65.972
181	N	LEU	185	11.459	85.554	64.047
182	CA	LEU	185	11.152	86.984	63.914
183	C	LEU	185	10.005	87.351	64.862
184	O	LEU	185	9.024	86.602	64.964
185	CB	LEU	185	10.723	87.233	62.467
186	CG	LEU	185	10.359	88.689	62.174
187	CD1	LEU	185	11.542	89.619	62.401
188	CD2	LEU	185	9.836	88.844	60.752
189	N	SER	186	10.168	88.432	65.608
190	CA	SER	186	9.072	88.956	66.429
191	C	SER	186	7.988	89.522	65.530
192	O	SER	186	8.222	90.527	64.850
193	CB	SER	186	9.584	90.084	67.313
194	OG	SER	186	8.447	90.667	67.941
195	N	SER	187	6.765	89.079	65.772
196	CA	SER	187	5.625	89.406	64.898
197	C	SER	187	5.096	90.844	64.988
198	O	SER	187	4.131	91.171	64.289
199	CB	SER	187	4.481	88.461	65.244
200	OG	SER	187	4.077	88.745	66.577
201	N	GLN	188	5.672	91.686	65.833
202	CA	GLN	188	5.230	93.079	65.870
203	C	GLN	188	6.164	94.015	65.104
204	O	GLN	188	5.799	95.173	64.863
205	CB	GLN	188	5.122	93.542	67.313
206	CG	GLN	188	4.070	92.739	68.064
207	CD	GLN	188	3.765	93.431	69.383
208	OE1	GLN	188	3.320	92.794	70.348
209	NE2	GLN	188	4.069	94.717	69.424
210	N	LEU	189	7.327	93.529	64.708
211	CA	LEU	189	8.278	94.390	63.995
212	C	LEU	189	8.751	93.780	62.684
213	O	LEU	189	8.687	92.566	62.469
214	CB	LEU	189	9.484	94.683	64.880
215	CG	LEU	189	9.141	95.714	65.947
216	CD1	LEU	189	10.361	96.046	66.793
217	CD2	LEU	189	8.595	96.977	65.294
218	N	SER	190	9.234	94.648	61.813
219	CA	SER	190	9.803	94.189	60.548
220	C	SER	190	11.118	93.473	60.816
221	O	SER	190	11.808	93.753	61.807
222	CB	SER	190	10.089	95.371	59.630
223	OG	SER	190	11.304	95.969	60.065
224	N	VAL	191	11.542	92.712	59.824
225	CA	VAL	191	12.812	91.991	59.914
226	C	VAL	191	14.015	92.938	59.856
227	O	VAL	191	14.974	92.729	60.608
228	CB	VAL	191	12.846	90.963	58.784
229	CG1	VAL	191	12.396	91.545	57.447
230	CG2	VAL	191	14.205	90.295	58.662
231	N	ALA	192	13.824	94.106	59.258
232	CA	ALA	192	14.866	95.133	59.257
233	C	ALA	192	15.029	95.761	60.639
234	O	ALA	192	16.162	95.816	61.133
235	CB	ALA	192	14.493	96.208	58.245
236	N	ASP	193	13.924	95.994	61.337
237	CA	ASP	193	13.993	96.527	62.709
238	C	ASP	193	14.726	95.572	63.639
239	O	ASP	193	15.752	95.954	64.214
240	CB	ASP	193	12.590	96.703	63.290
241	CG	ASP	193	11.721	97.657	62.480
242	OD1	ASP	193	12.270	98.521	61.811
243	OD2	ASP	193	10.524	97.399	62.425
244	N	GLN	194	14.371	94.299	63.574
245	CA	GLN	194	14.982	93.315	64.472
246	C	GLN	194	16.450	93.065	64.133
247	O	GLN	194	17.290	93.049	65.046
248	CB	GLN	194	14.192	92.020	64.348
249	CG	GLN	194	14.748	90.934	65.261
250	CD	GLN	194	13.896	89.677	65.137
251	OE1	GLN	194	12.702	89.690	65.459
252	NE2	GLN	194	14.511	88.615	64.651
253	N	ARG	195	16.784	93.175	62.858
254	CA	ARG	195	18.164	92.990	62.421
255	C	ARG	195	19.060	94.133	62.885
256	O	ARG	195	20.060	93.861	63.559
257	CB	ARG	195	18.178	92.949	60.900
258	CG	ARG	195	19.578	92.695	60.354
259	CD	ARG	195	19.612	92.878	58.843
260	NE	ARG	195	18.612	92.026	58.178
261	CZ	ARG	195	17.701	92.503	57.327
262	NH1	ARG	195	17.637	93.813	57.083
263	NH2	ARG	195	16.834	91.675	56.743
264	N	ARG	196	18.579	95.364	62.787
265	CA	ARG	196	19.430	96.504	63.147
266	C	ARG	196	19.501	96.737	64.658
267	O	ARG	196	20.558	97.160	65.147
268	CB	ARG	196	18.935	97.758	62.422
269	CG	ARG	196	17.545	98.204	62.869
270	CD	ARG	196	17.018	99.356	62.024
271	NE	ARG	196	16.824	98.931	60.628
272	CZ	ARG	196	17.271	99.624	59.580
273	NH1	ARG	196	17.943	100.762	59.768
274	NH2	ARG	196	17.056	99.171	58.342
275	N	ILE	197	18.542	96.206	65.401
276	CA	ILE	197	18.596	96.331	66.856
277	C	ILE	197	19.599	95.347	67.448
278	O	ILE	197	20.464	95.759	68.234
279	CB	ILE	197	17.207	96.061	67.420
280	CG1	ILE	197	16.230	97.149	66.991
281	CG2	ILE	197	17.259	95.962	68.938
282	CD1	ILE	197	14.804	96.824	67.421
283	N	VAL	198	19.667	94.157	66.873
284	CA	VAL	198	20.613	93.167	67.386
285	C	VAL	198	21.999	93.363	66.760
286	O	VAL	198	23.014	93.060	67.401
287	CB	VAL	198	20.050	91.775	67.119
288	CG1	VAL	198	20.838	90.724	67.887
289	CG2	VAL	198	18.590	91.701	67.548
290	N	ALA	199	22.038	94.118	65.674
291	CA	ALA	199	23.307	94.533	65.074
292	C	ALA	199	24.028	95.519	65.977
293	O	ALA	199	25.164	95.250	66.392
294	CB	ALA	199	23.004	95.236	63.759
295	N	LEU	200	23.280	96.487	66.488
296	CA	LEU	200	23.863	97.483	67.394
297	C	LEU	200	24.156	96.891	68.766
298	O	LEU	200	25.183	97.231	69.366
299	CB	LEU	200	22.886	98.642	67.543
300	CG	LEU	200	22.700	99.384	66.224
301	CD1	LEU	200	21.592	100.426	66.337
302	CD2	LEU	200	24.006	100.027	65.766
303	N	ALA	201	23.401	95.865	69.131
304	CA	ALA	201	23.661	95.111	70.358
305	C	ALA	201	25.060	94.509	70.358
306	O	ALA	201	25.902	94.928	71.164
307	CB	ALA	201	22.651	93.979	70.440
308	N	PHE	202	25.369	93.745	69.321
309	CA	PHE	202	26.686	93.110	69.254
310	C	PHE	202	27.792	94.115	68.973
311	O	PHE	202	28.834	94.036	69.633
312	CB	PHE	202	26.716	92.068	68.145
313	CG	PHE	202	25.934	90.781	68.373
314	CD1	PHE	202	26.512	89.739	69.084
315	CD2	PHE	202	24.664	90.631	67.835
316	CE1	PHE	202	25.806	88.562	69.289
317	CE2	PHE	202	23.957	89.455	68.040
318	CZ	PHE	202	24.526	88.423	68.772
319	N	ARG	203	27.465	95.183	68.267
320	CA	ARG	203	28.458	96.198	67.906
321	C	ARG	203	28.968	97.009	69.100
322	O	ARG	203	30.172	97.289	69.152
323	CB	ARG	203	27.805	97.140	66.902
324	CG	ARG	203	28.763	98.239	66.468
325	CD	ARG	203	28.121	99.171	65.451
326	NE	ARG	203	29.085	100.196	65.023
327	CZ	ARG	203	29.643	100.217	63.810
328	NH1	ARG	203	29.284	99.316	62.892
329	NH2	ARG	203	30.527	101.168	63.498
330	N	MET	204	28.165	97.140	70.146
331	CA	MET	204	28.635	97.864	71.332
332	C	MET	204	29.677	97.055	72.096
333	O	MET	204	30.795	97.545	72.311
334	CB	MET	204	27.451	98.143	72.252
335	CG	MET	204	26.410	99.027	71.579
336	SD	MET	204	26.981	100.658	71.048
337	CE	MET	204	25.455	101.241	70.272
338	N	TRP	205	29.438	95.756	72.195
339	CA	TRP	205	30.369	94.890	72.927
340	C	TRP	205	31.617	94.608	72.105
341	O	TRP	205	32.726	94.771	72.633
342	CB	TRP	205	29.660	93.586	73.277
343	CG	TRP	205	28.486	93.808	74.202
344	CD1	TRP	205	27.149	93.766	73.877
345	CD2	TRP	205	28.551	94.108	75.613
346	NE1	TRP	205	26.421	94.065	74.985
347	CE2	TRP	205	27.231	94.277	76.040
348	CE3	TRP	205	29.609	94.271	76.498
349	CZ2	TRP	205	26.973	94.628	77.361
350	CZ3	TRP	205	29.344	94.615	77.819
351	CH2	TRP	205	28.034	94.793	78.246
352	N	SER	206	31.425	94.627	70.796
353	CA	SER	206	32.505	94.417	69.834
354	C	SER	206	33.413	95.630	69.663
355	O	SER	206	34.480	95.482	69.068
356	CB	SER	206	31.876	94.129	68.474
357	OG	SER	206	31.135	92.922	68.565
358	N	GLU	207	33.026	96.787	70.181
359	CA	GLU	207	33.855	97.988	70.054
360	C	GLU	207	34.563	98.330	71.364
361	O	GLU	207	35.544	99.086	71.364
362	CB	GLU	207	32.935	99.141	69.653
363	CG	GLU	207	33.673	100.462	69.436
364	CD	GLU	207	34.511	100.435	68.158
365	OE1	GLU	207	34.203	99.591	67.327
366	OE2	GLU	207	35.120	101.465	67.893
367	N	VAL	208	34.080	97.790	72.469
368	CA	VAL	208	34.706	98.128	73.750
369	C	VAL	208	35.660	97.023	74.200
370	O	VAL	208	36.627	97.277	74.935
371	CB	VAL	208	33.612	98.418	74.779
372	CG1	VAL	208	32.746	99.580	74.302
373	CG2	VAL	208	32.731	97.213	75.092
374	N	THR	209	35.442	95.839	73.662
375	CA	THR	209	36.304	94.685	73.906
376	C	THR	209	36.395	93.935	72.579
377	O	THR	209	35.384	93.867	71.871
378	CB	THR	209	35.652	93.861	75.019
379	OG1	THR	209	35.767	94.615	76.221
380	CG2	THR	209	36.331	92.524	75.268
381	N	PRO	210	37.594	93.520	72.189
382	CA	PRO	210	37.835	93.002	70.829
383	C	PRO	210	37.137	91.667	70.562
384	O	PRO	210	37.676	90.593	70.859
385	CB	PRO	210	39.322	92.854	70.727
386	CG	PRO	210	39.964	93.206	72.060
387	CD	PRO	210	38.829	93.645	72.968
388	N	LEU	211	35.942	91.756	70.001
389	CA	LEU	211	35.105	90.580	69.733
390	C	LEU	211	34.385	90.712	68.394
391	O	LEU	211	33.552	91.603	68.210
392	CB	LEU	211	34.058	90.487	70.839
393	CG	LEU	211	34.654	90.146	72.201
394	CD1	LEU	211	33.666	90.439	73.322
395	CD2	LEU	211	35.135	88.699	72.245
396	N	ASP	212	34.637	89.782	67.495
397	CA	ASP	212	34.007	89.835	66.171
398	C	ASP	212	32.690	89.051	66.170
399	O	ASP	212	32.669	87.827	66.362
400	CB	ASP	212	35.002	89.261	65.170
401	CG	ASP	212	34.553	89.522	63.737
402	OD1	ASP	212	33.768	90.439	63.539
403	OD2	ASP	212	35.007	88.796	62.866
404	N	PHE	213	31.597	89.773	65.962
405	CA	PHE	213	30.254	89.171	66.035
406	C	PHE	213	29.314	89.556	64.887
407	O	PHE	213	29.659	89.534	63.694
408	CB	PHE	213	29.574	89.656	67.313
409	CG	PHE	213	30.151	89.196	68.649
410	CD1	PHE	213	30.616	87.898	68.812
411	CD2	PHE	213	30.170	90.080	69.718
412	CE1	PHE	213	31.125	87.494	70.038
413	CE2	PHE	213	30.676	89.676	70.947
414	CZ	PHE	213	31.156	88.381	71.105
415	N	ARG	214	28.150	90.022	65.335
416	CA	ARG	214	26.962	90.419	64.534
417	C	ARG	214	26.908	89.948	63.085
418	O	ARG	214	27.195	88.783	62.789
419	CB	ARG	214	26.733	91.933	64.613
420	CG	ARG	214	27.962	92.766	64.978
421	CD	ARG	214	28.933	93.003	63.825
422	NE	ARG	214	30.060	93.821	64.295
423	CZ	ARG	214	30.118	95.143	64.118
424	NH1	ARG	214	29.157	95.768	63.436
425	NH2	ARG	214	31.159	95.834	64.590
426	N	GLU	215	26.599	90.866	62.180
427	CA	GLU	215	26.351	90.498	60.775
428	C	GLU	215	27.592	90.096	59.980
429	O	GLU	215	27.434	89.480	58.923
430	CB	GLU	215	25.655	91.637	60.039
431	CG	GLU	215	24.227	91.856	60.522
432	CD	GLU	215	24.132	93.233	61.162
433	OE1	GLU	215	23.194	93.953	60.848
434	OE2	GLU	215	25.067	93.572	61.877
435	N	ASP	216	28.775	90.207	60.566
436	CA	ASP	216	29.978	89.731	59.885
437	C	ASP	216	30.122	88.221	60.079
438	O	ASP	216	30.820	87.549	59.312
439	CB	ASP	216	31.189	90.444	60.476
440	CG	ASP	216	31.054	91.951	60.281
441	OD1	ASP	216	31.111	92.658	61.279
442	OD2	ASP	216	30.727	92.354	59.175
443	N	LEU	217	29.406	87.695	61.062
444	CA	LEU	217	29.376	86.254	61.309
445	C	LEU	217	27.961	85.698	61.196
446	O	LEU	217	27.720	84.555	61.600
447	CB	LEU	217	29.895	85.949	62.714
448	CG	LEU	217	31.416	85.856	62.818
449	CD1	LEU	217	31.987	85.048	61.657
450	CD2	LEU	217	32.092	87.219	62.907
451	N	ALA	218	27.031	86.506	60.713
452	CA	ALA	218	25.619	86.117	60.739
453	C	ALA	218	25.171	85.291	59.540
454	O	ALA	218	24.931	85.813	58.447
455	CB	ALA	218	24.775	87.378	60.813
456	N	ALA	219	24.948	84.017	59.803
457	CA	ALA	219	24.378	83.112	58.813
458	C	ALA	219	22.856	83.140	58.917
459	O	ALA	219	22.297	83.460	59.976
460	CB	ALA	219	24.901	81.706	59.090
461	N	PRO	220	22.200	83.011	57.777
462	CA	PRO	220	20.744	82.885	57.773
463	C	PRO	220	20.299	81.625	58.515
464	O	PRO	220	19.610	81.726	59.537
465	CB	PRO	220	20.358	82.844	56.325
466	CG	PRO	220	21.616	82.885	55.465
467	CD	PRO	220	22.787	82.949	56.435
468	N	GLY	221	20.785	80.482	58.052
469	CA	GLY	221	20.480	79.188	58.668
470	C	GLY	221	21.695	78.635	59.410
471	O	GLY	221	22.637	79.383	59.698
472	N	ALA	222	21.653	77.336	59.680
473	CA	ALA	222	22.696	76.611	60.436
474	C	ALA	222	22.820	77.100	61.879
475	O	ALA	222	23.262	78.226	62.130
476	CB	ALA	222	24.042	76.709	59.721
477	N	ALA	223	22.465	76.228	62.811
478	CA	ALA	223	22.461	76.577	64.239
479	C	ALA	223	23.870	76.628	64.823
480	O	ALA	223	24.423	75.613	65.260
481	CB	ALA	223	21.635	75.534	64.984
482	N	VAL	224	24.424	77.828	64.839
483	CA	VAL	224	25.789	78.039	65.322
484	C	VAL	224	25.847	78.485	66.783
485	O	VAL	224	25.089	77.989	67.623
486	CB	VAL	224	26.454	79.036	64.383
487	CG1	VAL	224	27.138	78.314	63.228
488	CG2	VAL	224	25.435	80.042	63.862
489	N	ASP	225	26.730	79.430	67.076
490	CA	ASP	225	27.020	79.769	68.476
491	C	ASP	225	26.006	80.765	69.017
492	O	ASP	225	25.706	80.794	70.216
493	CB	ASP	225	28.390	80.442	68.542
494	CG	ASP	225	29.434	79.681	67.731
495	OD1	ASP	225	29.648	78.511	68.012
496	OD2	ASP	225	30.007	80.298	66.840
497	N	ILE	226	25.485	81.573	68.114
498	CA	ILE	226	24.522	82.605	68.478
499	C	ILE	226	23.222	82.423	67.700
500	O	ILE	226	23.060	83.010	66.630
501	CB	ILE	226	25.180	83.928	68.096
502	CG1	ILE	226	26.536	84.094	68.773
503	CG2	ILE	226	24.288	85.114	68.423
504	CD1	ILE	226	27.219	85.378	68.325
505	N	LYS	227	22.306	81.620	68.205
506	CA	LYS	227	21.042	81.399	67.479
507	C	LYS	227	20.054	82.530	67.797
508	O	LYS	227	20.390	83.379	68.624
509	CB	LYS	227	20.517	80.040	67.906
510	CG	LYS	227	21.589	78.973	67.715
511	CD	LYS	227	21.190	77.666	68.388
512	CE	LYS	227	19.871	77.142	67.843
513	NZ	LYS	227	19.475	75.897	68.521
514	N	LEU	228	18.924	82.616	67.106
515	CA	LEU	228	17.958	83.708	67.380
516	C	LEU	228	16.500	83.230	67.437
517	O	LEU	228	15.839	83.116	66.401
518	CB	LEU	228	18.169	84.780	66.303
519	CG	LEU	228	17.094	85.867	66.181
520	CD1	LEU	228	16.733	86.546	67.495
521	CD2	LEU	228	17.526	86.915	65.165
522	N	GLY	229	15.968	83.083	68.642
523	CA	GLY	229	14.619	82.531	68.792
524	C	GLY	229	13.696	83.225	69.793
525	O	GLY	229	14.088	83.691	70.870
526	N	PHE	230	12.434	83.242	69.400
527	CA	PHE	230	11.342	83.810	70.198
528	C	PHE	230	10.323	82.716	70.520
529	O	PHE	230	9.864	82.003	69.622
530	CB	PHE	230	10.634	84.904	69.392
531	CG	PHE	230	11.408	86.198	69.121
532	CD1	PHE	230	12.359	86.259	68.111
533	CD2	PHE	230	11.130	87.332	69.875
534	CE1	PHE	230	13.054	87.438	67.877
535	CE2	PHE	230	11.824	88.511	69.641
536	CZ	PHE	230	12.790	88.565	68.645
537	N	GLY	231	9.968	82.605	71.785
538	CA	GLY	231	8.952	81.640	72.213
539	C	GLY	231	7.973	82.274	73.198
540	O	GLY	231	7.897	83.504	73.335
541	N	ARG	232	7.138	81.428	73.772
542	CA	ARG	232	6.163	81.866	74.774
543	C	ARG	232	5.984	80.791	75.833
544	O	ARG	232	6.709	79.791	75.797
545	CB	ARG	232	4.843	82.167	74.086
546	CG	ARG	232	4.379	81.031	73.186
547	CD	ARG	232	3.182	81.492	72.368
548	NE	ARG	232	3.517	82.751	71.682
549	CZ	ARG	232	2.801	83.871	71.809
550	NH1	ARG	232	1.670	83.864	72.519
551	NH2	ARG	232	3.193	84.987	71.191
552	N	GLY	233	5.138	81.098	76.810
553	CA	GLY	233	4.765	80.230	77.953
554	C	GLY	233	5.433	78.858	78.057
555	O	GLY	233	6.665	78.732	78.136
556	N	ARG	234	4.598	77.836	78.089
557	CA	ARG	234	5.071	76.451	78.176
558	C	ARG	234	5.638	75.900	76.863
559	O	ARG	234	4.906	75.442	75.983
560	CB	ARG	234	3.926	75.577	78.702
561	CG	ARG	234	2.523	76.109	78.379
562	CD	ARG	234	2.038	75.833	76.953
563	NE	ARG	234	1.715	77.093	76.259
564	CZ	ARG	234	1.681	77.217	74.931
565	NH1	ARG	234	1.829	76.140	74.156
566	NH2	ARG	234	1.418	78.405	74.379
567	N	HIS	235	6.952	75.974	76.740
568	CA	HIS	235	7.630	75.333	75.610
569	C	HIS	235	8.662	74.305	76.083
570	O	HIS	235	9.246	73.596	75.257
571	CB	HIS	235	8.246	76.378	74.680
572	CG	HIS	235	9.367	77.239	75.231
573	ND1	HIS	235	9.237	78.363	75.961
574	CD2	HIS	235	10.714	77.032	75.050
575	CE1	HIS	235	10.461	78.849	76.249
576	NE2	HIS	235	11.373	78.029	75.682
577	N	LEU	236	8.820	74.209	77.399
578	CA	LEU	236	9.673	73.200	78.053
579	C	LEU	236	11.128	73.258	77.594
580	O	LEU	236	11.509	72.691	76.565
581	CB	LEU	236	9.110	71.802	77.798
582	CG	LEU	236	9.900	70.730	78.545
583	CD1	LEU	236	9.814	70.936	80.054
584	CD2	LEU	236	9.418	69.332	78.170
585	N	GLY	237	11.951	73.863	78.428
586	CA	GLY	237	13.380	73.931	78.139
587	C	GLY	237	14.195	73.415	79.312
588	O	GLY	237	15.299	73.909	79.578
589	N	CYS	238	13.581	72.497	80.042
590	CA	CYS	238	14.236	71.716	81.102
591	C	CYS	238	14.798	72.524	82.271
592	O	CYS	238	15.798	73.242	82.152
593	CB	CYS	238	15.349	70.881	80.478
594	SG	CYS	238	14.805	69.652	79.269
595	N	PRO	239	14.161	72.355	83.417
596	CA	PRO	239	12.826	71.750	83.500
597	C	PRO	239	11.649	72.715	83.261
598	O	PRO	239	10.501	72.258	83.222
599	CB	PRO	239	12.766	71.261	84.915
600	CG	PRO	239	13.825	71.992	85.731
601	CD	PRO	239	14.651	72.783	84.727
602	N	ARG	240	11.904	74.000	83.062
603	CA	ARG	240	10.806	74.969	83.122
604	C	ARG	240	10.493	75.647	81.787
605	O	ARG	240	10.789	75.126	80.703
606	CB	ARG	240	11.137	76.060	84.137
607	CG	ARG	240	12.001	75.627	85.324
608	CD	ARG	240	13.455	76.049	85.105
609	NE	ARG	240	14.285	75.957	86.312
610	CZ	ARG	240	15.439	76.621	86.424
611	NH1	ARG	240	16.230	76.412	87.477
612	NH2	ARG	240	15.847	77.414	85.432
613	N	ALA	241	9.701	76.703	81.910
614	CA	ALA	241	9.254	77.522	80.774
615	C	ALA	241	8.860	78.937	81.233
616	O	ALA	241	9.162	79.335	82.364
617	CB	ALA	241	8.062	76.826	80.139
618	N	PHE	242	8.229	79.689	80.345
619	CA	PHE	242	7.740	81.045	80.659
620	C	PHE	242	6.377	81.045	81.357
621	O	PHE	242	5.616	80.072	81.315
622	CB	PHE	242	7.522	81.848	79.378
623	CG	PHE	242	8.716	82.366	78.589
624	CD1	PHE	242	9.500	83.394	79.094
625	CD2	PHE	242	8.980	81.854	77.328
626	CE1	PHE	242	10.569	83.884	78.359
627	CE2	PHE	242	10.047	82.343	76.588
628	CZ	PHE	242	10.844	83.355	77.107
629	N	ASP	243	6.051	82.195	81.931
630	CA	ASP	243	4.698	82.440	82.452
631	C	ASP	243	3.903	83.297	81.458
632	O	ASP	243	2.672	83.364	81.538
633	CB	ASP	243	4.751	83.119	83.828
634	CG	ASP	243	4.871	84.642	83.736
635	OD1	ASP	243	5.923	85.104	83.320
636	OD2	ASP	243	3.864	85.304	83.937
637	N	GLY	244	4.592	83.904	80.502
638	CA	GLY	244	3.887	84.632	79.442
639	C	GLY	244	4.052	86.153	79.441
640	O	GLY	244	5.117	86.708	79.747
641	N	SER	245	2.943	86.800	79.116
642	CA	SER	245	2.901	88.236	78.789
643	C	SER	245	3.100	89.170	79.971
644	O	SER	245	2.728	88.855	81.108
645	CB	SER	245	1.532	88.550	78.191
646	OG	SER	245	1.355	87.764	77.021
647	N	GLY	246	3.770	90.278	79.695
648	CA	GLY	246	3.888	91.393	80.642
649	C	GLY	246	5.050	91.244	81.617
650	O	GLY	246	5.997	92.040	81.619
651	N	GLN	247	4.990	90.169	82.380
652	CA	GLN	247	5.900	89.945	83.493
653	C	GLN	247	7.280	89.401	83.172
654	O	GLN	247	7.969	89.145	84.162
655	CB	GLN	247	5.258	88.995	84.500
656	CG	GLN	247	4.246	89.705	85.392
657	CD	GLN	247	4.908	90.884	86.111
658	OE1	GLN	247	4.744	92.037	85.695
659	NE2	GLN	247	5.658	90.589	87.159
660	N	GLU	248	7.711	89.201	81.931
661	CA	GLU	248	9.103	88.735	81.796
662	C	GLU	248	9.851	88.817	80.466
663	O	GLU	248	9.342	88.723	79.340
664	CB	GLU	248	9.215	87.303	82.287
665	CG	GLU	248	8.282	86.350	81.579
666	CD	GLU	248	8.506	84.990	82.204
667	OE1	GLU	248	8.181	84.012	81.545
668	OE2	GLU	248	8.957	84.926	83.337
669	N	PHE	249	11.124	88.611	80.760
670	CA	PHE	249	12.323	88.534	79.908
671	C	PHE	249	12.382	87.686	78.644
672	O	PHE	249	11.414	87.372	77.943
673	CB	PHE	249	13.374	87.872	80.807
674	CG	PHE	249	13.045	86.515	81.478
675	CD1	PHE	249	13.800	86.126	82.579
676	CD2	PHE	249	12.065	85.652	80.998
677	CE1	PHE	249	13.525	84.932	83.237
678	CE2	PHE	249	11.786	84.463	81.656
679	CZ	PHE	249	12.507	84.109	82.783
680	N	ALA	250	13.650	87.586	78.277
681	CA	ALA	250	14.203	86.574	77.385
682	C	ALA	250	15.205	85.794	78.241
683	O	ALA	250	16.146	86.379	78.792
684	CB	ALA	250	14.936	87.272	76.255
685	N	HIS	251	14.954	84.513	78.422
686	CA	HIS	251	15.715	83.734	79.411
687	C	HIS	251	16.825	82.835	78.871
688	O	HIS	251	16.606	81.966	78.024
689	CB	HIS	251	14.729	82.911	80.214
690	CG	HIS	251	13.729	82.007	79.509
691	ND1	HIS	251	12.545	81.629	80.025
692	CD2	HIS	251	13.829	81.397	78.278
693	CE1	HIS	251	11.912	80.815	79.163
694	NE2	HIS	251	12.706	80.676	78.079
695	N	ALA	252	17.998	82.977	79.453
696	CA	ALA	252	19.161	82.213	78.984
697	C	ALA	252	19.548	81.052	79.878
698	O	ALA	252	18.698	80.337	80.427
699	CB	ALA	252	20.368	83.140	78.889
700	N	TRP	253	20.823	80.736	79.719
701	CA	TRP	253	21.608	79.883	80.627
702	C	TRP	253	23.053	79.802	80.146
703	O	TRP	253	23.408	80.390	79.114
704	CB	TRP	253	21.024	78.483	80.786
705	CG	TRP	253	20.413	78.263	82.160
706	CD1	TRP	253	19.095	77.977	82.441
707	CD2	TRP	253	21.098	78.318	83.432
708	NE1	TRP	253	18.956	77.852	83.785
709	CE2	TRP	253	20.130	78.047	84.415
710	CE3	TRP	253	22.412	78.560	83.802
711	CZ2	TRP	253	20.494	78.013	85.753
712	CZ3	TRP	253	22.770	78.526	85.144
713	CH2	TRP	253	21.815	78.254	86.115
714	N	ARG	254	23.804	78.899	80.761
715	CA	ARG	254	25.247	78.736	80.508
716	C	ARG	254	25.611	78.040	79.193
717	O	ARG	254	26.800	77.925	78.876
718	CB	ARG	254	25.870	77.974	81.669
719	CG	ARG	254	25.906	78.830	82.929
720	CD	ARG	254	26.750	80.083	82.717
721	NE	ARG	254	26.795	80.908	83.933
722	CZ	ARG	254	27.832	80.926	84.776
723	NH1	ARG	254	28.900	80.161	84.541
724	NH2	ARG	254	27.797	81.706	85.857
725	N	LEU	255	24.619	77.716	78.377
726	CA	LEU	255	24.887	77.247	77.015
727	C	LEU	255	25.200	78.444	76.119
728	O	LEU	255	25.676	78.287	74.992
729	CB	LEU	255	23.678	76.500	76.445
730	CG	LEU	255	23.665	75.001	76.754
731	CD1	LEU	255	23.350	74.688	78.214
732	CD2	LEU	255	22.654	74.294	75.858
733	N	GLY	256	24.905	79.630	76.632
734	CA	GLY	256	25.311	80.871	75.989
735	C	GLY	256	24.135	81.625	75.404
736	O	GLY	256	24.298	82.731	74.882
737	N	ASP	257	22.958	81.027	75.476
738	CA	ASP	257	21.829	81.600	74.750
739	C	ASP	257	20.513	81.738	75.532
740	O	ASP	257	20.193	80.901	76.389
741	CB	ASP	257	21.600	80.732	73.517
742	CG	ASP	257	22.672	80.867	72.429
743	OD1	ASP	257	22.339	81.398	71.372
744	OD2	ASP	257	23.700	80.232	72.590
745	N	ILE	258	19.819	82.842	75.273
746	CA	ILE	258	18.457	83.122	75.765
747	C	ILE	258	17.406	82.537	74.852
748	O	ILE	258	17.719	81.777	73.941
749	CB	ILE	258	18.144	84.625	75.793
750	CG1	ILE	258	18.451	85.280	74.461
751	CG2	ILE	258	18.811	85.375	76.929
752	CD1	ILE	258	18.159	86.773	74.476
753	N	HIS	259	16.176	82.667	75.298
754	CA	HIS	259	15.015	82.507	74.431
755	C	HIS	259	14.077	83.673	74.707
756	O	HIS	259	13.759	83.935	75.874
757	CB	HIS	259	14.329	81.201	74.790
758	CG	HIS	259	14.111	80.276	73.619
759	ND1	HIS	259	14.319	78.949	73.614
760	CD2	HIS	259	13.681	80.620	72.359
761	CE1	HIS	259	14.021	78.454	72.396
762	NE2	HIS	259	13.626	79.490	71.621
763	N	PHE	260	13.679	84.398	73.679
764	CA	PHE	260	12.818	85.574	73.889
765	C	PHE	260	11.382	85.221	74.243
766	O	PHE	260	10.908	84.133	73.905
767	CB	PHE	260	12.783	86.405	72.620
768	CG	PHE	260	14.008	87.265	72.362
769	CD1	PHE	260	14.930	86.901	71.388
770	CD2	PHE	260	14.179	88.438	73.080
771	CE1	PHE	260	16.037	87.708	71.151
772	CE2	PHE	260	15.284	89.239	72.845
773	CZ	PHE	260	16.213	88.876	71.882
774	N	ASP	261	10.741	86.084	75.010
775	CA	ASP	261	9.293	85.967	75.214
776	C	ASP	261	8.599	86.908	74.237
777	O	ASP	261	8.501	88.113	74.503
778	CB	ASP	261	8.980	86.357	76.658
779	CG	ASP	261	7.509	86.207	77.022
780	OD1	ASP	261	7.132	85.162	77.536
781	OD2	ASP	261	6.768	87.129	76.698
782	N	ASP	262	7.931	86.339	73.243
783	CA	ASP	262	7.285	87.134	72.179
784	C	ASP	262	5.882	87.615	72.582
785	O	ASP	262	5.116	88.134	71.763
786	CB	ASP	262	7.219	86.304	70.897
787	CG	ASP	262	7.115	87.222	69.674
788	OD1	ASP	262	7.918	88.140	69.587
789	OD2	ASP	262	6.291	86.950	68.811
790	N	ASP	263	5.555	87.416	73.849
791	CA	ASP	263	4.325	87.933	74.442
792	C	ASP	263	4.578	89.359	74.927
793	O	ASP	263	3.637	90.099	75.231
794	CB	ASP	263	3.982	87.097	75.666
795	CG	ASP	263	3.920	85.598	75.406
796	OD1	ASP	263	2.815	85.080	75.466
797	OD2	ASP	263	4.970	84.971	75.449
798	N	GLU	264	5.851	89.695	75.076
799	CA	GLU	264	6.246	91.085	75.289
800	C	GLU	264	5.957	91.844	74.003
801	O	GLU	264	6.333	91.394	72.914
802	CB	GLU	264	7.738	91.142	75.587
803	CG	GLU	264	8.135	90.410	76.863
804	CD	GLU	264	7.495	91.055	78.087
805	OE1	GLU	264	7.910	92.147	78.453
806	OE2	GLU	264	6.510	90.502	78.565
807	N	HIS	265	5.344	93.005	74.138
808	CA	HIS	265	4.854	93.739	72.966
809	C	HIS	265	5.883	94.675	72.350
810	O	HIS	265	5.651	95.885	72.281
811	CB	HIS	265	3.613	94.513	73.384
812	CG	HIS	265	2.498	93.575	73.792
813	ND1	HIS	265	2.005	92.571	73.044
814	CD2	HIS	265	1.809	93.569	74.980
815	CE1	HIS	265	1.029	91.942	73.729
816	NE2	HIS	265	0.910	92.559	74.928
817	N	PHE	266	6.821	94.056	71.652
818	CA	PHE	266	7.986	94.727	71.063
819	C	PHE	266	7.665	95.850	70.076
820	O	PHE	266	7.202	95.635	68.948
821	CB	PHE	266	8.817	93.655	70.367
822	CG	PHE	266	9.360	92.593	71.318
823	CD1	PHE	266	9.154	91.246	71.052
824	CD2	PHE	266	10.057	92.976	72.458
825	CE1	PHE	266	9.648	90.282	71.920
826	CE2	PHE	266	10.554	92.012	73.324
827	CZ	PHE	266	10.351	90.664	73.055
828	N	THR	267	7.874	97.055	70.575
829	CA	THR	267	7.864	98.292	69.801
830	C	THR	267	9.307	98.607	69.458
831	O	THR	267	10.217	98.070	70.101
832	CB	THR	267	7.348	99.428	70.683
833	OG1	THR	267	8.335	99.712	71.673
834	CG2	THR	267	6.035	99.080	71.370
835	N	PRO	268	9.529	99.366	68.402
836	CA	PRO	268	10.871	99.893	68.169
837	C	PRO	268	11.421	100.707	69.370
838	O	PRO	268	12.151	100.078	70.149
839	CB	PRO	268	10.794	100.613	66.853
840	CG	PRO	268	9.355	100.567	66.349
841	CD	PRO	268	8.552	99.836	67.415
842	N	PRO	269	10.971	101.930	69.670
843	CA	PRO	269	11.722	102.774	70.612
844	C	PRO	269	11.526	102.355	72.064
845	O	PRO	269	10.839	101.367	72.360
846	CB	PRO	269	11.219	104.168	70.410
847	CG	PRO	269	9.974	104.124	69.545
848	CD	PRO	269	9.823	102.676	69.120
849	N	THR	270	12.209	103.073	72.941
850	CA	THR	270	12.080	102.870	74.390
851	C	THR	270	10.668	103.139	74.907
852	O	THR	270	10.166	104.268	74.945
853	CB	THR	270	13.082	103.768	75.104
854	OG1	THR	270	14.361	103.187	74.898
855	CG2	THR	270	12.838	103.824	76.610
856	N	SER	271	10.023	102.033	75.223
857	CA	SER	271	8.725	102.006	75.884
858	C	SER	271	8.877	101.207	77.171
859	O	SER	271	9.920	100.585	77.399
860	CB	SER	271	7.699	101.361	74.959
861	OG	SER	271	8.160	100.058	74.630
862	N	ASP	272	7.825	101.203	77.972
863	CA	ASP	272	7.846	100.586	79.308
864	C	ASP	272	8.206	99.097	79.254
865	O	ASP	272	8.051	98.452	78.210
866	CB	ASP	272	6.456	100.771	79.914
867	CG	ASP	272	6.537	101.073	81.410
868	OD1	ASP	272	5.623	101.715	81.905
869	OD2	ASP	272	7.533	100.705	82.017
870	N	THR	273	8.699	98.586	80.378
871	CA	THR	273	9.075	97.170	80.578
872	C	THR	273	9.740	96.511	79.369
873	O	THR	273	10.442	97.169	78.590
874	CB	THR	273	7.825	96.401	81.004
875	OG1	THR	273	6.798	96.603	80.038
876	CG2	THR	273	7.300	96.917	82.341
877	N	GLY	274	9.435	95.242	79.144
878	CA	GLY	274	10.019	94.515	78.003
879	C	GLY	274	9.228	94.703	76.702
880	O	GLY	274	9.133	93.791	75.874
881	N	ILE	275	8.706	95.904	76.517
882	CA	ILE	275	7.943	96.271	75.335
883	C	ILE	275	8.849	97.060	74.393
884	O	ILE	275	8.506	97.286	73.232
885	CB	ILE	275	6.763	97.106	75.836
886	CG1	ILE	275	5.900	96.286	76.785
887	CG2	ILE	275	5.902	97.674	74.718
888	CD1	ILE	275	4.730	97.108	77.313
889	N	SER	276	10.014	97.450	74.876
890	CA	SER	276	10.972	98.145	73.998
891	C	SER	276	12.008	97.204	73.394
892	O	SER	276	13.059	96.991	74.008
893	CB	SER	276	11.687	99.237	74.784
894	OG	SER	276	12.153	98.729	76.028
895	N	LEU	277	11.864	96.901	72.113
896	CA	LEU	277	12.783	95.946	71.480
897	C	LEU	277	14.123	96.597	71.136
898	O	LEU	277	15.147	95.913	71.246
899	CB	LEU	277	12.146	95.356	70.224
900	CG	LEU	277	12.992	94.218	69.645
901	CD1	LEU	277	13.257	93.137	70.687
902	CD2	LEU	277	12.350	93.604	68.407
903	N	LEU	278	14.156	97.924	71.059
904	CA	LEU	278	15.437	98.621	70.867
905	C	LEU	278	16.263	98.629	72.153
906	O	LEU	278	17.496	98.600	72.089
907	CB	LEU	278	15.172	100.060	70.443
908	CG	LEU	278	15.872	100.419	69.137
909	CD1	LEU	278	15.401	101.776	68.627
910	CD2	LEU	278	17.392	100.389	69.273
911	N	LYS	279	15.603	98.426	73.280
912	CA	LYS	279	16.333	98.280	74.528
913	C	LYS	279	16.791	96.850	74.708
914	O	LYS	279	18.002	96.631	74.837
915	CB	LYS	279	15.421	98.601	75.701
916	CG	LYS	279	15.465	100.061	76.100
917	CD	LYS	279	14.698	100.253	77.400
918	CE	LYS	279	15.140	101.526	78.094
919	NZ	LYS	279	16.574	101.439	78.398
920	N	VAL	280	15.919	95.932	74.318
921	CA	VAL	280	16.003	94.516	74.708
922	C	VAL	280	17.194	93.728	74.148
923	O	VAL	280	17.653	92.799	74.826
924	CB	VAL	280	14.675	93.867	74.294
925	CG1	VAL	280	14.735	92.349	74.189
926	CG2	VAL	280	13.536	94.278	75.217
927	N	ALA	281	17.857	94.226	73.122
928	CA	ALA	281	18.981	93.462	72.592
929	C	ALA	281	20.369	93.927	73.040
930	O	ALA	281	21.274	93.085	73.106
931	CB	ALA	281	18.909	93.495	71.072
932	N	VAL	282	20.504	95.139	73.557
933	CA	VAL	282	21.848	95.739	73.600
934	C	VAL	282	22.752	95.352	74.785
935	O	VAL	282	23.944	95.673	74.751
936	CB	VAL	282	21.700	97.252	73.512
937	CG1	VAL	282	22.883	97.840	72.749
938	CG2	VAL	282	20.417	97.614	72.775
939	N	HIS	283	22.263	94.596	75.752
940	CA	HIS	283	23.158	94.141	76.828
941	C	HIS	283	23.428	92.649	76.715
942	O	HIS	283	24.424	92.171	77.281
943	CB	HIS	283	22.547	94.562	78.173
944	CG	HIS	283	22.351	93.608	79.356
945	ND1	HIS	283	22.098	94.019	80.613
946	CD2	HIS	283	22.345	92.228	79.401
947	CE1	HIS	283	21.962	92.959	81.423
948	NE2	HIS	283	22.111	91.847	80.670
949	N	GLU	284	22.713	92.007	75.799
950	CA	GLU	284	22.609	90.538	75.733
951	C	GLU	284	23.910	89.776	75.906
952	O	GLU	284	24.125	89.123	76.936
953	CB	GLU	284	22.049	90.139	74.374
954	CG	GLU	284	20.549	90.383	74.286
955	CD	GLU	284	19.984	89.762	73.015
956	OE1	GLU	284	20.649	88.896	72.453
957	OE2	GLU	284	19.004	90.302	72.512
958	N	ILE	285	24.854	90.114	75.050
959	CA	ILE	285	26.078	89.336	74.875
960	C	ILE	285	27.088	89.462	76.018
961	O	ILE	285	27.896	88.541	76.194
962	CB	ILE	285	26.698	89.835	73.574
963	CG1	ILE	285	25.631	89.909	72.487
964	CG2	ILE	285	27.836	88.927	73.126
965	CD1	ILE	285	25.326	91.346	72.070
966	N	GLY	286	26.877	90.404	76.922
967	CA	GLY	286	27.794	90.603	78.048
968	C	GLY	286	27.983	89.357	78.915
969	O	GLY	286	29.116	88.876	79.066
970	N	HIS	287	26.893	88.727	79.319
971	CA	HIS	287	27.021	87.630	80.285
972	C	HIS	287	27.539	86.302	79.716
973	O	HIS	287	28.266	85.626	80.452
974	CB	HIS	287	25.692	87.446	81.001
975	CG	HIS	287	25.371	88.628	81.892
976	ND1	HIS	287	26.050	88.997	82.994
977	CD2	HIS	287	24.353	89.537	81.730
978	CE1	HIS	287	25.491	90.105	83.518
979	NE2	HIS	287	24.440	90.437	82.736
980	N	VAL	288	27.510	86.113	78.402
981	CA	VAL	288	28.062	84.868	77.823
982	C	VAL	288	29.572	84.982	77.617
983	O	VAL	288	30.270	83.996	77.354
984	CB	VAL	288	27.392	84.569	76.481
985	CG1	VAL	288	27.684	85.638	75.439
986	CG2	VAL	288	27.786	83.209	75.930
987	N	LEU	289	30.082	86.182	77.837
988	CA	LEU	289	31.506	86.423	77.712
989	C	LEU	289	32.225	86.201	79.042
990	O	LEU	289	33.460	86.222	79.085
991	CB	LEU	289	31.672	87.856	77.226
992	CG	LEU	289	30.950	88.065	75.896
993	CD1	LEU	289	30.907	89.539	75.509
994	CD2	LEU	289	31.552	87.222	74.775
995	N	GLY	290	31.462	85.940	80.095
996	CA	GLY	290	32.045	85.669	81.409
997	C	GLY	290	31.651	86.726	82.430
998	O	GLY	290	32.132	86.702	83.574
999	N	LEU	291	30.769	87.627	82.026
1000	CA	LEU	291	30.389	88.743	82.899
1001	C	LEU	291	29.498	88.305	84.046
1002	O	LEU	291	28.516	87.576	83.864
1003	CB	LEU	291	29.672	89.828	82.101
1004	CG	LEU	291	30.664	90.692	81.341
1005	CD1	LEU	291	29.963	91.853	80.647
1006	CD2	LEU	291	31.706	91.230	82.304
1007	N	PRO	292	29.826	88.809	85.220
1008	CA	PRO	292	28.911	88.765	86.344
1009	C	PRO	292	27.913	89.894	86.212
1010	O	PRO	292	27.958	90.704	85.278
1011	CB	PRO	292	29.767	89.029	87.535
1012	CG	PRO	292	30.999	89.763	87.039
1013	CD	PRO	292	30.984	89.646	85.522
1014	N	HIS	293	26.997	89.912	87.152
1015	CA	HIS	293	26.067	91.019	87.259
1016	C	HIS	293	26.838	92.246	87.735
1017	O	HIS	293	27.814	92.093	88.475
1018	CB	HIS	293	25.055	90.603	88.309
1019	CG	HIS	293	23.788	91.388	88.218
1020	ND1	HIS	293	23.258	91.883	87.084
1021	CD2	HIS	293	22.957	91.758	89.245
1022	CE1	HIS	293	22.112	92.508	87.371
1023	NE2	HIS	293	21.929	92.442	88.707
1024	N	THR	294	26.509	93.428	87.249
1025	CA	THR	294	27.192	94.600	87.799
1026	C	THR	294	26.397	95.066	89.004
1027	O	THR	294	25.244	94.665	89.175
1028	CB	THR	294	27.357	95.709	86.769
1029	OG1	THR	294	26.076	96.171	86.374
1030	CG2	THR	294	28.115	95.221	85.539
1031	N	TYR	295	27.073	95.722	89.926
1032	CA	TYR	295	26.403	96.165	91.148
1033	C	TYR	295	26.607	97.668	91.375
1034	O	TYR	295	26.108	98.246	92.348
1035	CB	TYR	295	26.893	95.326	92.328
1036	CG	TYR	295	26.365	93.886	92.476
1037	CD1	TYR	295	25.662	93.536	93.618
1038	CD2	TYR	295	26.616	92.921	91.506
1039	CE1	TYR	295	25.175	92.245	93.774
1040	CE2	TYR	295	26.135	91.626	91.661
1041	CZ	TYR	295	25.407	91.293	92.794
1042	OH	TYR	295	24.861	90.032	92.914
1043	N	ARG	296	27.375	98.283	90.491
1044	CA	ARG	296	27.478	99.749	90.450
1045	C	ARG	296	26.258	100.349	89.750
1046	O	ARG	296	26.140	100.212	88.525
1047	CB	ARG	296	28.752	100.106	89.704
1048	CG	ARG	296	29.912	100.292	90.670
1049	CD	ARG	296	29.645	101.518	91.533
1050	NE	ARG	296	30.715	101.758	92.509
1051	CZ	ARG	296	31.583	102.767	92.432
1052	NH1	ARG	296	32.358	103.050	93.478
1053	NH2	ARG	296	31.548	103.601	91.392
1054	N	THR	297	25.591	101.263	90.444
1055	CA	THR	297	24.194	101.683	90.138
1056	C	THR	297	23.883	102.447	88.835
1057	O	THR	297	22.702	102.698	88.569
1058	CB	THR	297	23.709	102.562	91.284
1059	OG1	THR	297	24.433	103.782	91.241
1060	CG2	THR	297	23.911	101.908	92.648
1061	N	GLY	298	24.861	102.683	87.980
1062	CA	GLY	298	24.614	103.386	86.717
1063	C	GLY	298	24.901	102.480	85.526
1064	O	GLY	298	24.684	102.847	84.368
1065	N	SER	299	25.463	101.325	85.835
1066	CA	SER	299	25.779	100.298	84.834
1067	C	SER	299	24.513	99.771	84.162
1068	O	SER	299	23.401	100.076	84.599
1069	CB	SER	299	26.544	99.201	85.566
1070	OG	SER	299	27.123	98.298	84.645
1071	N	ILE	300	24.717	99.157	83.008
1072	CA	ILE	300	23.662	98.624	82.130
1073	C	ILE	300	23.331	97.146	82.423
1074	O	ILE	300	22.449	96.594	81.756
1075	CB	ILE	300	24.151	98.883	80.694
1076	CG1	ILE	300	23.971	100.372	80.401
1077	CG2	ILE	300	23.388	98.125	79.599
1078	CD1	ILE	300	22.503	100.700	80.475
1079	N	MET	301	24.002	96.523	83.386
1080	CA	MET	301	23.636	95.144	83.763
1081	C	MET	301	23.159	95.067	85.231
1082	O	MET	301	23.920	94.527	86.045
1083	CB	MET	301	24.859	94.224	83.669
1084	CG	MET	301	25.916	94.670	82.654
1085	SD	MET	301	25.603	94.459	80.889
1086	CE	MET	301	26.043	92.719	80.675
1087	N	GLN	302	21.897	95.398	85.499
1088	CA	GLN	302	21.335	95.540	86.864
1089	C	GLN	302	19.780	95.571	86.942
1090	O	GLN	302	19.092	95.665	85.923
1091	CB	GLN	302	21.947	96.809	87.426
1092	CG	GLN	302	23.221	96.548	88.193
1093	CD	GLN	302	23.852	97.890	88.441
1094	OE1	GLN	302	24.379	98.180	89.521
1095	NE2	GLN	302	23.675	98.744	87.455
1096	N	PRO	303	19.237	95.419	88.148
1097	CA	PRO	303	17.795	95.154	88.360
1098	C	PRO	303	16.801	96.323	88.395
1099	O	PRO	303	15.689	96.100	88.886
1100	CB	PRO	303	17.724	94.579	89.725
1101	CG	PRO	303	18.986	94.898	90.470
1102	CD	PRO	303	19.979	95.244	89.396
1103	N	ASN	304	17.189	97.548	88.109
1104	CA	ASN	304	16.212	98.651	88.106
1105	C	ASN	304	16.073	99.308	86.727
1106	O	ASN	304	16.972	99.233	85.883
1107	CB	ASN	304	16.600	99.686	89.167
1108	CG	ASN	304	15.871	99.509	90.509
1109	OD1	ASN	304	15.844	100.451	91.311
1110	ND2	ASN	304	15.285	98.351	90.747
1111	N	TYR	305	14.914	99.902	86.494
1112	CA	TYR	305	14.583	100.466	85.177
1113	C	TYR	305	15.017	101.919	85.032
1114	O	TYR	305	15.006	102.665	86.011
1115	CB	TYR	305	13.065	100.364	85.021
1116	CG	TYR	305	12.475	100.726	83.651
1117	CD1	TYR	305	11.853	101.949	83.456
1118	CD2	TYR	305	12.559	99.803	82.603
1119	CE1	TYR	305	11.318	102.239	82.208
1120	CE2	TYR	305	12.035	100.106	81.352
1121	CZ	TYR	305	11.419	101.330	81.159
1122	OH	TYR	305	10.862	101.620	79.936
1123	N	ILE	306	15.514	102.260	83.851
1124	CA	ILE	306	15.818	103.654	83.480
1125	C	ILE	306	15.835	103.813	81.952
1126	O	ILE	306	16.570	103.097	81.262
1127	CB	ILE	306	17.155	104.062	84.100
1128	CG1	ILE	306	17.560	105.484	83.720
1129	CG2	ILE	306	18.257	103.071	83.754
1130	CD1	ILE	306	16.661	106.530	84.375
1131	N	PRO	307	14.968	104.678	81.444
1132	CA	PRO	307	14.881	104.923	80.002
1133	C	PRO	307	16.172	105.487	79.413
1134	O	PRO	307	16.823	106.361	79.993
1135	CB	PRO	307	13.747	105.885	79.833
1136	CG	PRO	307	13.190	106.249	81.195
1137	CD	PRO	307	13.998	105.463	82.210
1138	N	GLN	308	16.572	104.856	78.320
1139	CA	GLN	308	17.761	105.207	77.530
1140	C	GLN	308	17.871	104.225	76.365
1141	O	GLN	308	16.876	103.595	75.993
1142	CB	GLN	308	19.024	105.119	78.381
1143	CG	GLN	308	19.136	103.785	79.110
1144	CD	GLN	308	20.565	103.601	79.596
1145	OE1	GLN	308	21.467	103.362	78.787
1146	NE2	GLN	308	20.746	103.666	80.903
1147	N	GLU	309	19.016	104.194	75.712
1148	CA	GLU	309	19.273	103.106	74.766
1149	C	GLU	309	20.543	102.378	75.191
1150	O	GLU	309	21.655	102.857	74.942
1151	CB	GLU	309	19.381	103.637	73.339
1152	CG	GLU	309	18.059	104.202	72.809
1153	CD	GLU	309	16.956	103.142	72.696
1154	OE1	GLU	309	15.833	103.539	72.399
1155	OE2	GLU	309	17.277	101.962	72.718
1156	N	PRO	310	20.350	101.179	75.720
1157	CA	PRO	310	21.363	100.506	76.543
1158	C	PRO	310	22.671	100.273	75.800
1159	O	PRO	310	22.695	100.250	74.565
1160	CB	PRO	310	20.741	99.219	76.975
1161	CG	PRO	310	19.298	99.186	76.508
1162	CD	PRO	310	19.066	100.485	75.759
1163	N	ALA	311	23.750	100.323	76.567
1164	CA	ALA	311	25.135	100.213	76.067
1165	C	ALA	311	25.592	101.445	75.272
1166	O	ALA	311	26.625	101.415	74.593
1167	CB	ALA	311	25.325	98.931	75.261
1168	N	PHE	312	24.796	102.497	75.349
1169	CA	PHE	312	25.184	103.841	74.943
1170	C	PHE	312	24.699	104.711	76.093
1171	O	PHE	312	23.573	104.531	76.564
1172	CB	PHE	312	24.555	104.208	73.605
1173	CG	PHE	312	25.595	104.690	72.597
1174	CD1	PHE	312	25.203	105.425	71.484
1175	CD2	PHE	312	26.934	104.383	72.789
1176	CE1	PHE	312	26.163	105.859	70.570
1177	CE2	PHE	312	27.887	104.815	71.883
1178	CZ	PHE	312	27.506	105.553	70.775
1179	N	GLU	313	25.547	105.639	76.513
1180	CA	GLU	313	25.549	106.156	77.899
1181	C	GLU	313	26.098	105.007	78.740
1182	O	GLU	313	25.571	104.655	79.801
1183	CB	GLU	313	24.167	106.566	78.432
1184	CG	GLU	313	23.489	107.664	77.619
1185	CD	GLU	313	24.297	108.952	77.696
1186	OE1	GLU	313	25.129	109.150	76.819
1187	OE2	GLU	313	24.112	109.685	78.657
1188	N	LEU	314	27.162	104.420	78.211
1189	CA	LEU	314	27.739	103.197	78.765
1190	C	LEU	314	28.565	103.541	79.996
1191	O	LEU	314	29.507	104.342	79.945
1192	CB	LEU	314	28.579	102.557	77.656
1193	CG	LEU	314	28.842	101.060	77.840
1194	CD1	LEU	314	29.199	100.397	76.513
1195	CD2	LEU	314	29.916	100.767	78.879
1196	N	ASP	315	28.108	103.019	81.117
1197	CA	ASP	315	28.766	103.246	82.399
1198	C	ASP	315	30.034	102.400	82.530
1199	O	ASP	315	30.097	101.255	82.056
1200	CB	ASP	315	27.734	102.892	83.457
1201	CG	ASP	315	28.193	103.229	84.867
1202	OD1	ASP	315	28.799	102.357	85.486
1203	OD2	ASP	315	27.920	104.329	85.320
1204	N	TRP	316	30.985	102.924	83.289
1205	CA	TRP	316	32.304	102.300	83.440
1206	C	TRP	316	32.307	100.924	84.116
1207	O	TRP	316	33.140	100.100	83.725
1208	CB	TRP	316	33.211	103.250	84.226
1209	CG	TRP	316	33.036	103.336	85.732
1210	CD1	TRP	316	31.885	103.570	86.452
1211	CD2	TRP	316	34.101	103.202	86.696
1212	NE1	TRP	316	32.193	103.556	87.773
1213	CE2	TRP	316	33.510	103.342	87.961
1214	CE3	TRP	316	35.464	102.974	86.579
1215	CZ2	TRP	316	34.295	103.250	89.100
1216	CZ3	TRP	316	36.245	102.887	87.724
1217	CH2	TRP	316	35.662	103.023	88.979
1218	N	SER	317	31.253	100.567	84.836
1219	CA	SER	317	31.191	99.238	85.451
1220	C	SER	317	30.895	98.126	84.441
1221	O	SER	317	31.387	97.005	84.618
1222	CB	SER	317	30.098	99.263	86.509
1223	OG	SER	317	29.968	97.951	87.040
1224	N	ASP	318	30.314	98.475	83.300
1225	CA	ASP	318	30.083	97.469	82.259
1226	C	ASP	318	31.386	97.189	81.548
1227	O	ASP	318	31.882	96.053	81.523
1228	CB	ASP	318	29.158	98.013	81.175
1229	CG	ASP	318	27.791	98.391	81.720
1230	OD1	ASP	318	27.014	97.486	81.966
1231	OD2	ASP	318	27.573	99.561	81.995
1232	N	ARG	319	32.054	98.280	81.219
1233	CA	ARG	319	33.183	98.181	80.315
1234	C	ARG	319	34.448	97.720	81.015
1235	O	ARG	319	35.096	96.812	80.488
1236	CB	ARG	319	33.401	99.530	79.668
1237	CG	ARG	319	34.282	99.338	78.453
1238	CD	ARG	319	34.531	100.655	77.752
1239	NE	ARG	319	33.261	101.278	77.373
1240	CZ	ARG	319	33.204	102.587	77.166
1241	NH1	ARG	319	34.298	103.315	77.361
1242	NH2	ARG	319	32.060	103.171	76.805
1243	N	LYS	320	34.571	98.031	82.293
1244	CA	LYS	320	35.735	97.571	83.052
1245	C	LYS	320	35.662	96.071	83.335
1246	O	LYS	320	36.681	95.383	83.199
1247	CB	LYS	320	35.773	98.348	84.361
1248	CG	LYS	320	36.916	97.905	85.263
1249	CD	LYS	320	36.917	98.718	86.550
1250	CE	LYS	320	35.568	98.622	87.251
1251	NZ	LYS	320	35.557	99.430	88.479
1252	N	ALA	321	34.455	95.536	83.411
1253	CA	ALA	321	34.318	94.101	83.642
1254	C	ALA	321	34.474	93.290	82.352
1255	O	ALA	321	35.174	92.270	82.367
1256	CB	ALA	321	32.959	93.845	84.282
1257	N	ILE	322	34.073	93.848	81.217
1258	CA	ILE	322	34.234	93.099	79.959
1259	C	ILE	322	35.652	93.245	79.387
1260	O	ILE	322	36.194	92.270	78.842
1261	CB	ILE	322	33.150	93.515	78.958
1262	CG1	ILE	322	33.235	92.702	77.673
1263	CG2	ILE	322	33.192	95.002	78.630
1264	CD1	ILE	322	33.049	91.214	77.944
1265	N	GLN	323	36.343	94.297	79.800
1266	CA	GLN	323	37.751	94.468	79.443
1267	C	GLN	323	38.669	93.742	80.421
1268	O	GLN	323	39.845	93.515	80.115
1269	CB	GLN	323	38.074	95.954	79.429
1270	CG	GLN	323	37.274	96.658	78.342
1271	CD	GLN	323	37.541	98.157	78.377
1272	OE1	GLN	323	37.702	98.755	79.449
1273	NE2	GLN	323	37.488	98.762	77.204
1274	N	LYS	324	38.098	93.251	81.509
1275	CA	LYS	324	38.838	92.394	82.432
1276	C	LYS	324	38.782	90.941	81.962
1277	O	LYS	324	39.670	90.141	82.282
1278	CB	LYS	324	38.186	92.543	83.802
1279	CG	LYS	324	38.970	91.858	84.911
1280	CD	LYS	324	38.332	92.156	86.260
1281	CE	LYS	324	38.226	93.661	86.491
1282	NZ	LYS	324	37.604	93.961	87.792
1283	N	LEU	325	37.838	90.657	81.076
1284	CA	LEU	325	37.749	89.330	80.467
1285	C	LEU	325	38.660	89.197	79.254
1286	O	LEU	325	39.629	88.431	79.286
1287	CB	LEU	325	36.315	89.089	80.027
1288	CG	LEU	325	35.390	88.884	81.216
1289	CD1	LEU	325	33.943	88.992	80.773
1290	CD2	LEU	325	35.658	87.541	81.886
1291	N	TYR	326	38.353	89.935	78.199
1292	CA	TYR	326	39.109	89.764	76.950
1293	C	TYR	326	40.023	90.938	76.619
1294	O	TYR	326	40.552	91.002	75.504
1295	CB	TYR	326	38.148	89.568	75.786
1296	CG	TYR	326	37.137	88.442	75.962
1297	CD1	TYR	326	37.558	87.122	76.072
1298	CD2	TYR	326	35.785	88.749	76.012
1299	CE1	TYR	326	36.623	86.107	76.228
1300	CE2	TYR	326	34.852	87.737	76.167
1301	CZ	TYR	326	35.272	86.419	76.273
1302	OH	TYR	326	34.336	85.421	76.426
1303	N	GLY	327	40.157	91.883	77.532
1304	CA	GLY	327	41.002	93.048	77.261
1305	C	GLY	327	40.217	94.229	76.697
1306	O	GLY	327	39.082	94.100	76.218
1307	N	SER	328	40.821	95.394	76.844
1308	CA	SER	328	40.293	96.634	76.268
1309	C	SER	328	40.692	96.743	74.804
1310	O	SER	328	41.735	96.184	74.449
1311	CB	SER	328	40.919	97.797	77.038
1312	OG	SER	328	40.478	99.022	76.462
1313	N	CYS	329	39.770	97.242	73.982
1314	CA	CYS	329	40.009	97.698	72.584
1315	C	CYS	329	38.942	97.288	71.581
1316	O	CYS	329	37.904	96.696	71.898
1317	CB	CYS	329	41.360	97.333	71.967
1318	SG	CYS	329	42.744	98.423	72.379
1319	N	GLU	330	39.368	97.517	70.348
1320	CA	GLU	330	38.622	97.415	69.080
1321	C	GLU	330	38.276	98.844	68.701
1322	O	GLU	330	37.113	99.259	68.650
1323	CB	GLU	330	37.376	96.547	69.121
1324	CG	GLU	330	36.937	96.178	67.708
1325	CD	GLU	330	37.957	95.234	67.092
1326	OE1	GLU	330	38.045	94.110	67.569
1327	OE2	GLU	330	38.788	95.727	66.340

TABLE VI

Atom	Atom		Residue
No	Name	Residue	No	X coord	Y coord	Z coord
1	N	GLN	346	31.981	116.323	91.895
2	CA	GLN	346	31.747	117.708	91.457
3	C	GLN	346	30.383	117.854	90.782
4	O	GLN	346	30.162	117.329	89.685
5	CB	GLN	346	32.844	118.087	90.473
6	CG	GLN	346	34.223	117.902	91.094
7	CD	GLN	346	35.302	118.254	90.079
8	OE1	GLN	346	36.464	118.484	90.439
9	NE2	GLN	346	34.896	118.314	88.822
10	N	TYR	347	29.502	118.606	91.419
11	CA	TYR	347	28.129	118.765	90.913
12	C	TYR	347	27.909	120.137	90.283
13	O	TYR	347	28.820	120.975	90.248
14	CB	TYR	347	27.144	118.558	92.061
15	CG	TYR	347	27.180	117.164	92.686
16	CD1	TYR	347	28.046	116.889	93.737
17	CD2	TYR	347	26.340	116.169	92.208
18	CE1	TYR	347	28.081	115.619	94.290
19	CE2	TYR	347	26.372	114.897	92.764
20	CZ	TYR	347	27.244	114.625	93.809
21	OH	TYR	347	27.246	113.388	94.414
22	N	GLY	348	26.711	120.337	89.759
23	CA	GLY	348	26.358	121.630	89.158
24	C	GLY	348	25.055	122.192	89.721
25	O	GLY	348	24.944	123.398	89.984
26	N	GLU	349	24.133	121.300	90.029
27	CA	GLU	349	22.818	121.717	90.533
28	C	GLU	349	22.418	121.009	91.834
29	O	GLU	349	22.411	119.774	91.881
30	CB	GLU	349	21.833	121.413	89.414
31	CG	GLU	349	22.258	120.174	88.636
32	CD	GLU	349	21.193	119.714	87.646
33	OE1	GLU	349	20.114	119.403	88.141
34	OE2	GLU	349	21.606	119.251	86.591
35	N	VAL	350	22.152	121.784	92.884
36	CA	VAL	350	21.776	121.222	94.204
37	C	VAL	350	20.769	122.099	94.969
38	O	VAL	350	21.187	122.972	95.741
39	CB	VAL	350	23.014	121.072	95.100
40	CG1	VAL	350	22.648	120.486	96.462
41	CG2	VAL	350	24.104	120.218	94.468
42	N	MET	351	19.479	121.840	94.779
43	CA	MET	351	18.390	122.507	95.543
44	C	MET	351	17.046	121.789	95.389
45	O	MET	351	16.668	121.470	94.258
46	CB	MET	351	18.151	123.941	95.056
47	CG	MET	351	19.049	124.989	95.703
48	SD	MET	351	18.619	126.705	95.346
49	CE	MET	351	17.090	126.824	96.301
50	N	VAL	352	16.344	121.556	96.496
51	CA	VAL	352	14.927	121.106	96.462
52	C	VAL	352	14.159	121.521	97.722
53	O	VAL	352	14.591	122.390	98.484
54	CB	VAL	352	14.730	119.603	96.237
55	CG1	VAL	352	14.806	119.177	94.777
56	CG2	VAL	352	15.609	118.740	97.115
57	N	ARG	353	12.987	120.920	97.887
58	CA	ARG	353	12.070	121.237	98.996
59	C	ARG	353	12.492	120.590	100.314
60	O	ARG	353	12.086	119.452	100.585
61	CB	ARG	353	10.705	120.674	98.618
62	CG	ARG	353	10.351	121.062	97.190
63	CD	ARG	353	9.004	120.515	96.737
64	NE	ARG	353	8.733	120.973	95.366
65	CZ	ARG	353	7.782	120.460	94.582
66	NH1	ARG	353	7.036	119.439	95.010
67	NH2	ARG	353	7.603	120.950	93.355
68	N	PHE	354	13.301	121.307	101.094
69	CA	PHE	354	13.872	120.866	102.401
70	C	PHE	354	14.986	119.816	102.274
71	O	PHE	354	16.010	119.885	102.965
72	CB	PHE	354	12.786	120.299	103.316
73	CG	PHE	354	11.785	121.300	103.884
74	CD1	PHE	354	12.223	122.333	104.702
75	CD2	PHE	354	10.431	121.161	103.605
76	CE1	PHE	354	11.309	123.234	105.233
77	CE2	PHE	354	9.517	122.063	104.134
78	CZ	PHE	354	9.956	123.099	104.948
79	N	SER	355	14.742	118.824	101.439
80	CA	SER	355	15.708	117.796	101.077
81	C	SER	355	16.856	118.415	100.281
82	O	SER	355	16.733	119.515	99.730
83	CB	SER	355	14.930	116.790	100.229
84	OG	SER	355	15.774	115.717	99.857
85	N	THR	356	17.988	117.739	100.275
86	CA	THR	356	19.141	118.217	99.516
87	C	THR	356	19.408	117.298	98.327
88	O	THR	356	19.884	116.173	98.499
89	CB	THR	356	20.339	118.214	100.452
90	OG1	THR	356	19.912	118.729	101.706
91	CG2	THR	356	21.476	119.073	99.917
92	N	TYR	357	19.064	117.763	97.138
93	CA	TYR	357	19.280	116.949	95.928
94	C	TYR	357	20.508	117.387	95.146
95	O	TYR	357	20.530	118.480	94.567
96	CB	TYR	357	18.085	117.034	94.987
97	CG	TYR	357	16.895	116.124	95.283
98	CD1	TYR	357	16.818	115.405	96.469
99	CD2	TYR	357	15.888	116.002	94.333
100	CE1	TYR	357	15.714	114.602	96.721
101	CE2	TYR	357	14.786	115.201	94.583
102	CZ	TYR	357	14.697	114.509	95.783
103	OH	TYR	357	13.575	113.763	96.062
104	N	PHE	358	21.469	116.486	95.058
105	CA	PHE	358	22.681	116.717	94.268
106	C	PHE	358	22.531	116.094	92.877
107	O	PHE	358	22.048	114.963	92.733
108	CB	PHE	358	23.872	116.093	94.994
109	CG	PHE	358	24.160	116.632	96.396
110	CD1	PHE	358	23.619	116.012	97.514
111	CD2	PHE	358	24.999	117.728	96.552
112	CE1	PHE	358	23.879	116.510	98.782
113	CE2	PHE	358	25.259	118.229	97.822
114	CZ	PHE	358	24.694	117.622	98.935
115	N	PHE	359	22.808	116.894	91.859
116	CA	PHE	359	22.753	116.402	90.472
117	C	PHE	359	24.025	116.701	89.670
118	O	PHE	359	24.591	117.808	89.720
119	CB	PHE	359	21.605	117.064	89.723
120	CG	PHE	359	20.155	116.709	90.043
121	CD1	PHE	359	19.373	116.136	89.050
122	CD2	PHE	359	19.595	117.010	91.277
123	CE1	PHE	359	18.045	115.829	89.301
124	CE2	PHE	359	18.267	116.696	91.532
125	CZ	PHE	359	17.494	116.103	90.543
126	N	ARG	360	24.388	115.720	88.856
127	CA	ARG	360	25.512	115.840	87.909
128	C	ARG	360	25.422	114.720	86.875
129	O	ARG	360	24.911	113.644	87.197
130	CB	ARG	360	26.854	115.825	88.650
131	CG	ARG	360	27.214	114.502	89.325
132	CD	ARG	360	28.130	113.651	88.454
133	NE	ARG	360	29.245	114.472	87.961
134	CZ	ARG	360	30.506	114.043	87.885
135	NH1	ARG	360	30.812	112.800	88.262
136	NH2	ARG	360	31.458	114.856	87.424
137	N	ASN	361	26.068	114.903	85.732
138	CA	ASN	361	25.926	113.979	84.585
139	C	ASN	361	24.483	113.522	84.416
140	O	ASN	361	23.569	114.347	84.296
141	CB	ASN	361	26.798	112.738	84.776
142	CG	ASN	361	28.287	113.047	84.656
143	OD1	ASN	361	28.688	114.180	84.364
144	ND2	ASN	361	29.088	112.013	84.838
145	N	SER	362	24.301	112.211	84.421
146	CA	SER	362	22.971	111.595	84.302
147	C	SER	362	22.400	111.132	85.650
148	O	SER	362	21.483	110.302	85.662
149	CB	SER	362	23.068	110.393	83.367
150	OG	SER	362	23.913	109.423	83.976
151	N	TRP	363	22.957	111.625	86.746
152	CA	TRP	363	22.572	111.208	88.104
153	C	TRP	363	21.635	112.190	88.812
154	O	TRP	363	21.883	113.404	88.830
155	CB	TRP	363	23.858	111.118	88.913
156	CG	TRP	363	24.836	110.088	88.397
157	CD1	TRP	363	26.080	110.300	87.844
158	CD2	TRP	363	24.624	108.665	88.404
159	NE1	TRP	363	26.624	109.094	87.538
160	CE2	TRP	363	25.789	108.090	87.870
161	CE3	TRP	363	23.578	107.865	88.839
162	CZ2	TRP	363	25.897	106.712	87.797
163	CZ3	TRP	363	23.694	106.486	88.758
164	CH2	TRP	363	24.851	105.914	88.243
165	N	TYR	364	20.603	111.646	89.445
166	CA	TYR	364	19.666	112.472	90.223
167	C	TYR	364	19.674	112.126	91.705
168	O	TYR	364	20.129	111.042	92.108
169	CB	TYR	364	18.233	112.388	89.675
170	CG	TYR	364	17.304	111.181	89.913
171	CD1	TYR	364	17.662	110.072	90.670
172	CD2	TYR	364	16.033	111.236	89.353
173	CE1	TYR	364	16.763	109.029	90.849
174	CE2	TYR	364	15.136	110.192	89.523
175	CZ	TYR	364	15.505	109.087	90.270
176	OH	TYR	364	14.646	108.017	90.380
177	N	TRP	365	19.185	113.087	92.483
178	CA	TRP	365	18.942	112.983	93.942
179	C	TRP	365	20.200	113.021	94.806
180	O	TRP	365	20.331	113.906	95.661
181	CB	TRP	365	18.202	111.692	94.290
182	CG	TRP	365	16.703	111.633	94.048
183	CD1	TRP	365	15.980	112.090	92.972
184	CD2	TRP	365	15.749	111.046	94.958
185	NE1	TRP	365	14.674	111.798	93.172
186	CE2	TRP	365	14.492	111.173	94.349
187	CE3	TRP	365	15.869	110.429	96.196
188	CZ2	TRP	365	13.360	110.683	94.991
189	CZ3	TRP	365	14.732	109.947	96.831
190	CH2	TRP	365	13.485	110.071	96.232
191	N	LEU	366	21.099	112.085	94.543
192	CA	LEU	366	22.322	111.842	95.310
193	C	LEU	366	22.131	112.083	96.789
194	O	LEU	366	22.487	113.153	97.296
195	CB	LEU	366	23.492	112.668	94.789
196	CG	LEU	366	24.229	111.949	93.666
197	CD1	LEU	366	24.259	110.459	93.969
198	CD2	LEU	366	23.610	112.188	92.293
199	N	TYR	367	21.674	111.033	97.456
200	CA	TYR	367	21.398	111.023	98.904
201	C	TYR	367	20.799	112.335	99.402
202	O	TYR	367	21.518	113.211	99.894
203	CB	TYR	367	22.653	110.623	99.688
204	CG	TYR	367	24.022	111.048	99.141
205	CD1	TYR	367	24.772	110.152	98.386
206	CD2	TYR	367	24.532	112.312	99.417
207	CE1	TYR	367	26.016	110.524	97.892
208	CE2	TYR	367	25.775	112.684	98.924
209	CZ	TYR	367	26.515	111.790	98.163
210	OH	TYR	367	27.760	112.158	97.696
211	N	GLU	368	19.484	112.283	99.564
212	CA	GLU	368	18.651	113.475	99.787
213	C	GLU	368	18.725	114.134	101.178
214	O	GLU	368	17.993	115.104	101.408
215	CB	GLU	368	17.213	113.085	99.416
216	CG	GLU	368	16.695	111.779	100.025
217	CD	GLU	368	16.389	111.958	101.514
218	OE1	GLU	368	17.251	111.487	102.241
219	OE2	GLU	368	15.741	112.953	101.768
220	N	ASN	369	19.531	113.594	102.086
221	CA	ASN	369	19.811	114.158	103.429
222	C	ASN	369	18.678	114.063	104.480
223	O	ASN	369	18.963	113.757	105.647
224	CB	ASN	369	20.271	115.600	103.205
225	CG	ASN	369	20.455	116.364	104.504
226	OD1	ASN	369	19.694	117.294	104.793
227	ND2	ASN	369	21.490	116.009	105.245
228	N	ARG	370	17.428	114.255	104.089
229	CA	ARG	370	16.291	114.087	105.004
230	C	ARG	370	16.038	112.626	105.370
231	O	ARG	370	15.539	111.845	104.551
232	CB	ARG	370	15.037	114.636	104.331
233	CG	ARG	370	14.908	116.143	104.485
234	CD	ARG	370	14.743	116.486	105.960
235	NE	ARG	370	14.373	117.893	106.163
236	CZ	ARG	370	13.209	118.250	106.711
237	NH1	ARG	370	12.288	117.323	106.982
238	NH2	ARG	370	12.936	119.541	106.918
239	N	ASN	371	16.355	112.309	106.617
240	CA	ASN	371	16.098	110.992	107.236
241	C	ASN	371	16.408	109.818	106.314
242	O	ASN	371	15.495	109.166	105.790
243	CB	ASN	371	14.632	110.938	107.656
244	CG	ASN	371	14.356	111.970	108.747
245	OD1	ASN	371	14.061	113.138	108.469
246	ND2	ASN	371	14.443	111.513	109.984
247	N	ASN	372	17.686	109.541	106.135
248	CA	ASN	372	18.067	108.499	105.184
249	C	ASN	372	18.982	107.444	105.807
250	O	ASN	372	19.951	107.763	106.505
251	CB	ASN	372	18.758	109.188	104.017
252	CG	ASN	372	18.877	108.224	102.848
253	OD1	ASN	372	18.180	107.203	102.795
254	ND2	ASN	372	19.821	108.506	101.972
255	N	ARG	373	18.655	106.190	105.534
256	CA	ARG	373	19.463	105.056	105.995
257	C	ARG	373	20.370	104.514	104.888
258	O	ARG	373	21.339	103.803	105.176
259	CB	ARG	373	18.533	103.931	106.446
260	CG	ARG	373	17.696	104.328	107.653
261	CD	ARG	373	18.582	104.684	108.843
262	NE	ARG	373	17.764	105.092	109.994
263	CZ	ARG	373	17.530	106.369	110.303
264	NH1	ARG	373	16.777	106.671	111.362
265	NH2	ARG	373	18.056	107.344	109.558
266	N	THR	374	20.063	104.838	103.640
267	CA	THR	374	20.840	104.292	102.514
268	C	THR	374	20.925	105.301	101.373
269	O	THR	374	19.892	105.737	100.857
270	CB	THR	374	20.134	103.031	102.023
271	OG1	THR	374	20.004	102.138	103.122
272	CG2	THR	374	20.904	102.317	100.916
273	N	ARG	375	22.131	105.684	100.989
274	CA	ARG	375	22.278	106.695	99.931
275	C	ARG	375	21.678	106.251	98.595
276	O	ARG	375	21.873	105.127	98.118
277	CB	ARG	375	23.745	107.069	99.766
278	CG	ARG	375	24.646	105.861	99.561
279	CD	ARG	375	26.075	106.300	99.259
280	NE	ARG	375	26.582	107.215	100.296
281	CZ	ARG	375	27.797	107.107	100.839
282	NH1	ARG	375	28.205	108.002	101.741
283	NH2	ARG	375	28.614	106.123	100.458
284	N	TYR	376	20.873	107.148	98.052
285	CA	TYR	376	20.185	106.904	96.777
286	C	TYR	376	20.949	107.581	95.641
287	O	TYR	376	21.250	108.776	95.730
288	CB	TYR	376	18.776	107.492	96.869
289	CG	TYR	376	17.966	107.036	98.086
290	CD1	TYR	376	17.832	105.684	98.373
291	CD2	TYR	376	17.346	107.980	98.896
292	CE1	TYR	376	17.109	105.276	99.486
293	CE2	TYR	376	16.620	107.574	100.008
294	CZ	TYR	376	16.513	106.222	100.306
295	OH	TYR	376	15.925	105.825	101.487
296	N	GLY	377	21.194	106.855	94.562
297	CA	GLY	377	21.969	107.417	93.443
298	C	GLY	377	21.582	106.774	92.115
299	O	GLY	377	22.265	105.871	91.616
300	N	ASP	378	20.504	107.275	91.534
301	CA	ASP	378	19.942	106.655	90.327
302	C	ASP	378	19.990	107.645	89.158
303	O	ASP	378	20.162	108.845	89.389
304	CB	ASP	378	18.481	106.275	90.595
305	CG	ASP	378	18.203	105.814	92.032
306	OD1	ASP	378	18.975	105.029	92.570
307	OD2	ASP	378	17.233	106.310	92.592
308	N	PRO	379	19.973	107.151	87.931
309	CA	PRO	379	19.783	108.031	86.765
310	C	PRO	379	18.486	108.851	86.842
311	O	PRO	379	17.487	108.410	87.426
312	CB	PRO	379	19.835	107.126	85.576
313	CG	PRO	379	20.042	105.692	86.042
314	CD	PRO	379	20.066	105.737	87.560
315	N	ILE	380	18.485	109.998	86.174
316	CA	ILE	380	17.442	111.026	86.392
317	C	ILE	380	16.060	110.799	85.762
318	O	ILE	380	15.104	111.465	86.169
319	CB	ILE	380	17.937	112.368	85.855
320	CG1	ILE	380	19.414	112.583	86.109
321	CG2	ILE	380	17.179	113.510	86.519
322	CD1	ILE	380	19.878	113.937	85.584
323	N	GLN	381	15.895	109.837	84.876
324	CA	GLN	381	14.610	109.793	84.166
325	C	GLN	381	13.547	108.878	84.788
326	O	GLN	381	12.485	108.680	84.187
327	CB	GLN	381	14.850	109.524	82.686
328	CG	GLN	381	15.641	110.703	82.115
329	CD	GLN	381	15.660	110.704	80.590
330	OE1	GLN	381	15.567	109.650	79.951
331	NE2	GLN	381	15.775	111.896	80.026
332	N	ILE	382	13.799	108.352	85.977
333	CA	ILE	382	12.732	107.644	86.696
334	C	ILE	382	11.876	108.673	87.443
335	O	ILE	382	12.408	109.496	88.194
336	CB	ILE	382	13.357	106.596	87.615
337	CG1	ILE	382	13.990	105.500	86.768
338	CG2	ILE	382	12.340	105.990	88.578
339	CD1	ILE	382	12.960	104.866	85.837
340	N	LEU	383	10.574	108.629	87.172
341	CA	LEU	383	9.557	109.617	87.616
342	C	LEU	383	9.447	109.931	89.115
343	O	LEU	383	8.452	109.559	89.745
344	CB	LEU	383	8.192	109.061	87.218
345	CG	LEU	383	8.021	108.889	85.716
346	CD1	LEU	383	6.834	107.981	85.406
347	CD2	LEU	383	7.872	110.236	85.021
348	N	THR	384	10.344	110.756	89.629
349	CA	THR	384	10.218	111.231	91.012
350	C	THR	384	9.736	112.679	91.090
351	O	THR	384	9.710	113.266	92.175
352	CB	THR	384	11.545	111.077	91.746
353	OG1	THR	384	12.528	111.918	91.153
354	CG2	THR	384	12.033	109.634	91.700
355	N	GLY	385	9.420	113.264	89.948
356	CA	GLY	385	8.862	114.618	89.952
357	C	GLY	385	8.985	115.332	88.612
358	O	GLY	385	8.322	116.355	88.383
359	N	TRP	386	9.848	114.810	87.755
360	CA	TRP	386	10.132	115.486	86.482
361	C	TRP	386	9.988	114.539	85.299
362	O	TRP	386	10.983	113.959	84.847
363	CB	TRP	386	11.576	115.981	86.508
364	CG	TRP	386	12.064	116.393	87.880
365	CD1	TRP	386	11.738	117.536	88.574
366	CD2	TRP	386	12.969	115.648	88.723
367	NE1	TRP	386	12.360	117.498	89.777
368	CE2	TRP	386	13.090	116.379	89.916
369	CE3	TRP	386	13.629	114.439	88.573
370	CZ2	TRP	386	13.848	115.877	90.961
371	CZ3	TRP	386	14.398	113.951	89.620
372	CH2	TRP	386	14.503	114.663	90.810
373	N	PRO	387	8.792	114.483	84.728
374	CA	PRO	387	8.511	113.566	83.613
375	C	PRO	387	9.115	113.989	82.265
376	O	PRO	387	9.055	113.219	81.301
377	CB	PRO	387	7.016	113.532	83.519
378	CG	PRO	387	6.428	114.603	84.424
379	CD	PRO	387	7.607	115.240	85.140
380	N	GLY	388	9.713	115.167	82.208
381	CA	GLY	388	10.375	115.624	80.986
382	C	GLY	388	11.661	116.373	81.325
383	O	GLY	388	11.967	117.398	80.707
384	N	ILE	389	12.393	115.843	82.292
385	CA	ILE	389	13.668	116.425	82.736
386	C	ILE	389	14.658	116.458	81.560
387	O	ILE	389	14.612	115.580	80.689
388	CB	ILE	389	14.131	115.537	83.910
389	CG1	ILE	389	15.265	116.108	84.768
390	CG2	ILE	389	14.491	114.136	83.427
391	CD1	ILE	389	16.661	115.865	84.198
392	N	PRO	390	15.466	117.510	81.489
393	CA	PRO	390	16.485	117.642	80.440
394	C	PRO	390	17.381	116.415	80.305
395	O	PRO	390	17.445	115.540	81.180
396	CB	PRO	390	17.300	118.840	80.822
397	CG	PRO	390	16.685	119.498	82.045
398	CD	PRO	390	15.476	118.655	82.409
399	N	THR	391	18.005	116.318	79.146
400	CA	THR	391	18.955	115.237	78.908
401	C	THR	391	20.305	115.558	79.547
402	O	THR	391	21.166	116.216	78.948
403	CB	THR	391	19.068	115.000	77.404
404	OG1	THR	391	20.217	114.196	77.170
405	CG2	THR	391	19.229	116.299	76.618
406	N	HIS	392	20.442	115.072	80.776
407	CA	HIS	392	21.645	115.183	81.620
408	C	HIS	392	22.434	116.491	81.497
409	O	HIS	392	21.872	117.546	81.174
410	CB	HIS	392	22.547	113.963	81.380
411	CG	HIS	392	22.739	113.479	79.951
412	ND1	HIS	392	22.165	112.396	79.391
413	CD2	HIS	392	23.552	114.037	78.994
414	CE1	HIS	392	22.571	112.289	78.110
415	NE2	HIS	392	23.429	113.304	77.865
416	N	ASN	393	23.613	116.449	82.097
417	CA	ASN	393	24.643	117.500	81.979
418	C	ASN	393	24.461	118.687	82.916
419	O	ASN	393	23.983	119.742	82.476
420	CB	ASN	393	24.742	118.047	80.559
421	CG	ASN	393	25.250	117.004	79.577
422	OD1	ASN	393	26.029	116.111	79.931
423	ND2	ASN	393	24.763	117.118	78.354
424	N	ILE	394	24.712	118.416	84.192
425	CA	ILE	394	25.113	119.335	85.301
426	C	ILE	394	24.505	120.744	85.557
427	O	ILE	394	24.471	121.131	86.731
428	CB	ILE	394	26.622	119.486	85.061
429	CG1	ILE	394	27.283	118.113	85.033
430	CG2	ILE	394	27.315	120.363	86.094
431	CD1	ILE	394	28.780	118.204	84.763
432	N	ASP	395	23.927	121.427	84.584
433	CA	ASP	395	23.584	122.855	84.710
434	C	ASP	395	22.489	123.170	85.720
435	O	ASP	395	21.770	122.287	86.212
436	CB	ASP	395	23.166	123.409	83.356
437	CG	ASP	395	24.286	123.313	82.329
438	OD1	ASP	395	23.953	123.369	81.154
439	OD2	ASP	395	25.448	123.341	82.720
440	N	ALA	396	22.377	124.469	85.961
441	CA	ALA	396	21.535	125.084	86.999
442	C	ALA	396	20.181	124.439	87.277
443	O	ALA	396	19.530	123.829	86.418
444	CB	ALA	396	21.324	126.546	86.624
445	N	PHE	397	19.789	124.583	88.529
446	CA	PHE	397	18.529	124.034	89.026
447	C	PHE	397	18.071	124.851	90.228
448	O	PHE	397	18.307	124.473	91.383
449	CB	PHE	397	18.773	122.585	89.421
450	CG	PHE	397	17.592	121.786	89.958
451	CD1	PHE	397	17.826	120.727	90.824
452	CD2	PHE	397	16.293	122.101	89.579
453	CE1	PHE	397	16.764	119.983	91.315
454	CE2	PHE	397	15.230	121.358	90.073
455	CZ	PHE	397	15.466	120.298	90.940
456	N	VAL	398	17.440	125.972	89.934
457	CA	VAL	398	16.972	126.874	90.986
458	C	VAL	398	15.641	126.388	91.542
459	O	VAL	398	14.813	125.813	90.825
460	CB	VAL	398	16.879	128.296	90.423
461	CG1	VAL	398	16.171	128.355	89.082
462	CG2	VAL	398	16.270	129.292	91.401
463	N	HIS	399	15.520	126.455	92.854
464	CA	HIS	399	14.263	126.075	93.474
465	C	HIS	399	13.551	127.237	94.145
466	O	HIS	399	14.066	127.860	95.080
467	CB	HIS	399	14.540	125.001	94.500
468	CG	HIS	399	13.820	123.724	94.169
469	ND1	HIS	399	14.161	122.848	93.213
470	CD2	HIS	399	12.686	123.248	94.771
471	CE1	HIS	399	13.282	121.827	93.221
472	NE2	HIS	399	12.370	122.075	94.188
473	N	ILE	400	12.323	127.448	93.711
474	CA	ILE	400	11.423	128.387	94.380
475	C	ILE	400	10.290	127.552	94.971
476	O	ILE	400	9.094	127.767	94.732
477	CB	ILE	400	10.902	129.424	93.389
478	CG1	ILE	400	12.048	129.995	92.560
479	CG2	ILE	400	10.212	130.561	94.131
480	CD1	ILE	400	11.559	131.091	91.622
481	N	TRP	401	10.707	126.625	95.818
482	CA	TRP	401	9.825	125.602	96.387
483	C	TRP	401	8.982	126.075	97.552
484	O	TRP	401	8.079	125.341	97.965
485	CB	TRP	401	10.683	124.455	96.899
486	CG	TRP	401	11.727	124.819	97.943
487	CD1	TRP	401	13.031	125.202	97.708
488	CD2	TRP	401	11.559	124.811	99.380
489	NE1	TRP	401	13.635	125.439	98.899
490	CE2	TRP	401	12.788	125.223	99.923
491	CE3	TRP	401	10.493	124.508	100.210
492	CZ2	TRP	401	12.928	125.341	101.298
493	CZ3	TRP	401	10.642	124.624	101.587
494	CH2	TRP	401	11.855	125.041	102.127
495	N	THR	402	9.208	127.305	97.993
496	CA	THR	402	8.504	127.887	99.140
497	C	THR	402	7.013	127.618	99.087
498	O	THR	402	6.426	127.540	98.001
499	CB	THR	402	8.718	129.392	99.111
500	OG1	THR	402	8.113	129.890	97.922
501	CG2	THR	402	10.200	129.745	99.088
502	N	TRP	403	6.397	127.725	100.250
503	CA	TRP	403	4.970	127.432	100.474
504	C	TRP	403	3.973	128.419	99.844
505	O	TRP	403	2.765	128.315	100.079
506	CB	TRP	403	4.763	127.374	101.991
507	CG	TRP	403	5.789	128.158	102.798
508	CD1	TRP	403	6.817	127.625	103.547
509	CD2	TRP	403	5.877	129.594	102.951
510	NE1	TRP	403	7.529	128.643	104.093
511	CE2	TRP	403	7.009	129.838	103.750
512	CE3	TRP	403	5.131	130.653	102.456
513	CZ2	TRP	403	7.387	131.142	104.027
514	CZ3	TRP	403	5.514	131.957	102.744
515	CH2	TRP	403	6.640	132.201	103.526
516	N	LYS	404	4.475	129.367	99.069
517	CA	LYS	404	3.638	130.295	98.325
518	C	LYS	404	3.684	129.983	96.823
519	O	LYS	404	2.759	130.347	96.088
520	CB	LYS	404	4.214	131.685	98.584
521	CG	LYS	404	3.481	132.789	97.832
522	CD	LYS	404	4.189	134.126	98.013
523	CE	LYS	404	3.516	135.229	97.206
524	NZ	LYS	404	4.238	136.503	97.355
525	N	ARG	405	4.710	129.262	96.381
526	CA	ARG	405	4.925	129.098	94.933
527	C	ARG	405	5.127	127.645	94.476
528	O	ARG	405	4.572	127.260	93.439
529	CB	ARG	405	6.132	129.946	94.530
530	CG	ARG	405	5.903	131.421	94.860
531	CD	ARG	405	7.038	132.324	94.395
532	NE	ARG	405	7.125	132.342	92.927
533	CZ	ARG	405	7.141	133.461	92.201
534	NH1	ARG	405	7.126	134.651	92.805
535	NH2	ARG	405	7.209	133.389	90.869
536	N	ASP	406	5.875	126.862	95.244
537	CA	ASP	406	6.178	125.446	94.931
538	C	ASP	406	6.615	125.225	93.475
539	O	ASP	406	5.867	124.674	92.658
540	CB	ASP	406	4.928	124.625	95.242
541	CG	ASP	406	5.218	123.127	95.180
542	OD1	ASP	406	5.840	122.633	96.108
543	OD2	ASP	406	4.820	122.500	94.204
544	N	GLU	407	7.801	125.708	93.140
545	CA	GLU	407	8.284	125.636	91.749
546	C	GLU	407	9.713	125.105	91.604
547	O	GLU	407	10.666	125.704	92.124
548	CB	GLU	407	8.269	127.057	91.195
549	CG	GLU	407	6.885	127.691	91.236
550	CD	GLU	407	7.002	129.192	91.001
551	OE1	GLU	407	5.973	129.854	91.016
552	OE2	GLU	407	8.131	129.658	90.926
553	N	ARG	408	9.859	124.022	90.858
554	CA	ARG	408	11.195	123.555	90.438
555	C	ARG	408	11.643	124.258	89.156
556	O	ARG	408	10.784	124.720	88.400
557	CB	ARG	408	11.110	122.059	90.185
558	CG	ARG	408	10.615	121.354	91.438
559	CD	ARG	408	10.570	119.847	91.276
560	NE	ARG	408	10.678	119.181	92.583
561	CZ	ARG	408	10.000	118.079	92.909
562	NH1	ARG	408	9.081	117.591	92.074
563	NH2	ARG	408	10.173	117.526	94.111
564	N	TYR	409	12.941	124.412	88.943
565	CA	TYR	409	13.430	125.008	87.676
566	C	TYR	409	14.760	124.439	87.173
567	O	TYR	409	15.832	124.848	87.636
568	CB	TYR	409	13.649	126.504	87.853
569	CG	TYR	409	12.421	127.399	87.935
570	CD1	TYR	409	11.880	127.737	89.169
571	CD2	TYR	409	11.867	127.908	86.768
572	CE1	TYR	409	10.766	128.562	89.234
573	CE2	TYR	409	10.756	128.737	86.833
574	CZ	TYR	409	10.206	129.057	88.066
575	OH	TYR	409	9.070	129.832	88.131
576	N	PHE	410	14.689	123.654	86.108
577	CA	PHE	410	15.903	123.109	85.472
578	C	PHE	410	16.436	124.009	84.357
579	O	PHE	410	15.687	124.793	83.760
580	CB	PHE	410	15.612	121.738	84.880
581	CG	PHE	410	15.580	120.600	85.891
582	CD1	PHE	410	16.776	120.050	86.337
583	CD2	PHE	410	14.371	120.110	86.359
584	CE1	PHE	410	16.761	119.009	87.256
585	CE2	PHE	410	14.358	119.070	87.276
586	CZ	PHE	410	15.550	118.519	87.726
587	N	PHE	411	17.739	123.932	84.137
588	CA	PHE	411	18.390	124.670	83.042
589	C	PHE	411	19.327	123.781	82.226
590	O	PHE	411	20.130	123.018	82.778
591	CB	PHE	411	19.215	125.818	83.619
592	CG	PHE	411	18.440	127.063	84.043
593	CD1	PHE	411	17.787	127.112	85.270
594	CD2	PHE	411	18.409	128.163	83.197
595	CE1	PHE	411	17.084	128.252	85.636
596	CE2	PHE	411	17.712	129.305	83.567
597	CZ	PHE	411	17.045	129.347	84.784
598	N	GLN	412	19.268	123.930	80.915
599	CA	GLN	412	20.226	123.224	80.055
600	C	GLN	412	20.809	124.169	79.009
601	O	GLN	412	20.196	124.447	77.971
602	CB	GLN	412	19.566	121.995	79.424
603	CG	GLN	412	20.474	121.259	78.434
604	CD	GLN	412	21.888	121.068	78.987
605	OE1	GLN	412	22.839	121.656	78.460
606	NE2	GLN	412	22.022	120.235	79.999
607	N	GLY	413	22.050	124.560	79.259
608	CA	GLY	413	22.726	125.561	78.439
609	C	GLY	413	21.925	126.850	78.424
610	O	GLY	413	21.537	127.385	79.473
611	N	ASN	414	21.529	127.212	77.221
612	CA	ASN	414	20.747	128.422	76.981
613	C	ASN	414	19.244	128.149	76.868
614	O	ASN	414	18.646	128.411	75.818
615	CB	ASN	414	21.294	129.080	75.713
616	CG	ASN	414	21.630	128.073	74.601
617	OD1	ASN	414	22.798	127.725	74.394
618	ND2	ASN	414	20.605	127.592	73.920
619	N	GLN	415	18.653	127.599	77.916
620	CA	GLN	415	17.192	127.386	77.948
621	C	GLN	415	16.723	126.926	79.329
622	O	GLN	415	17.298	126.011	79.939
623	CB	GLN	415	16.765	126.381	76.873
624	CG	GLN	415	17.296	124.980	77.134
625	CD	GLN	415	17.374	124.176	75.844
626	OE1	GLN	415	16.374	123.963	75.151
627	NE2	GLN	415	18.579	123.704	75.574
628	N	TYR	416	15.740	127.640	79.850
629	CA	TYR	416	15.139	127.258	81.132
630	C	TYR	416	13.900	126.407	80.898
631	O	TYR	416	13.201	126.547	79.886
632	CB	TYR	416	14.797	128.486	81.978
633	CG	TYR	416	13.679	129.405	81.491
634	CD1	TYR	416	13.918	130.317	80.473
635	CD2	TYR	416	12.429	129.357	82.099
636	CE1	TYR	416	12.903	131.155	80.038
637	CE2	TYR	416	11.411	130.198	81.666
638	CZ	TYR	416	11.653	131.091	80.631
639	OH	TYR	416	10.635	131.872	80.136
640	N	TRP	417	13.684	125.482	81.814
641	CA	TRP	417	12.546	124.576	81.708
642	C	TRP	417	11.231	125.271	82.017
643	O	TRP	417	11.187	126.276	82.739
644	CB	TRP	417	12.779	123.339	82.558
645	CG	TRP	417	13.004	122.135	81.670
646	CD1	TRP	417	12.237	120.994	81.636
647	CD2	TRP	417	14.048	121.958	80.683
648	NE1	TRP	417	12.718	120.183	80.661
649	CE2	TRP	417	13.777	120.737	80.043
650	CE3	TRP	417	15.108	122.747	80.260
651	CZ2	TRP	417	14.539	120.347	78.950
652	CZ3	TRP	417	15.882	122.336	79.182
653	CH2	TRP	417	15.595	121.144	78.526
654	N	ARG	418	10.177	124.647	81.511
655	CA	ARG	418	8.844	125.247	81.335
656	C	ARG	418	8.370	125.926	82.605
657	O	ARG	418	7.969	125.228	83.535
658	CB	ARG	418	7.848	124.135	81.000
659	CG	ARG	418	8.361	122.979	80.130
660	CD	ARG	418	8.588	123.290	78.646
661	NE	ARG	418	9.926	123.849	78.405
662	CZ	ARG	418	11.023	123.101	78.282
663	NH1	ARG	418	10.910	121.793	78.039
664	NH2	ARG	418	12.216	123.689	78.184
665	N	TYR	419	8.275	127.249	82.557
666	CA	TYR	419	8.081	128.120	83.735
667	C	TYR	419	7.189	127.499	84.806
668	O	TYR	419	6.025	127.167	84.561
669	CB	TYR	419	7.496	129.444	83.254
670	CG	TYR	419	7.573	130.575	84.277
671	CD1	TYR	419	8.785	131.216	84.500
672	CD2	TYR	419	6.440	130.959	84.983
673	CE1	TYR	419	8.866	132.247	85.427
674	CE2	TYR	419	6.518	131.990	85.910
675	CZ	TYR	419	7.731	132.632	86.128
676	OH	TYR	419	7.808	133.659	87.041
677	N	ASP	420	7.758	127.423	86.003
678	CA	ASP	420	7.234	126.630	87.133
679	C	ASP	420	7.448	125.166	86.770
680	O	ASP	420	6.605	124.330	87.121
681	CB	ASP	420	5.746	126.888	87.399
682	CG	ASP	420	5.469	128.372	87.621
683	OD1	ASP	420	6.344	129.026	88.168
684	OD2	ASP	420	4.376	128.816	87.296
685	N	SER	421	8.704	124.873	86.451
686	CA	SER	421	9.095	123.693	85.647
687	C	SER	421	8.423	122.418	86.083
688	O	SER	421	7.707	121.785	85.302
689	CB	SER	421	10.604	123.498	85.670
690	OG	SER	421	11.207	124.686	85.178
691	N	ASP	422	8.690	121.997	87.300
692	CA	ASP	422	7.950	120.854	87.819
693	C	ASP	422	7.255	121.244	89.114
694	O	ASP	422	7.892	121.677	90.084
695	CB	ASP	422	8.866	119.646	87.984
696	CG	ASP	422	9.388	119.186	86.619
697	OD1	ASP	422	8.724	118.374	85.988
698	OD2	ASP	422	10.481	119.610	86.264
699	N	LYS	423	5.937	121.185	89.078
700	CA	LYS	423	5.134	121.525	90.250
701	C	LYS	423	4.311	120.317	90.688
702	O	LYS	423	3.336	119.931	90.035
703	CB	LYS	423	4.231	122.699	89.902
704	CG	LYS	423	3.490	123.199	91.135
705	CD	LYS	423	2.684	124.456	90.834
706	CE	LYS	423	2.021	124.987	92.099
707	NZ	LYS	423	1.151	123.965	92.701
708	N	ASP	424	4.713	119.767	91.823
709	CA	ASP	424	4.130	118.535	92.376
710	C	ASP	424	4.097	117.412	91.335
711	O	ASP	424	3.035	117.070	90.802
712	CB	ASP	424	2.724	118.845	92.887
713	CG	ASP	424	2.173	117.669	93.686
714	OD1	ASP	424	2.971	117.013	94.343
715	OD2	ASP	424	0.963	117.499	93.693
716	N	GLN	425	5.272	116.842	91.095
717	CA	GLN	425	5.491	115.756	90.116
718	C	GLN	425	4.794	116.022	88.771
719	O	GLN	425	3.972	115.215	88.324
720	CB	GLN	425	4.997	114.447	90.746
721	CG	GLN	425	5.483	113.189	90.019
722	CD	GLN	425	5.005	111.937	90.756
723	OE1	GLN	425	3.925	111.934	91.358
724	NE2	GLN	425	5.818	110.891	90.727
725	N	ALA	426	5.219	117.075	88.087
726	CA	ALA	426	4.524	117.493	86.854
727	C	ALA	426	5.214	118.643	86.121
728	O	ALA	426	5.323	119.756	86.648
729	CB	ALA	426	3.106	117.930	87.211
730	N	LEU	427	5.623	118.369	84.894
731	CA	LEU	427	6.248	119.382	84.032
732	C	LEU	427	5.190	120.344	83.471
733	O	LEU	427	4.127	119.898	83.019
734	CB	LEU	427	6.948	118.636	82.894
735	CG	LEU	427	7.863	119.529	82.061
736	CD1	LEU	427	9.007	120.075	82.908
737	CD2	LEU	427	8.418	118.770	80.862
738	N	THR	428	5.455	121.638	83.564
739	CA	THR	428	4.541	122.667	83.037
740	C	THR	428	4.317	122.508	81.536
741	O	THR	428	5.252	122.328	80.747
742	CB	THR	428	5.133	124.052	83.290
743	OG1	THR	428	5.309	124.232	84.684
744	CG2	THR	428	4.254	125.195	82.784
745	N	GLU	429	3.054	122.559	81.154
746	CA	GLU	429	2.660	122.538	79.740
747	C	GLU	429	2.869	123.904	79.063
748	O	GLU	429	1.913	124.681	78.958
749	CB	GLU	429	1.173	122.175	79.737
750	CG	GLU	429	0.543	122.076	78.352
751	CD	GLU	429	−0.961	121.864	78.516
752	OE1	GLU	429	−1.597	121.489	77.542
753	OE2	GLU	429	−1.427	122.000	79.639
754	N	ASP	430	4.096	124.214	78.654
755	CA	ASP	430	4.326	125.458	77.891
756	C	ASP	430	5.716	125.550	77.240
757	O	ASP	430	6.727	125.705	77.929
758	CB	ASP	430	4.124	126.660	78.812
759	CG	ASP	430	3.722	127.873	77.979
760	OD1	ASP	430	3.331	127.667	76.837
761	OD2	ASP	430	3.821	128.982	78.488
762	N	GLU	431	5.679	125.651	75.916
763	CA	GLU	431	6.827	125.841	74.994
764	C	GLU	431	8.199	125.325	75.435
765	O	GLU	431	8.896	125.954	76.243
766	CB	GLU	431	6.961	127.332	74.684
767	CG	GLU	431	8.028	127.608	73.621
768	CD	GLU	431	7.601	127.033	72.267
769	OE1	GLU	431	6.896	127.741	71.563
770	OE2	GLU	431	7.800	125.843	72.064
771	N	GLN	432	8.630	124.263	74.776
772	CA	GLN	432	10.001	123.766	74.936
773	C	GLN	432	11.007	124.753	74.345
774	O	GLN	432	10.772	125.362	73.295
775	CB	GLN	432	10.154	122.382	74.300
776	CG	GLN	432	9.574	122.279	72.889
777	CD	GLN	432	8.193	121.619	72.929
778	OE1	GLN	432	7.183	122.278	73.206
779	NE2	GLN	432	8.186	120.311	72.743
780	N	GLY	433	12.077	124.969	75.093
781	CA	GLY	433	13.131	125.911	74.704
782	C	GLY	433	12.638	127.354	74.787
783	O	GLY	433	12.899	128.159	73.884
784	N	LYS	434	11.921	127.670	75.851
785	CA	LYS	434	11.337	129.008	75.977
786	C	LYS	434	12.353	130.098	76.293
787	O	LYS	434	13.489	129.826	76.704
788	CB	LYS	434	10.238	128.991	77.024
789	CG	LYS	434	10.625	128.200	78.263
790	CD	LYS	434	9.548	128.404	79.312
791	CE	LYS	434	8.172	128.322	78.669
792	NZ	LYS	434	7.112	128.661	79.627
793	N	SER	435	11.920	131.304	75.946
794	CA	SER	435	12.619	132.607	76.061
795	C	SER	435	13.623	132.778	77.204
796	O	SER	435	13.385	133.556	78.139
797	CB	SER	435	11.511	133.634	76.240
798	OG	SER	435	10.702	133.195	77.328
799	N	TYR	436	14.841	132.351	76.928
800	CA	TYR	436	15.894	132.296	77.942
801	C	TYR	436	16.373	133.679	78.416
802	O	TYR	436	16.005	134.013	79.545
803	CB	TYR	436	17.013	131.313	77.541
804	CG	TYR	436	17.346	131.096	76.052
805	CD1	TYR	436	16.493	130.368	75.228
806	CD2	TYR	436	18.549	131.572	75.548
807	CE1	TYR	436	16.812	130.167	73.895
808	CE2	TYR	436	18.871	131.374	74.208
809	CZ	TYR	436	17.999	130.674	73.383
810	OH	TYR	436	18.348	130.423	72.078
811	N	PRO	437	16.939	134.558	77.595
812	CA	PRO	437	17.220	135.909	78.097
813	C	PRO	437	16.010	136.851	78.042
814	O	PRO	437	16.074	137.957	78.591
815	CB	PRO	437	18.281	136.420	77.171
816	CG	PRO	437	18.282	135.576	75.907
817	CD	PRO	437	17.297	134.453	76.173
818	N	LYS	438	14.886	136.356	77.550
819	CA	LYS	438	13.818	137.231	77.084
820	C	LYS	438	12.646	137.309	78.056
821	O	LYS	438	11.964	138.340	78.112
822	CB	LYS	438	13.388	136.645	75.742
823	CG	LYS	438	12.327	137.451	75.007
824	CD	LYS	438	12.041	136.807	73.651
825	CE	LYS	438	10.998	137.583	72.856
826	NZ	LYS	438	10.774	136.962	71.541
827	N	LEU	439	12.452	136.280	78.859
828	CA	LEU	439	11.358	136.355	79.825
829	C	LEU	439	11.881	136.461	81.246
830	O	LEU	439	11.232	137.093	82.087
831	CB	LEU	439	10.464	135.129	79.694
832	CG	LEU	439	9.205	135.256	80.549
833	CD1	LEU	439	8.425	136.519	80.190
834	CD2	LEU	439	8.316	134.026	80.415
835	N	ILE	440	13.031	135.871	81.518
836	CA	ILE	440	13.559	135.971	82.878
837	C	ILE	440	14.624	137.055	83.000
838	O	ILE	440	15.331	137.372	82.034
839	CB	ILE	440	14.070	134.615	83.356
840	CG1	ILE	440	15.163	134.068	82.455
841	CG2	ILE	440	12.915	133.622	83.456
842	CD1	ILE	440	15.642	132.701	82.931
843	N	SER	441	14.483	137.770	84.107
844	CA	SER	441	15.381	138.832	84.598
845	C	SER	441	15.417	140.051	83.687
846	O	SER	441	16.350	140.853	83.800
847	CB	SER	441	16.803	138.308	84.780
848	OG	SER	441	17.452	138.283	83.516
849	N	GLU	442	14.297	140.321	83.040
850	CA	GLU	442	14.190	141.386	82.032
851	C	GLU	442	15.479	141.595	81.228
852	O	GLU	442	16.187	142.585	81.450
853	CB	GLU	442	13.831	142.676	82.768
854	CG	GLU	442	13.477	143.820	81.820
855	CD	GLU	442	13.302	145.108	82.618
856	OE1	GLU	442	13.235	146.158	81.995
857	OE2	GLU	442	13.110	145.003	83.822
858	N	GLY	443	15.827	140.640	80.379
859	CA	GLY	443	16.932	140.860	79.440
860	C	GLY	443	18.317	140.429	79.925
861	O	GLY	443	19.165	141.274	80.237
862	N	PHE	444	18.558	139.128	79.920
863	CA	PHE	444	19.927	138.619	80.102
864	C	PHE	444	20.770	138.951	78.872
865	O	PHE	444	20.244	139.030	77.757
866	CB	PHE	444	19.904	137.102	80.271
867	CG	PHE	444	19.702	136.592	81.688
868	CD1	PHE	444	20.458	137.116	82.728
869	CD2	PHE	444	18.787	135.577	81.935
870	CE1	PHE	444	20.286	136.641	84.019
871	CE2	PHE	444	18.615	135.101	83.228
872	CZ	PHE	444	19.363	135.635	84.270
873	N	PRO	445	22.057	139.179	79.081
874	CA	PRO	445	22.979	139.324	77.952
875	C	PRO	445	23.018	138.046	77.114
876	O	PRO	445	22.963	136.935	77.649
877	CB	PRO	445	24.310	139.633	78.565
878	CG	PRO	445	24.193	139.561	80.081
879	CD	PRO	445	22.740	139.225	80.377
880	N	GLY	446	23.187	138.223	75.813
881	CA	GLY	446	23.173	137.095	74.866
882	C	GLY	446	24.482	136.306	74.818
883	O	GLY	446	24.514	135.168	74.333
884	N	ILE	447	25.559	136.928	75.267
885	CA	ILE	447	26.859	136.246	75.336
886	C	ILE	447	26.895	135.074	76.347
887	O	ILE	447	27.365	134.005	75.937
888	CB	ILE	447	27.941	137.278	75.654
889	CG1	ILE	447	27.879	138.438	74.668
890	CG2	ILE	447	29.326	136.639	75.627
891	CD1	ILE	447	28.932	139.492	74.990
892	N	PRO	448	26.507	135.233	77.614
893	CA	PRO	448	26.389	134.052	78.481
894	C	PRO	448	25.324	133.079	77.996
895	O	PRO	448	24.121	133.364	78.024
896	CB	PRO	448	26.022	134.564	79.835
897	CG	PRO	448	25.830	136.065	79.775
898	CD	PRO	448	26.133	136.458	78.343
899	N	SER	449	25.787	131.905	77.615
900	CA	SER	449	24.880	130.840	77.191
901	C	SER	449	24.521	129.850	78.316
902	O	SER	449	23.333	129.797	78.654
903	CB	SER	449	25.463	130.152	75.960
904	OG	SER	449	24.809	128.902	75.822
905	N	PRO	450	25.440	129.059	78.868
906	CA	PRO	450	25.032	128.115	79.910
907	C	PRO	450	24.914	128.779	81.277
908	O	PRO	450	25.823	129.488	81.724
909	CB	PRO	450	26.107	127.074	79.937
910	CG	PRO	450	27.316	127.604	79.182
911	CD	PRO	450	26.881	128.933	78.588
912	N	LEU	451	23.781	128.565	81.922
913	CA	LEU	451	23.648	129.003	83.319
914	C	LEU	451	24.256	127.956	84.248
915	O	LEU	451	23.834	126.790	84.273
916	CB	LEU	451	22.186	129.258	83.681
917	CG	LEU	451	21.742	130.697	83.398
918	CD1	LEU	451	22.753	131.688	83.969
919	CD2	LEU	451	21.500	130.982	81.917
920	N	ASP	452	25.199	128.401	85.063
921	CA	ASP	452	25.946	127.463	85.905
922	C	ASP	452	25.188	127.169	87.188
923	O	ASP	452	24.663	126.061	87.351
924	CB	ASP	452	27.310	128.052	86.236
925	CG	ASP	452	28.089	128.391	84.973
926	OD1	ASP	452	28.088	127.583	84.054
927	OD2	ASP	452	28.652	129.481	84.938
928	N	THR	453	25.056	128.163	88.045
929	CA	THR	453	24.306	127.963	89.289
930	C	THR	453	23.238	129.035	89.481
931	O	THR	453	23.511	130.231	89.383
932	CB	THR	453	25.292	127.983	90.451
933	OG1	THR	453	26.204	126.907	90.268
934	CG2	THR	453	24.601	127.789	91.794
935	N	ALA	454	22.011	128.606	89.706
936	CA	ALA	454	20.946	129.565	90.004
937	C	ALA	454	20.296	129.215	91.336
938	O	ALA	454	19.862	128.074	91.530
939	CB	ALA	454	19.921	129.529	88.878
940	N	PHE	455	20.278	130.161	92.259
941	CA	PHE	455	19.649	129.885	93.558
942	C	PHE	455	18.694	131.004	93.975
943	O	PHE	455	18.970	132.193	93.779
944	CB	PHE	455	20.727	129.656	94.623
945	CG	PHE	455	21.082	130.843	95.518
946	CD1	PHE	455	21.951	131.829	95.070
947	CD2	PHE	455	20.539	130.926	96.794
948	CE1	PHE	455	22.265	132.904	95.894
949	CE2	PHE	455	20.851	132.000	97.615
950	CZ	PHE	455	21.712	132.990	97.166
951	N	TYR	456	17.556	130.617	94.519
952	CA	TYR	456	16.598	131.614	94.999
953	C	TYR	456	16.882	131.976	96.453
954	O	TYR	456	16.842	131.119	97.342
955	CB	TYR	456	15.184	131.057	94.871
956	CG	TYR	456	14.130	131.904	95.582
957	CD1	TYR	456	13.893	133.210	95.172
958	CD2	TYR	456	13.415	131.366	96.643
959	CE1	TYR	456	12.951	133.984	95.836
960	CE2	TYR	456	12.471	132.139	97.307
961	CZ	TYR	456	12.247	133.449	96.906
962	OH	TYR	456	11.401	134.254	97.636
963	N	ASP	457	17.195	133.235	96.698
964	CA	ASP	457	17.354	133.665	98.082
965	C	ASP	457	16.010	134.116	98.642
966	O	ASP	457	15.526	135.223	98.370
967	CB	ASP	457	18.369	134.792	98.187
968	CG	ASP	457	18.559	135.090	99.666
969	OD1	ASP	457	18.176	136.185	100.045
970	OD2	ASP	457	18.626	134.114	100.401
971	N	ARG	458	15.560	133.341	99.616
972	CA	ARG	458	14.245	133.532	100.234
973	C	ARG	458	14.157	134.783	101.106
974	O	ARG	458	13.186	135.532	100.963
975	CB	ARG	458	13.991	132.293	101.090
976	CG	ARG	458	12.746	132.407	101.962
977	CD	ARG	458	12.585	131.153	102.816
978	NE	ARG	458	11.491	131.293	103.789
979	CZ	ARG	458	11.437	130.573	104.912
980	NH1	ARG	458	12.384	129.668	105.168
981	NH2	ARG	458	10.435	130.754	105.776
982	N	ARG	459	15.282	135.177	101.684
983	CA	ARG	459	15.293	136.315	102.614
984	C	ARG	459	15.329	137.684	101.931
985	O	ARG	459	15.214	138.707	102.613
986	CB	ARG	459	16.517	136.177	103.512
987	CG	ARG	459	16.435	134.915	104.363
988	CD	ARG	459	15.223	134.959	105.287
989	NE	ARG	459	15.133	133.739	106.103
990	CZ	ARG	459	13.972	133.225	106.513
991	NH1	ARG	459	13.959	132.165	107.321
992	NH2	ARG	459	12.825	133.816	106.166
993	N	GLN	460	15.529	137.707	100.622
994	CA	GLN	460	15.506	138.966	99.877
995	C	GLN	460	14.505	138.899	98.726
996	O	GLN	460	14.343	139.886	98.001
997	CB	GLN	460	16.898	139.222	99.301
998	CG	GLN	460	17.987	139.354	100.365
999	CD	GLN	460	17.804	140.623	101.194
1000	OE1	GLN	460	17.613	141.715	100.649
1001	NE2	GLN	460	17.943	140.472	102.501
1002	N	LYS	461	13.824	137.761	98.610
1003	CA	LYS	461	12.999	137.369	97.438
1004	C	LYS	461	13.679	137.707	96.102
1005	O	LYS	461	13.074	138.238	95.155
1006	CB	LYS	461	11.564	137.902	97.554
1007	CG	LYS	461	11.430	139.420	97.629
1008	CD	LYS	461	10.029	139.840	98.055
1009	CE	LYS	461	9.905	141.358	98.096
1010	NZ	LYS	461	10.903	141.941	99.006
1011	N	LEU	462	14.923	137.257	96.018
1012	CA	LEU	462	15.797	137.525	94.866
1013	C	LEU	462	16.533	136.263	94.443
1014	O	LEU	462	17.305	135.685	95.218
1015	CB	LEU	462	16.871	138.550	95.236
1016	CG	LEU	462	16.333	139.927	95.607
1017	CD1	LEU	462	17.464	140.845	96.051
1018	CD2	LEU	462	15.560	140.559	94.461
1019	N	ILE	463	16.349	135.875	93.198
1020	CA	ILE	463	17.083	134.731	92.659
1021	C	ILE	463	18.374	135.213	91.996
1022	O	ILE	463	18.377	136.145	91.182
1023	CB	ILE	463	16.168	133.979	91.696
1024	CG1	ILE	463	16.904	132.948	90.854
1025	CG2	ILE	463	15.400	134.939	90.808
1026	CD1	ILE	463	15.960	132.352	89.818
1027	N	TYR	464	19.477	134.645	92.446
1028	CA	TYR	464	20.792	135.039	91.941
1029	C	TYR	464	21.311	134.041	90.911
1030	O	TYR	464	21.390	132.827	91.150
1031	CB	TYR	464	21.768	135.166	93.106
1032	CG	TYR	464	21.420	136.275	94.100
1033	CD1	TYR	464	20.729	135.977	95.270
1034	CD2	TYR	464	21.810	137.581	93.839
1035	CE1	TYR	464	20.414	136.988	96.167
1036	CE2	TYR	464	21.496	138.593	94.738
1037	CZ	TYR	464	20.796	138.294	95.897
1038	OH	TYR	464	20.455	139.300	96.775
1039	N	PHE	465	21.616	134.586	89.747
1040	CA	PHE	465	22.168	133.807	88.636
1041	C	PHE	465	23.677	133.935	88.541
1042	O	PHE	465	24.231	135.020	88.330
1043	CB	PHE	465	21.550	134.271	87.327
1044	CG	PHE	465	20.191	133.655	87.042
1045	CD1	PHE	465	20.125	132.438	86.380
1046	CD2	PHE	465	19.026	134.295	87.440
1047	CE1	PHE	465	18.892	131.861	86.106
1048	CE2	PHE	465	17.795	133.718	87.165
1049	CZ	PHE	465	17.725	132.503	86.498
1050	N	PHE	466	24.320	132.793	88.652
1051	CA	PHE	466	25.770	132.708	88.602
1052	C	PHE	466	26.265	132.161	87.263
1053	O	PHE	466	26.094	130.981	86.916
1054	CB	PHE	466	26.214	131.791	89.733
1055	CG	PHE	466	26.041	132.326	91.150
1056	CD1	PHE	466	27.075	133.039	91.739
1057	CD2	PHE	466	24.874	132.080	91.862
1058	CE1	PHE	466	26.935	133.527	93.031
1059	CE2	PHE	466	24.732	132.570	93.152
1060	CZ	PHE	466	25.761	133.296	93.736
1061	N	LYS	467	26.928	133.041	86.534
1062	CA	LYS	467	27.579	132.661	85.279
1063	C	LYS	467	29.055	133.032	85.401
1064	O	LYS	467	29.410	134.211	85.281
1065	CB	LYS	467	26.904	133.405	84.127
1066	CG	LYS	467	27.104	132.737	82.763
1067	CD	LYS	467	28.510	132.898	82.188
1068	CE	LYS	467	28.882	134.367	82.017
1069	NZ	LYS	467	30.241	134.511	81.471
1070	N	GLU	468	29.901	132.013	85.418
1071	CA	GLU	468	31.321	132.159	85.782
1072	C	GLU	468	31.503	133.103	86.967
1073	O	GLU	468	31.038	132.802	88.072
1074	CB	GLU	468	32.117	132.645	84.577
1075	CG	GLU	468	32.111	131.607	83.457
1076	CD	GLU	468	32.693	130.287	83.961
1077	OE1	GLU	468	31.906	129.436	84.356
1078	OE2	GLU	468	33.909	130.182	84.003
1079	N	SER	469	32.110	134.252	86.715
1080	CA	SER	469	32.377	135.226	87.779
1081	C	SER	469	31.287	136.282	87.946
1082	O	SER	469	31.485	137.215	88.733
1083	CB	SER	469	33.684	135.945	87.472
1084	OG	SER	469	33.462	136.771	86.338
1085	N	LEU	470	30.201	136.189	87.196
1086	CA	LEU	470	29.145	137.209	87.261
1087	C	LEU	470	27.913	136.738	88.035
1088	O	LEU	470	27.389	135.641	87.805
1089	CB	LEU	470	28.723	137.552	85.835
1090	CG	LEU	470	29.906	137.970	84.967
1091	CD1	LEU	470	29.480	138.124	83.512
1092	CD2	LEU	470	30.565	139.249	85.474
1093	N	VAL	471	27.473	137.587	88.949
1094	CA	VAL	471	26.247	137.366	89.729
1095	C	VAL	471	25.156	138.357	89.331
1096	O	VAL	471	25.285	139.569	89.550
1097	CB	VAL	471	26.559	137.573	91.207
1098	CG1	VAL	471	25.331	137.328	92.079
1099	CG2	VAL	471	27.707	136.687	91.658
1100	N	PHE	472	24.089	137.822	88.766
1101	CA	PHE	472	22.918	138.617	88.385
1102	C	PHE	472	21.821	138.533	89.445
1103	O	PHE	472	21.343	137.439	89.768
1104	CB	PHE	472	22.380	138.099	87.054
1105	CG	PHE	472	23.367	138.172	85.890
1106	CD1	PHE	472	23.575	139.381	85.238
1107	CD2	PHE	472	24.049	137.035	85.478
1108	CE1	PHE	472	24.472	139.453	84.180
1109	CE2	PHE	472	24.947	137.107	84.420
1110	CZ	PHE	472	25.158	138.317	83.773
1111	N	ALA	473	21.431	139.676	89.981
1112	CA	ALA	473	20.366	139.698	91.001
1113	C	ALA	473	19.003	139.971	90.379
1114	O	ALA	473	18.744	141.079	89.905
1115	CB	ALA	473	20.670	140.780	92.027
1116	N	PHE	474	18.126	138.985	90.431
1117	CA	PHE	474	16.811	139.100	89.788
1118	C	PHE	474	15.650	139.026	90.794
1119	O	PHE	474	15.505	138.067	91.560
1120	CB	PHE	474	16.819	137.996	88.719
1121	CG	PHE	474	15.542	137.320	88.209
1122	CD1	PHE	474	15.674	136.064	87.632
1123	CD2	PHE	474	14.289	137.914	88.266
1124	CE1	PHE	474	14.560	135.379	87.167
1125	CE2	PHE	474	13.174	137.227	87.810
1126	CZ	PHE	474	13.306	135.958	87.267
1127	N	ASP	475	14.836	140.071	90.787
1128	CA	ASP	475	13.612	140.102	91.599
1129	C	ASP	475	12.571	139.188	90.968
1130	O	ASP	475	12.008	139.525	89.916
1131	CB	ASP	475	13.081	141.534	91.652
1132	CG	ASP	475	11.740	141.583	92.383
1133	OD1	ASP	475	11.744	141.608	93.604
1134	OD2	ASP	475	10.726	141.412	91.712
1135	N	VAL	476	12.155	138.182	91.721
1136	CA	VAL	476	11.336	137.103	91.149
1137	C	VAL	476	9.904	137.517	90.830
1138	O	VAL	476	9.419	137.225	89.731
1139	CB	VAL	476	11.271	135.987	92.181
1140	CG1	VAL	476	10.720	134.711	91.556
1141	CG2	VAL	476	12.640	135.728	92.783
1142	N	ASN	477	9.383	138.437	91.621
1143	CA	ASN	477	7.965	138.807	91.548
1144	C	ASN	477	7.622	139.608	90.294
1145	O	ASN	477	6.593	139.355	89.657
1146	CB	ASN	477	7.670	139.679	92.766
1147	CG	ASN	477	8.325	139.091	94.015
1148	OD1	ASN	477	8.081	137.935	94.385
1149	ND2	ASN	477	9.179	139.888	94.635
1150	N	ARG	478	8.502	140.518	89.915
1151	CA	ARG	478	8.250	141.350	88.734
1152	C	ARG	478	9.104	140.933	87.543
1153	O	ARG	478	8.986	141.514	86.457
1154	CB	ARG	478	8.523	142.798	89.105
1155	CG	ARG	478	7.627	143.224	90.259
1156	CD	ARG	478	7.890	144.668	90.665
1157	NE	ARG	478	7.067	145.048	91.821
1158	CZ	ARG	478	7.539	145.105	93.068
1159	NH1	ARG	478	8.820	144.821	93.315
1160	NH2	ARG	478	6.729	145.457	94.071
1161	N	ASN	479	9.972	139.961	87.783
1162	CA	ASN	479	10.829	139.353	86.762
1163	C	ASN	479	11.825	140.352	86.168
1164	O	ASN	479	12.074	140.357	84.955
1165	CB	ASN	479	9.930	138.757	85.686
1166	CG	ASN	479	10.577	137.513	85.101
1167	OD1	ASN	479	11.808	137.407	85.044
1168	ND2	ASN	479	9.740	136.552	84.755
1169	N	ARG	480	12.505	141.072	87.047
1170	CA	ARG	480	13.450	142.119	86.616
1171	C	ARG	480	14.816	141.960	87.281
1172	O	ARG	480	14.878	141.751	88.499
1173	CB	ARG	480	12.913	143.479	87.049
1174	CG	ARG	480	11.497	143.766	86.567
1175	CD	ARG	480	11.021	145.086	87.155
1176	NE	ARG	480	11.251	145.087	88.609
1177	CZ	ARG	480	10.993	146.128	89.403
1178	NH1	ARG	480	10.435	147.231	88.898
1179	NH2	ARG	480	11.253	146.049	90.710
1180	N	VAL	481	15.889	142.083	86.516
1181	CA	VAL	481	17.221	142.113	87.135
1182	C	VAL	481	17.421	143.516	87.712
1183	O	VAL	481	16.903	144.505	87.175
1184	CB	VAL	481	18.308	141.750	86.118
1185	CG1	VAL	481	18.529	142.864	85.100
1186	CG2	VAL	481	19.624	141.371	86.796
1187	N	LEU	482	18.060	143.580	88.863
1188	CA	LEU	482	18.196	144.840	89.602
1189	C	LEU	482	19.268	145.749	89.017
1190	O	LEU	482	20.444	145.670	89.394
1191	CB	LEU	482	18.539	144.507	91.047
1192	CG	LEU	482	17.438	143.678	91.698
1193	CD1	LEU	482	17.841	143.269	93.109
1194	CD2	LEU	482	16.111	144.432	91.716
1195	N	ASN	483	18.809	146.668	88.178
1196	CA	ASN	483	19.661	147.650	87.483
1197	C	ASN	483	20.956	147.008	87.004
1198	O	ASN	483	22.038	147.308	87.526
1199	CB	ASN	483	19.948	148.809	88.434
1200	CG	ASN	483	18.641	149.512	88.800
1201	OD1	ASN	483	18.000	149.187	89.807
1202	ND2	ASN	483	18.264	150.470	87.973
1203	N	SER	484	20.799	146.129	86.020
1204	CA	SER	484	21.809	145.194	85.454
1205	C	SER	484	22.448	144.177	86.433
1206	O	SER	484	22.346	142.976	86.153
1207	CB	SER	484	22.892	145.945	84.663
1208	OG	SER	484	23.749	146.693	85.516
1209	N	TYR	485	22.928	144.622	87.588
1210	CA	TYR	485	23.686	143.829	88.585
1211	C	TYR	485	24.365	142.571	88.028
1212	O	TYR	485	23.779	141.489	88.138
1213	CB	TYR	485	22.693	143.429	89.676
1214	CG	TYR	485	23.268	143.028	91.038
1215	CD1	TYR	485	24.063	141.896	91.177
1216	CD2	TYR	485	22.966	143.799	92.153
1217	CE1	TYR	485	24.558	141.537	92.424
1218	CE2	TYR	485	23.458	143.443	93.402
1219	CZ	TYR	485	24.252	142.312	93.533
1220	OH	TYR	485	24.712	141.936	94.775
1221	N	PRO	486	25.459	142.727	87.289
1222	CA	PRO	486	26.309	141.605	86.864
1223	C	PRO	486	27.534	141.407	87.766
1224	O	PRO	486	28.555	140.897	87.291
1225	CB	PRO	486	26.791	142.065	85.525
1226	CG	PRO	486	26.781	143.590	85.537
1227	CD	PRO	486	26.046	143.985	86.811
1228	N	LYS	487	27.426	141.752	89.039
1229	CA	LYS	487	28.615	141.994	89.871
1230	C	LYS	487	29.491	140.765	90.095
1231	O	LYS	487	29.034	139.622	90.032
1232	CB	LYS	487	28.158	142.583	91.196
1233	CG	LYS	487	27.456	143.913	90.947
1234	CD	LYS	487	27.000	144.573	92.243
1235	CE	LYS	487	26.270	145.881	91.960
1236	NZ	LYS	487	25.790	146.497	93.206
1237	N	ARG	488	30.772	141.036	90.276
1238	CA	ARG	488	31.785	139.991	90.469
1239	C	ARG	488	31.458	139.127	91.682
1240	O	ARG	488	31.213	139.660	92.775
1241	CB	ARG	488	33.108	140.711	90.711
1242	CG	ARG	488	34.319	139.787	90.757
1243	CD	ARG	488	35.535	140.550	91.270
1244	NE	ARG	488	35.658	141.840	90.574
1245	CZ	ARG	488	36.656	142.146	89.742
1246	NH1	ARG	488	36.668	143.329	89.123
1247	NH2	ARG	488	37.626	141.259	89.505
1248	N	ILE	489	31.610	137.819	91.526
1249	CA	ILE	489	31.345	136.867	92.614
1250	C	ILE	489	32.141	137.164	93.878
1251	O	ILE	489	31.512	137.388	94.915
1252	CB	ILE	489	31.687	135.448	92.161
1253	CG1	ILE	489	30.687	134.912	91.153
1254	CG2	ILE	489	31.768	134.494	93.343
1255	CD1	ILE	489	30.849	133.405	91.018
1256	N	THR	490	33.397	137.550	93.728
1257	CA	THR	490	34.239	137.787	94.905
1258	C	THR	490	33.947	139.122	95.610
1259	O	THR	490	34.246	139.244	96.804
1260	CB	THR	490	35.693	137.720	94.443
1261	OG1	THR	490	35.895	136.441	93.857
1262	CG2	THR	490	36.684	137.868	95.592
1263	N	GLU	491	33.196	140.016	94.980
1264	CA	GLU	491	32.889	141.288	95.642
1265	C	GLU	491	31.476	141.290	96.231
1266	O	GLU	491	31.127	142.225	96.961
1267	CB	GLU	491	33.055	142.459	94.670
1268	CG	GLU	491	31.898	142.575	93.684
1269	CD	GLU	491	32.104	143.744	92.727
1270	OE1	GLU	491	31.666	143.614	91.589
1271	OE2	GLU	491	32.778	144.689	93.106
1272	N	VAL	492	30.663	140.297	95.898
1273	CA	VAL	492	29.326	140.223	96.498
1274	C	VAL	492	29.215	139.006	97.410
1275	O	VAL	492	28.291	138.898	98.220
1276	CB	VAL	492	28.254	140.211	95.409
1277	CG1	VAL	492	28.130	141.579	94.752
1278	CG2	VAL	492	28.495	139.133	94.360
1279	N	PHE	493	30.155	138.094	97.246
1280	CA	PHE	493	30.317	136.939	98.132
1281	C	PHE	493	31.812	136.673	98.325
1282	O	PHE	493	32.411	135.866	97.598
1283	CB	PHE	493	29.662	135.709	97.502
1284	CG	PHE	493	28.154	135.804	97.271
1285	CD1	PHE	493	27.281	135.676	98.341
1286	CD2	PHE	493	27.656	136.016	95.991
1287	CE1	PHE	493	25.910	135.769	98.135
1288	CE2	PHE	493	26.287	136.111	95.786
1289	CZ	PHE	493	25.412	135.988	96.857
1290	N	PRO	494	32.411	137.388	99.267
1291	CA	PRO	494	33.867	137.367	99.413
1292	C	PRO	494	34.408	135.984	99.751
1293	O	PRO	494	33.874	135.248	100.590
1294	CB	PRO	494	34.184	138.368	100.478
1295	CG	PRO	494	32.897	139.020	100.949
1296	CD	PRO	494	31.776	138.348	100.174
1297	N	ALA	495	35.416	135.624	98.971
1298	CA	ALA	495	36.163	134.366	99.105
1299	C	ALA	495	35.350	133.089	98.881
1300	O	ALA	495	35.592	132.086	99.563
1301	CB	ALA	495	36.829	134.318	100.478
1302	N	VAL	496	34.428	133.106	97.931
1303	CA	VAL	496	33.776	131.852	97.528
1304	C	VAL	496	34.274	131.378	96.162
1305	O	VAL	496	33.688	130.452	95.586
1306	CB	VAL	496	32.257	132.003	97.498
1307	CG1	VAL	496	31.708	132.382	98.865
1308	CG2	VAL	496	31.810	133.003	96.445
1309	N	ILE	497	35.322	132.022	95.660
1310	CA	ILE	497	35.932	131.700	94.352
1311	C	ILE	497	35.025	132.075	93.173
1312	O	ILE	497	33.850	131.696	93.111
1313	CB	ILE	497	36.347	130.221	94.343
1314	CG1	ILE	497	37.535	130.003	95.273
1315	CG2	ILE	497	36.670	129.694	92.950
1316	CD1	ILE	497	38.035	128.564	95.204
1317	N	PRO	498	35.589	132.838	92.241
1318	CA	PRO	498	34.854	133.407	91.093
1319	C	PRO	498	34.414	132.427	89.982
1320	O	PRO	498	34.080	132.879	88.884
1321	CB	PRO	498	35.775	134.440	90.518
1322	CG	PRO	498	37.128	134.351	91.204
1323	CD	PRO	498	36.981	133.297	92.287
1324	N	GLN	499	34.491	131.128	90.213
1325	CA	GLN	499	33.956	130.160	89.249
1326	C	GLN	499	33.034	129.187	89.981
1327	O	GLN	499	33.422	128.095	90.411
1328	CB	GLN	499	35.084	129.458	88.504
1329	CG	GLN	499	36.179	128.929	89.421
1330	CD	GLN	499	37.200	128.189	88.569
1331	OE1	GLN	499	38.398	128.164	88.875
1332	NE2	GLN	499	36.705	127.607	87.491
1333	N	ASN	500	31.781	129.600	90.023
1334	CA	ASN	500	30.742	128.989	90.857
1335	C	ASN	500	30.364	127.546	90.515
1336	O	ASN	500	30.345	127.109	89.357
1337	CB	ASN	500	29.486	129.857	90.741
1338	CG	ASN	500	28.833	129.735	89.359
1339	OD1	ASN	500	28.029	128.827	89.121
1340	ND2	ASN	500	29.108	130.682	88.481
1341	N	HIS	501	30.139	126.809	91.588
1342	CA	HIS	501	29.462	125.512	91.553
1343	C	HIS	501	28.194	125.670	92.411
1344	O	HIS	501	27.942	126.801	92.844
1345	CB	HIS	501	30.440	124.455	92.068
1346	CG	HIS	501	31.434	123.955	91.040
1347	ND1	HIS	501	31.312	122.836	90.299
1348	CD2	HIS	501	32.622	124.546	90.681
1349	CE1	HIS	501	32.392	122.713	89.502
1350	NE2	HIS	501	33.200	123.771	89.738
1351	N	PRO	502	27.380	124.632	92.602
1352	CA	PRO	502	26.060	124.787	93.237
1353	C	PRO	502	26.052	125.515	94.575
1354	O	PRO	502	27.071	125.670	95.262
1355	CB	PRO	502	25.510	123.414	93.405
1356	CG	PRO	502	26.428	122.439	92.710
1357	CD	PRO	502	27.543	123.267	92.104
1358	N	PHE	503	24.842	125.897	94.936
1359	CA	PHE	503	24.611	126.795	96.061
1360	C	PHE	503	23.329	126.400	96.795
1361	O	PHE	503	22.230	126.810	96.405
1362	CB	PHE	503	24.464	128.175	95.434
1363	CG	PHE	503	24.713	129.369	96.338
1364	CD1	PHE	503	24.000	129.530	97.515
1365	CD2	PHE	503	25.659	130.314	95.962
1366	CE1	PHE	503	24.237	130.633	98.319
1367	CE2	PHE	503	25.897	131.418	96.768
1368	CZ	PHE	503	25.186	131.578	97.947
1369	N	ARG	504	23.486	125.630	97.857
1370	CA	ARG	504	22.334	125.146	98.634
1371	C	ARG	504	21.964	126.124	99.764
1372	O	ARG	504	22.844	126.703	100.410
1373	CB	ARG	504	22.704	123.761	99.167
1374	CG	ARG	504	21.543	123.045	99.845
1375	CD	ARG	504	20.348	122.845	98.915
1376	NE	ARG	504	19.188	122.364	99.681
1377	CZ	ARG	504	18.049	123.051	99.807
1378	NH1	ARG	504	17.849	124.148	99.074
1379	NH2	ARG	504	17.069	122.582	100.580
1380	N	ASN	505	20.669	126.272	100.005
1381	CA	ASN	505	20.113	127.221	100.994
1382	C	ASN	505	19.846	126.670	102.405
1383	O	ASN	505	19.156	127.354	103.176
1384	CB	ASN	505	18.755	127.692	100.486
1385	CG	ASN	505	18.825	128.596	99.261
1386	OD1	ASN	505	19.278	128.205	98.179
1387	ND2	ASN	505	18.302	129.794	99.445
1388	N	ILE	506	20.322	125.475	102.730
1389	CA	ILE	506	19.920	124.780	103.979
1390	C	ILE	506	20.039	125.638	105.244
1391	O	ILE	506	21.093	126.206	105.549
1392	CB	ILE	506	20.760	123.512	104.126
1393	CG1	ILE	506	20.371	122.482	103.079
1394	CG2	ILE	506	20.649	122.896	105.517
1395	CD1	ILE	506	21.221	121.225	103.214
1396	N	ASP	507	18.895	125.799	105.903
1397	CA	ASP	507	18.754	126.561	107.163
1398	C	ASP	507	19.199	128.017	107.042
1399	O	ASP	507	19.864	128.535	107.948
1400	CB	ASP	507	19.560	125.882	108.268
1401	CG	ASP	507	19.002	124.496	108.571
1402	OD1	ASP	507	17.792	124.338	108.477
1403	OD2	ASP	507	19.799	123.602	108.815
1404	N	SER	508	18.867	128.635	105.915
1405	CA	SER	508	19.215	130.031	105.600
1406	C	SER	508	20.727	130.270	105.566
1407	O	SER	508	21.196	131.379	105.853
1408	CB	SER	508	18.564	130.957	106.622
1409	OG	SER	508	17.169	130.693	106.606
1410	N	ALA	509	21.471	129.241	105.199
1411	CA	ALA	509	22.919	129.356	105.083
1412	C	ALA	509	23.307	129.207	103.626
1413	O	ALA	509	22.637	128.515	102.849
1414	CB	ALA	509	23.582	128.276	105.925
1415	N	TYR	510	24.372	129.889	103.256
1416	CA	TYR	510	24.788	129.905	101.857
1417	C	TYR	510	25.880	128.880	101.597
1418	O	TYR	510	27.073	129.153	101.778
1419	CB	TYR	510	25.278	131.305	101.498
1420	CG	TYR	510	24.194	132.372	101.304
1421	CD1	TYR	510	24.576	133.681	101.045
1422	CD2	TYR	510	22.844	132.039	101.338
1423	CE1	TYR	510	23.613	134.664	100.858
1424	CE2	TYR	510	21.879	133.021	101.152
1425	CZ	TYR	510	22.268	134.332	100.918
1426	OH	TYR	510	21.316	135.318	100.780
1427	N	TYR	511	25.474	127.720	101.116
1428	CA	TYR	511	26.433	126.653	100.810
1429	C	TYR	511	26.880	126.713	99.357
1430	O	TYR	511	26.449	125.904	98.521
1431	CB	TYR	511	25.801	125.298	101.096
1432	CG	TYR	511	25.432	125.095	102.560
1433	CD1	TYR	511	24.110	125.203	102.964
1434	CD2	TYR	511	26.423	124.793	103.488
1435	CE1	TYR	511	23.777	125.032	104.299
1436	CE2	TYR	511	26.090	124.617	104.824
1437	CZ	TYR	511	24.767	124.744	105.226
1438	OH	TYR	511	24.434	124.618	106.556
1439	N	SER	512	27.734	127.682	99.078
1440	CA	SER	512	28.312	127.841	97.745
1441	C	SER	512	29.564	126.996	97.646
1442	O	SER	512	30.486	127.148	98.453
1443	CB	SER	512	28.702	129.298	97.542
1444	OG	SER	512	29.376	129.382	96.293
1445	N	TYR	513	29.601	126.079	96.704
1446	CA	TYR	513	30.812	125.280	96.646
1447	C	TYR	513	31.561	125.346	95.328
1448	O	TYR	513	31.227	126.128	94.428
1449	CB	TYR	513	30.548	123.863	97.121
1450	CG	TYR	513	29.579	122.904	96.442
1451	CD1	TYR	513	29.829	122.425	95.164
1452	CD2	TYR	513	28.470	122.462	97.151
1453	CE1	TYR	513	28.992	121.464	94.614
1454	CE2	TYR	513	27.627	121.510	96.597
1455	CZ	TYR	513	27.904	120.998	95.340
1456	OH	TYR	513	27.237	119.878	94.909
1457	N	ALA	514	32.722	124.717	95.363
1458	CA	ALA	514	33.615	124.668	94.206
1459	C	ALA	514	34.350	123.334	94.155
1460	O	ALA	514	35.148	123.017	95.050
1461	CB	ALA	514	34.619	125.813	94.298
1462	N	TYR	515	34.223	122.685	93.008
1463	CA	TYR	515	34.776	121.346	92.747
1464	C	TYR	515	34.484	120.364	93.874
1465	O	TYR	515	33.361	119.861	93.969
1466	CB	TYR	515	36.278	121.456	92.500
1467	CG	TYR	515	36.633	122.301	91.280
1468	CD1	TYR	515	36.375	121.814	90.005
1469	CD2	TYR	515	37.203	123.559	91.442
1470	CE1	TYR	515	36.690	122.581	88.891
1471	CE2	TYR	515	37.516	124.328	90.329
1472	CZ	TYR	515	37.259	123.837	89.057
1473	OH	TYR	515	37.577	124.593	87.952
1474	N	ASN	516	35.481	120.092	94.701
1475	CA	ASN	516	35.334	119.132	95.803
1476	C	ASN	516	35.174	119.765	97.197
1477	O	ASN	516	34.841	119.051	98.150
1478	CB	ASN	516	36.543	118.200	95.768
1479	CG	ASN	516	36.519	117.393	94.469
1480	OD1	ASN	516	35.596	116.600	94.245
1481	ND2	ASN	516	37.541	117.569	93.646
1482	N	SER	517	35.302	121.080	97.315
1483	CA	SER	517	35.149	121.733	98.635
1484	C	SER	517	33.861	122.557	98.730
1485	O	SER	517	33.322	122.957	97.691
1486	CB	SER	517	36.345	122.648	98.855
1487	OG	SER	517	37.515	121.844	98.776
1488	N	ILE	518	33.315	122.691	99.934
1489	CA	ILE	518	32.135	123.557	100.151
1490	C	ILE	518	32.463	124.730	101.083
1491	O	ILE	518	33.282	124.607	102.005
1492	CB	ILE	518	30.965	122.742	100.727
1493	CG1	ILE	518	29.616	123.405	100.472
1494	CG2	ILE	518	31.098	122.531	102.227
1495	CD1	ILE	518	28.483	122.580	101.070
1496	N	PHE	519	31.904	125.886	100.761
1497	CA	PHE	519	32.018	127.055	101.633
1498	C	PHE	519	30.703	127.322	102.368
1499	O	PHE	519	29.671	127.613	101.750
1500	CB	PHE	519	32.373	128.276	100.787
1501	CG	PHE	519	33.691	128.174	100.023
1502	CD1	PHE	519	34.897	128.234	100.711
1503	CD2	PHE	519	33.687	128.026	98.642
1504	CE1	PHE	519	36.097	128.144	100.018
1505	CE2	PHE	519	34.888	127.935	97.948
1506	CZ	PHE	519	36.093	127.994	98.639
1507	N	PHE	520	30.751	127.189	103.683
1508	CA	PHE	520	29.621	127.572	104.543
1509	C	PHE	520	29.669	129.073	104.799
1510	O	PHE	520	30.418	129.565	105.656
1511	CB	PHE	520	29.707	126.809	105.864
1512	CG	PHE	520	28.732	127.276	106.945
1513	CD1	PHE	520	27.375	127.015	106.823
1514	CD2	PHE	520	29.207	127.969	108.052
1515	CE1	PHE	520	26.494	127.443	107.808
1516	CE2	PHE	520	28.327	128.398	109.036
1517	CZ	PHE	520	26.968	128.136	108.913
1518	N	PHE	521	28.886	129.782	104.010
1519	CA	PHE	521	28.856	131.242	104.028
1520	C	PHE	521	27.643	131.779	104.794
1521	O	PHE	521	26.524	131.254	104.694
1522	CB	PHE	521	28.790	131.631	102.557
1523	CG	PHE	521	28.959	133.093	102.183
1524	CD1	PHE	521	27.862	133.943	102.151
1525	CD2	PHE	521	30.215	133.566	101.844
1526	CE1	PHE	521	28.028	135.268	101.780
1527	CE2	PHE	521	30.384	134.889	101.471
1528	CZ	PHE	521	29.289	135.737	101.436
1529	N	LYS	522	27.880	132.796	105.602
1530	CA	LYS	522	26.775	133.467	106.291
1531	C	LYS	522	26.363	134.716	105.519
1532	O	LYS	522	27.034	135.757	105.564
1533	CB	LYS	522	27.189	133.819	107.714
1534	CG	LYS	522	26.037	134.439	108.497
1535	CD	LYS	522	26.360	134.501	109.984
1536	CE	LYS	522	26.529	133.096	110.553
1537	NZ	LYS	522	26.904	133.135	111.975
1538	N	GLY	523	25.191	134.610	104.910
1539	CA	GLY	523	24.643	135.661	104.042
1540	C	GLY	523	24.510	137.014	104.725
1541	O	GLY	523	25.229	137.953	104.370
1542	N	ASN	524	23.752	137.048	105.811
1543	CA	ASN	524	23.474	138.304	106.527
1544	C	ASN	524	24.560	138.723	107.538
1545	O	ASN	524	24.251	139.461	108.483
1546	CB	ASN	524	22.118	138.177	107.220
1547	CG	ASN	524	21.257	139.421	106.982
1548	OD1	ASN	524	20.387	139.427	106.101
1549	ND2	ASN	524	21.517	140.459	107.760
1550	N	ALA	525	25.773	138.202	107.418
1551	CA	ALA	525	26.881	138.746	108.210
1552	C	ALA	525	27.177	140.123	107.639
1553	O	ALA	525	27.022	140.311	106.427
1554	CB	ALA	525	28.108	137.857	108.065
1555	N	TYR	526	27.736	141.025	108.428
1556	CA	TYR	526	27.888	142.412	107.951
1557	C	TYR	526	29.165	142.692	107.137
1558	O	TYR	526	29.775	143.761	107.250
1559	CB	TYR	526	27.770	143.381	109.116
1560	CG	TYR	526	27.278	144.737	108.624
1561	CD1	TYR	526	26.399	144.777	107.549
1562	CD2	TYR	526	27.703	145.914	109.225
1563	CE1	TYR	526	25.943	145.996	107.069
1564	CE2	TYR	526	27.246	147.136	108.745
1565	CZ	TYR	526	26.370	147.172	107.667
1566	OH	TYR	526	25.935	148.383	107.176
1567	N	TRP	527	29.546	141.711	106.336
1568	CA	TRP	527	30.623	141.815	105.351
1569	C	TRP	527	30.623	140.518	104.552
1570	O	TRP	527	31.480	140.316	103.683
1571	CB	TRP	527	31.987	142.089	105.990
1572	CG	TRP	527	32.631	140.970	106.785
1573	CD1	TRP	527	33.499	140.020	106.297
1574	CD2	TRP	527	32.490	140.708	108.199
1575	NE1	TRP	527	33.868	139.207	107.319
1576	CE2	TRP	527	33.288	139.583	108.474
1577	CE3	TRP	527	31.776	141.323	109.217
1578	CZ2	TRP	527	33.349	139.079	109.764
1579	CZ3	TRP	527	31.846	140.816	110.508
1580	CH2	TRP	527	32.628	139.697	110.780
1581	N	LYS	528	29.569	139.737	104.770
1582	CA	LYS	528	29.373	138.418	104.141
1583	C	LYS	528	30.609	137.514	104.206
1584	O	LYS	528	31.523	137.630	103.383
1585	CB	LYS	528	28.962	138.658	102.696
1586	CG	LYS	528	27.580	139.285	102.592
1587	CD	LYS	528	27.140	139.346	101.136
1588	CE	LYS	528	25.725	139.891	100.987
1589	NZ	LYS	528	25.328	139.923	99.569
1590	N	VAL	529	30.580	136.541	105.100
1591	CA	VAL	529	31.801	135.752	105.351
1592	C	VAL	529	31.618	134.231	105.252
1593	O	VAL	529	30.574	133.686	105.633
1594	CB	VAL	529	32.285	136.152	106.746
1595	CG1	VAL	529	31.206	135.940	107.799
1596	CG2	VAL	529	33.592	135.477	107.160
1597	N	VAL	530	32.580	133.578	104.615
1598	CA	VAL	530	32.684	132.111	104.688
1599	C	VAL	530	33.283	131.727	106.040
1600	O	VAL	530	34.468	131.979	106.287
1601	CB	VAL	530	33.630	131.605	103.601
1602	CG1	VAL	530	33.799	130.091	103.677
1603	CG2	VAL	530	33.183	132.021	102.208
1604	N	ASN	531	32.505	131.048	106.864
1605	CA	ASN	531	32.978	130.699	108.204
1606	C	ASN	531	33.580	129.303	108.252
1607	O	ASN	531	34.527	129.050	109.008
1608	CB	ASN	531	31.796	130.714	109.164
1609	CG	ASN	531	31.086	132.057	109.139
1610	OD1	ASN	531	31.714	133.115	109.018
1611	ND2	ASN	531	29.774	131.998	109.284
1612	N	ASP	532	33.047	128.392	107.458
1613	CA	ASP	532	33.536	127.004	107.547
1614	C	ASP	532	33.738	126.338	106.189
1615	O	ASP	532	32.805	126.214	105.389
1616	CB	ASP	532	32.554	126.157	108.361
1617	CG	ASP	532	32.491	126.578	109.832
1618	OD1	ASP	532	33.049	125.860	110.648
1619	OD2	ASP	532	31.705	127.473	110.120
1620	N	LYS	533	34.956	125.887	105.953
1621	CA	LYS	533	35.237	125.071	104.765
1622	C	LYS	533	35.063	123.595	105.120
1623	O	LYS	533	35.628	123.115	106.110
1624	CB	LYS	533	36.659	125.359	104.303
1625	CG	LYS	533	36.826	126.838	103.977
1626	CD	LYS	533	38.251	127.163	103.546
1627	CE	LYS	533	38.401	128.641	103.205
1628	NZ	LYS	533	39.778	128.946	102.781
1629	N	ASP	534	34.229	122.904	104.363
1630	CA	ASP	534	33.881	121.518	104.718
1631	C	ASP	534	33.693	120.652	103.463
1632	O	ASP	534	33.887	121.125	102.334
1633	CB	ASP	534	32.582	121.614	105.536
1634	CG	ASP	534	32.301	120.389	106.411
1635	OD1	ASP	534	31.190	120.290	106.911
1636	OD2	ASP	534	33.225	119.613	106.623
1637	N	LYS	535	33.518	119.358	103.675
1638	CA	LYS	535	33.035	118.457	102.625
1639	C	LYS	535	31.624	118.890	102.239
1640	O	LYS	535	30.884	119.405	103.082
1641	CB	LYS	535	32.943	117.052	103.210
1642	CG	LYS	535	34.213	116.634	103.939
1643	CD	LYS	535	34.032	115.266	104.590
1644	CE	LYS	535	35.280	114.836	105.354
1645	NZ	LYS	535	36.441	114.742	104.456
1646	N	GLN	536	31.253	118.695	100.986
1647	CA	GLN	536	29.896	119.064	100.558
1648	C	GLN	536	28.925	117.933	100.834
1649	O	GLN	536	28.578	117.615	101.977
1650	CB	GLN	536	29.888	119.235	99.056
1651	CG	GLN	536	31.093	119.991	98.544
1652	CD	GLN	536	31.166	119.697	97.061
1653	OE1	GLN	536	30.430	118.836	96.565
1654	NE2	GLN	536	32.019	120.425	96.377
1655	N	GLN	537	28.698	117.190	99.765
1656	CA	GLN	537	27.824	116.009	99.767
1657	C	GLN	537	28.342	114.842	100.606
1658	O	GLN	537	27.525	114.156	101.231
1659	CB	GLN	537	27.649	115.567	98.316
1660	CG	GLN	537	28.736	116.100	97.379
1661	CD	GLN	537	30.065	115.347	97.470
1662	OE1	GLN	537	30.109	114.151	97.782
1663	NE2	GLN	537	31.131	116.059	97.153
1664	N	ASN	538	29.635	114.848	100.885
1665	CA	ASN	538	30.245	113.842	101.749
1666	C	ASN	538	29.959	114.114	103.231
1667	O	ASN	538	30.115	113.212	104.061
1668	CB	ASN	538	31.747	113.906	101.496
1669	CG	ASN	538	32.408	112.577	101.827
1670	OD1	ASN	538	32.563	112.203	102.996
1671	ND2	ASN	538	32.841	111.906	100.774
1672	N	SER	539	29.490	115.312	103.553
1673	CA	SER	539	29.096	115.619	104.927
1674	C	SER	539	27.572	115.693	105.052
1675	O	SER	539	27.040	115.586	106.162
1676	CB	SER	539	29.711	116.949	105.350
1677	OG	SER	539	29.014	118.007	104.707
1678	N	TRP	540	26.884	115.789	103.922
1679	CA	TRP	540	25.413	115.787	103.932
1680	C	TRP	540	24.864	114.368	103.946
1681	O	TRP	540	23.709	114.137	104.334
1682	CB	TRP	540	24.889	116.555	102.725
1683	CG	TRP	540	25.103	118.050	102.846
1684	CD1	TRP	540	25.746	118.871	101.948
1685	CD2	TRP	540	24.664	118.894	103.932
1686	NE1	TRP	540	25.733	120.138	102.435
1687	CE2	TRP	540	25.098	120.195	103.622
1688	CE3	TRP	540	23.969	118.653	105.108
1689	CZ2	TRP	540	24.836	121.236	104.499
1690	CZ3	TRP	540	23.707	119.702	105.979
1691	CH2	TRP	540	24.141	120.989	105.677
1692	N	LEU	541	25.689	113.443	103.492
1693	CA	LEU	541	25.507	112.028	103.815
1694	C	LEU	541	26.897	111.498	104.135
1695	O	LEU	541	27.644	111.122	103.223
1696	CB	LEU	541	24.891	111.289	102.633
1697	CG	LEU	541	24.109	110.037	103.041
1698	CD1	LEU	541	24.995	108.878	103.483
1699	CD2	LEU	541	23.055	110.363	104.094
1700	N	PRO	542	27.220	111.488	105.420
1701	CA	PRO	542	28.606	111.335	105.872
1702	C	PRO	542	29.199	109.996	105.465
1703	O	PRO	542	28.696	108.932	105.858
1704	CB	PRO	542	28.562	111.483	107.362
1705	CG	PRO	542	27.135	111.770	107.796
1706	CD	PRO	542	26.308	111.807	106.524
1707	N	ALA	543	30.259	110.062	104.674
1708	CA	ALA	543	30.923	108.837	104.229
1709	C	ALA	543	31.562	108.148	105.420
1710	O	ALA	543	32.441	108.695	106.097
1711	CB	ALA	543	31.974	109.161	103.181
1712	N	ASN	544	31.073	106.952	105.687
1713	CA	ASN	544	31.501	106.212	106.876
1714	C	ASN	544	32.966	105.822	106.740
1715	O	ASN	544	33.393	105.260	105.727
1716	CB	ASN	544	30.603	104.991	107.066
1717	CG	ASN	544	29.449	105.256	108.047
1718	OD1	ASN	544	29.261	104.474	108.984
1719	ND2	ASN	544	28.702	106.333	107.851

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Claims as granted

9 claims

Classifications

3 codes

IPC · International Patent Classification

Section A — Human necessities

A61K38/17

Section C — Chemistry; metallurgy

C12N9/64

USPC · US Patent Classification

530/350

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Pendency

1.8 y

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Examiner

James Martinell

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