Recombinant factor X with no glycosylation and method for preparing the same

This application is a divisional of U.S. Ser. No. 12/682,445 filed Apr. 9, 2010, now U.S. Pat. No. 8,173,777, which is a National Stage Entry of PCT/JP08/68271 filed Oct. 8, 2008.

The present invention relates to a recombinant Factor X (hereinafter also referred to as “FX”) that may be expressed as a two-chain protein where a high enzymatic activity and an efficient recombinant expression are possible. Specifically, the present invention relates to a method for efficiently preparing a two-chain, recombinant FX by intervening glycosylation at such an amino acid sequence that is essential for glycosylation, said amino acid sequence being present within an activation peptide domain of FX, to allow for expression of a recombinant FX with no glycosylation for improving expression efficiency.

It is widely known that FX is a vitamin K dependent blood coagulation factor. Like the other vitamin K dependent factors, FX possesses a Gla domain consisting of 11 γ-carboxyglutamic acids (hereinafter also referred to as “Gla”) in the amino acid sequence beginning from the N-terminal to the 39th residue (Non-patent reference 1). In vitro, FX is converted into activated Factor X (hereinafter also referred to as “FXa”) by an activated Factor VII (hereinafter also referred to as “FVIIa”) or an activated Factor IX (hereinafter also referred to as “FIXa”). FX is used for the treatment of hemophilia patients with inhibitor where an inhibitor to FVIII or FIX is produced as a consequence of substitution therapy with said FVIII or FIX.

Human FX, in the course of its biosynthesis, is subject to posttranslational modification such as generation of Gla, cleavage of a prepro sequence (the sequence of FX after this cleavage is shown in SEQ ID NO: 1), .beta.-hydroxylation of aspartic acid at position 63 in SEQ ID NO: 1, asparagine-type glycosylation at positions 181 and 191, serine/threonine-type glycosylation at positions 159, 171 and 443, and the like. It is thought that FX, after being synthesized as a single-chain protein, is subject to limited degradation with furin, a signal peptidase, at the cleavage motif Arg-Arg-Lys-Arg (SEQ ID NO: 18) at positions 139 to 142 in SEQ ID NO: 1 to thereby secrete a two-chain protein.

For expression of a recombinant FX, the expression as a two-chain protein is the most important. It is known that a recombinant expression from an expression vector to which cDNA (SEQ ID NO: 3) encoding the amino acid sequence of FX (the amino acid sequence of FX including the prepro sequence is shown in SEQ ID NO: 2) is simply ligated results in expressed products, most of which are secreted into culture supernatant as a single-chain protein and which have a low specific activity.

In general, in recombinant factors, their expression level is often the matter. For genetic recombination of Factor X in the present invention, in addition to its expression level, the process for generating a two-chain protein was thought to be a rate-determining (Non-patent reference 2). In Non-patent reference 2, Himmelspach et al. co-expressed FX with furin so as to promote generation of a two-chain protein with as high an expression level of FX as 120 μg/ml or more but with a low activity of 25%.

Non-patent reference 1: Journal of Thrombosis and Haemostasis, 3: 2633-2648 (2005) Non-patent reference 2: Thrombosis Research 97: 51-67 (2000)

Technical Problem to be Solved by the Invention

A problem to be solved by the present invention is to prepare and provide a recombinant, two-chain FX with a high activity.

Means for Solving the Problems

Under the circumstances, the present inventors have assiduously investigated so as to prepare a recombinant, two-chain FX with a high activity, and as a result, having regard to a sugar chain of FX, have succeeded in preparing a secreted, two-chain. FX by intervening glycosylation, to thereby complete the present invention.

Thus, the present invention includes the following (1) to (13):

(1) A method for efficiently preparing a recombinant, two-chain Factor X (hereinafter also referred to as “FX”) which comprises intervening glycosylation at such an amino acid sequence that is essential for glycosylation in FX to thereby allow for expression of a recombinant FX with no glycosylation.

(2) The method of (1) above wherein the recombinant FX with no glycosylation is a recombinant FX with no glycosylation at asparagine at position 181 (Asn181) and/or asparagine at position 191 (Asn191) in SEQ ID NO: 1.

(3) The method of (2) above wherein the recombinant FX with no glycosylation at Asn181 and/or Asn191 is obtained by substituting Asn181 and/or Asn191 with a protein-constituting amino acid other than Asn.

(4) The method of (2) above wherein the recombinant FX with no glycosylation at Asn181 and/or Asn191 is obtained by substituting threonine at position 183 (Thr183) and/or threonine at position 193 (Thr193) with a protein-constituting amino acid other than threonine (Thr) or serine (Ser).

(5) The method of (1) above wherein intervening glycosylation at such an amino acid sequence that is essential for glycosylation in FX is carried out by adding an inhibitor to glycosyltransferase during cell culture.

(6) The method of (5) above wherein the inhibitor to glycosyltransferase is tunicamycin, RNAi, or an antisense DNA.

(7) The method of (1) above wherein intervening glycosylation at such an amino acid sequence that is essential for glycosylation in FX is carried out by using a glycosyltransferase-deficient cell strain as a host cell.

(8) A recombinant FX with no glycosylation obtained by the method of any of (1) to (7) above.

(9) A gene fragment comprising a nucleotide sequence encoding the recombinant FX with no glycosylation of (8) above.

(10) An expression vector comprising the gene fragment of (9) above.

(11) A transformed cell in which the expression vector of (10) above is introduced.

(12) A pharmaceutical composition comprising the recombinant FX with no glycosylation of (8) above as an active ingredient.

(13) A therapeutic agent effective for the treatment of a hemophilia patient comprising the pharmaceutical composition of (12) above.

More Efficacious Effects than Prior Art

The recombinant FX with no glycosylation obtained in accordance with the present invention may efficiently be expressed as a two-chain protein. Accordingly, the recombinant FX of the present invention may be used as a medicament quite useful for substitution therapy to hemophilia patients, in particular, those patients possessing an inhibitor.

FIG. 1 shows results of Western blot with antibodies to various recombinant FXs obtained by the present invention, showing expression patterns of the recombinant FXs in culture supernatant with BHK cells as a host. Lane 1: Variant supernatant (D63A) where aspartic acid at position 63 is substituted with alanine; Lane 2: Variant supernatant (T159A) where threonine at position 159 is substituted with alanine; Lane 3: Variant supernatant (T171A) where threonine at position 171 is substituted with alanine; Lane 4: Variant supernatant (N181A) where asparagine at position 181 is substituted with alanine; Lane 5: Variant supernatant (N191A) where asparagine at position 191 is substituted with alanine; Lane 6: Variant supernatant (T443A) where threonine at position 443 is substituted with alanine; Lane 7: Wild-type recombinant FX supernatant; and Lane 8: Negative control supernatant.

The present inventors have focused on sites of posttranslational modification such as glycosylation and carried out amino acid substitution in FX to successfully prepare a recombinant FX having a high enzymatic activity. The recombinant FX according to the present invention is explained in detail hereinbelow.

In general, asparagine-type glycosylation is initiated at the site: Asn-X-Thr/Ser where X is any amino acid other than Pro by various glycosyltransferases in the endoplasmic reticulum followed by further modification in the Golgi body (Molecular Biology of The Cell 2nd edition, Chapter 8, Bruce Alberts et al. Garland Publishing, Inc.). Accordingly, the Asn or Thr/Ser may be substituted with other amino acids for intervening glycosylation to allow for efficient generation of a two-chain, recombinant FX and expression of the recombinant FX having a high activity.

For an amino acid to be used the purpose of amino acid substitution, alanine (Ala) is selected herein by way of example but any amino acid may be used insofar as it does not cause any significant disturbance such as loss of an enzymatic activity.

The variant with glycosylation being intervened may be obtained by using genetic recombination technique. A host cell is preferably a eukaryote such as animal cells. The variant of the present invention may be obtained by incorporating cDNA encoding an amino acid sequence of the variants into a suitable expression vector, transfecting a host cell with the vector, cloning cells that express the desired gene, culturing the obtained stable culture cells, followed by purification.

In addition to amino acid substitution, asparagine-type glycosylation may also be intervened by adding an inhibitor to glycosyltransferase such as tunicamycin to a culture medium of cells (Current Protocols in Protein Science VOL. 2 Chapter 12, John E. Coligan et al. John Wiley & Sons, Inc.).

Furthermore, intervention of asparagine-type glycosylation may also be possible by intervening expression of glycosyltransferase by the use of RNAi, an antisense DNA, and the like.

Besides, the glycosylation may also be intervened by using a glycosyltransferase-deficient cell strain as a host cell.

The FX variant of the present invention may be formulated into a pharmaceutical formulation for use in therapy, diagnosis, and the like. For preparing a formulation for intravenous administration, the composition may usually be dissolved in an aqueous solution containing a physiologically acceptable substance, e.g. sodium chloride, glycine, etc. and having a buffered pH acceptable to physiological conditions. To ensure long-term stability, a lyophilized form of the formulation may also be considered as a final dosage form. Guidelines for a composition for intravenous administration are established by government regulations such as “Minimum Requirements for Biological Products”.

Specific use of a pharmaceutical composition comprising the FX variant of the present invention may include the use for the treatment of hemophilia patients with inhibitor where an inhibitor to FVIII or FIX is produced as a consequence of substitution therapy with said FVIII or FIX.

The present invention is explained by means of the following Examples but should not be construed to be limited thereto. In Examples, the variants were those expressed in culture supernatant of animal cells (BHK). Reagents for genetic recombination were purchased from TAKARA SHUZO CO., LTD., TOYOBO, Perkin Elmer Applied, and New England Biolabs unless otherwise instructed.