CX3CR1-targeting imaging agents and their use in the diagnosis and treatment of disease

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 28, 2014, is named 09-0622-US-2_SL.txt and is 227,056 bytes in size.

The present invention relates to CX3CR1-binding polypeptides and their uses in in vivo imaging for the diagnosis and treatment of diseases including atherosclerosis.

Cardiovascular diseases are a major cause of death in the United States and other developed countries. Atherosclerosis is a progressive disease of the arterial wall where lipid deposition and chronic inflammation lead to the development of plaque. While most plaques will remain asymptomatic, some may become susceptible to thrombosis (vulnerable) and rupture resulting in myocardial infarctions or strokes. Various imaging modalities have been developed to view the vessel wall (Verjans, 2013 ; J. of Cardiovasc. Trans. Res . ePub June, 2013). Several technologies such as optical coherence tomography (OCT) and intravascular ultrasound (IVUS) can provide information on plaque composition and stability however they require invasive procedures. Another technology utilizes 18 F-fluorodeoxyglucose, a substrate that is taken up by actively metabolizing cells such as plaque macrophages, and can be detected by positron emission tomography (PET). While 18 F-FDG PET has shown clinical utility for monitoring plaque inflammation, it can also be taken up into many tissues nonselectively. New molecular imaging tools are needed to provide insight into the active cellular and molecular processes that drive the progression of atherosclerotic disease and the development of vulnerable plaques. Tools that support the detection of highly inflamed and/or rupture-prone lesions would provide a valuable mechanism for the identification of at-risk patients and for the assessment of the efficacy of novel therapies.

The present invention provides novel CX3CR1-targeted imaging agents. In another aspect, these imaging agents are useful for diagnosing atherosclerotic disease. In another aspect these imaging agents are useful in selecting or stratifying patients with atherosclerosis who would benefit by treatment with a CX3CR1 antagonist therapeutic or other known treatments for atherosclerotic disease. In a further aspect these imaging agents are useful in diagnosis of diseases characterized by increased tissue expression of CX3CR1.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 : Normalized binding of 99m Tc-VHH domains (1 nM) to CHO-hCX3CR1 and CHO-WT cells.

FIG. 2 : Representative in vivo SPECT/CT whole-body maximum intensity projection images of the biodistribution of monovalent VHH domain 99m Tc-CX3CR1BII66B02 and bivalent VHH domain 99m Tc-CX3CR1BII318 in C57BL/6 and hCX3CR1 KI mice obtained 3 hours after intravenous injection. L=liver, Kd=Kidney, B1=Bladder, LN=lymph node, St=Stomach, B=Bone, Int=Intestines, Spl=Spleen, i.s.=injection site.

FIG. 3 : Total body SPECT/CT images of the biodistribution of 99m Tc-CX3CR1BII66B02 and 99m Tc-CX3CR1BII318 in hCX3CR1 KI ApoE −/− mice that were fed a high fat, high cholesterol diet for 16 weeks. Transverse, coronal and sagittal views enable visualization of the uptake of 99m Tc-VHH domains into atherosclerotic lesions at the base of the aorta (white arrows). An excess of unlabeled VHH domain was co-administered to demonstrate specificity (+ blocking).

FIGS. 4A and 4B : Ex vivo quantitation of the uptake of 99m Tc-CX3CR1BII66B02 ( FIG. 4A ) and 99m Tc-CX3CR1BII318 ( FIG. 4B ) into aorta segments from ApoE −/− mice, hCX3CR1 KI ApoE −/− mice and hCX3CR1KI ApoE −/− mice co-administered an excess of unlabeled VHH domain ranked according to lesion score.

FIG. 5 : Representative color-scaled autoradiographic images from isolated aortic segments from 99m Tc-CX3CR1BII66B02 injected hCX3CR1 KI ApoE −/− mice. White arrows point to small, individual plaques in segments with lesion score 1.

The present invention relates to imaging agents, also known as imaging tracers, based on immunoglobulin single variable domain polypeptides that specifically bind CX3CR1 and their use as diagnostic tools. The imaging agents are comprised of a CX3CR1-targeting single variable domain polypeptide linked to a detection label. The single variable domain polypeptides comprising the imaging agents of the present invention are preferably, but not limited to, VHH domains (or simply “VHHs”) from camelids, as defined hereinafter.

CX3CR1 is a G-protein coupled integral membrane protein, and a member of the chemokine receptor family. It has a unique ligand, fractalkine, which is produced as an integral membrane protein. It can also be released into the circulation by proteolytic cleavage. In humans, a CX3CR1 variant (V249I/T280M) with decreased activity has been shown to be associated with a lower risk of cardiovascular disease (coronary heart disease, cerebrovascular disease or peripheral vascular disease) (McDermott, 2001 ; Circ. Res. 89:401), coronary artery disease (angiographic evidence of stenosis) (McDermott, 2003 ; J. Clin. Invest. 111:1241), and carotid artery occlusive disease (Ghilardi, 2004 ; Stroke 35:1276). Several independent mouse genetic studies have shown a beneficial effect of CX3CR1 deficiency on atherosclerosis. A reduction in lesion area in the aortic arch and thoracic aorta as well as a decrease in monocyte/macrophage accumulation in plaques were seen in two independently derived strains of CX3CR1 −/− apoE −/− mice fed a high fat diet (Combadiëre, 2003 ; Circulation, 107:1009, Lesnik, 2003 ; J. Clin. Invest. 111:333).

CX3CR1 is predominantly expressed on cell types such as monocytes, dendritic cells and T cells that have been associated with the initiation and progression of atherosclerotic plaques. It is highly expressed on circulating human intermediate (CD14 + CD16 + ) and non-classical (CD14 dim CD16 + ) monocytes (Cros, 2010 ; Immunity 33:375). Increased numbers of circulating CD16 + CX3CR1 + monocytes were observed in patients with unstable angina pectoris with evidence of ruptured plaques as determined by intravascular OCT (Ikejima, 2010 ; Circ. J. 74:337). Similarly, increased circulating CD16 + monocytes levels correlated with vulnerable plaque as measured by multidetector computed tomography in patients with stable angina pectoris (Kashiwagii, 2010 ; Atherosclerosis 212:71) and CD14 + CD16 + monocyte levels independently predicted cardiovascular events in patients undergoing elective coronary angiography (Ragacev, 2012 ; J. Am. Coll. Cardiol. 60:1512). By immunohistochemistry, CX3CR1 has also been shown to be expressed in human carotid plaques with the number of CX3CR1 + cells increasing with lesion development (Stolla, 2012 ; PLOS One 7:e43572). CX3CR1 appears to be a marker for plaques with elevated levels of inflammation.

Immunoglobulin single variable (VHH) domains are well suited for use as imaging agents (De Vos, 2013 ; Expert Opin. Biol. Ther. 8:1149). One type of VHH is derived from the antigen binding domain of camelid single chain antibodies. Due to their small size (<15 kDa) which leads to rapid clearance from the blood and their high affinity which allows specific target binding, imaging with good signal to background can be carried out shortly after administration enabling the use of short-lived radioisotopes which minimizes patient exposure. VHH domains have also good physicochemical properties and are stable in blood and under conditions required for labeling for use in various imaging modalities. A VHH domain specific for CX3CR1 could provide a valuable non-invasive imaging tool identifying inflamed or unstable plaques and could be utilized for patient selection, stratification, diagnosis, prognosis or monitoring treatment success for new atherosclerosis therapies. It could also be used for in vivo imaging in other diseases characterized by elevated CX3CR1 tissue expression.

Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990), and Roitt et al., “Immunology” (2 nd Ed.), Gower Medical Publishing, London, New York (1989), as well as to the general background art cited herein; Furthermore, unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks, to the general background art referred to above and to the further references cited therein;

Unless indicated otherwise, the terms “immunoglobulin” and “immunoglobulin sequence”—whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody—are used as general terms to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH/VL domains, respectively). In addition, the term “sequence” as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “(single) variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acid sequences or nucleotide sequences encoding the same, unless the context requires a more limited interpretation;

The term “domain” (of a polypeptide or protein) as used herein refers to a folded protein structure which has the ability to retain its tertiary structure independently of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins, and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.

The term “immunoglobulin domain” as used herein refers to a globular region of an antibody chain (such as e.g. a chain of a conventional 4-chain antibody or of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are characterized in that they retain the immunoglobulin fold characteristic of antibody molecules, which consists of a 2-layer sandwich of about 7 antiparallel beta-strands arranged in two beta-sheets, optionally stabilized by a conserved disulphide bond.

The term “immunoglobulin variable domain” as used herein means an immunoglobulin domain essentially consisting of four “framework regions” which are referred to in the art and herein below as “framework region 1” or “FR1”; as “framework region 2” or“FR2”; as “framework region 3” or “FR3”; and as “framework region 4” or “FR4”, respectively; which framework regions are interrupted by three “complementarity determining regions” or “CDRs”, which are referred to in the art and herein below as “complementarity determining region 1” or “CDR1”; as “complementarity determining region 2” or “CDR2”; and as “complementarity determining region 3” or “CDR3”, respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain(s) that confer specificity to an antibody for the antigen by carrying the antigen-binding site.

The terms “immunoglobulin single variable domain” and “single variable domain” as used herein mean an immunoglobulin variable domain which is capable of specifically binding to an epitope of the antigen without pairing with an additional variable immunoglobulin domain. One example of immunoglobulin single variable domains in the meaning of the present invention are “domain antibodies”, such as the immunoglobulin single variable domains VH and VL (VH domains and VL domains). Another example of immunoglobulin single variable domains are “VHH domains” (or simply “VHHs”) from camelids, as defined hereinafter.

In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e. by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

“VHH domains”, also known as VHHs, V H H domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (variable) domain of “heavy chain antibodies” (i.e. of “antibodies devoid of light chains”; C. Hamers-Casterman et al., 1993 ; Nature 363: 446). The term “VHH domain” has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “V H domains” or “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “V L domains” or “VL domains”). VHH domains can specifically bind to an epitope without an additional antigen binding domain (as opposed to VH or VL domains in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain together with a VH domain). VHH domains are small, robust and efficient antigen recognition units formed by a single immunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH, V H H domain, VHH antibody fragment, VHH antibody, as well as “Nanobody®” and “Nanobody® domain” (“Nanobody” being a trademark of the company Ablynx N.V.; Ghent; Belgium) are used interchangeably and are representatives of immunoglobulin single variable domains (having the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to an epitope without requiring the presence of a second immunoglobulin variable domain), and which are distinguished from VH domains by the so-called “hallmark residues”, as defined in e.g. WO2009/109635, FIG. 1.

The amino acid residues of a VHH domain are numbered according to the general numbering for V H domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids, as shown e.g. in FIG. 2 of Riechmann and Muyldermans, 1999 ; J. Immunol. Methods, 231: 25. According to this numbering,

FR1 comprises the amino acid residues at positions 1-30, CDR1 comprises the amino acid residues at positions 31-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-65, FR3 comprises the amino acid residues at positions 66-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that—as is well known in the art for V H domains and for VHH domains—the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

Alternative methods for numbering the amino acid residues of V H domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the present description, claims and figures, the numbering according to Kabat and applied to VHH domains as described above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

Determination of CDR regions may also be done according to different methods. In the CDR determination according to Kabat, FR1 of a VHH comprises the amino acid residues at positions 1-30, CDR1 of a VHH comprises the amino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positions 50-65, FR3 of a VHH comprises the amino acid residues at positions 66-94, CDR3 of a VHH comprises the amino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113.

In the present application, however, CDR sequences were determined according to Kontermann and Dübel (Eds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

Further structural characteristics and functional properties of VHH domains and polypeptides containing the same can be summarized as follows:

VHH domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) can function as a single, relatively small, functional antigen-binding structural unit, domain or polypeptide. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or immunoglobulin single variable domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in scFv's, which consist of a VH domain covalently linked to a VL domain).

Because of these unique properties, the use of VHH domains—either alone or as part of a larger polypeptide—offers a number of significant advantages over the use of conventional VH and VL domains, scFv's or conventional antibody fragments (such as Fab- or F(ab′)2-fragments):

only a single domain is required to bind an antigen with high affinity and with high selectivity, so that there is no need to have two separate domains present, nor to assure that these two domains are present in the right spacial conformation and configuration (i.e. through the use of especially designed linkers, as with scFv's); VHH domains can be expressed from a single gene and require no post-translational folding or modifications; VHH domains can easily be engineered into multivalent and multispecific formats (as further discussed herein); VHH domains are highly soluble and do not have a tendency to aggregate (as with the mouse-derived antigen-binding domains described by Ward et al., 1989 ; Nature, 341: 544; VHH domains are highly stable to heat, pH, proteases and other denaturing agents or conditions and, thus, may be prepared, stored or transported without the use of refrigeration equipment, conveying a cost, time and environmental savings; VHH domains are easy and relatively cheap to prepare, even on a scale required for production. For example, VHH domains and polypeptides containing the same can be produced using microbial fermentation (e.g. as further described below) and do not require the use of mammalian expression systems, as with for example conventional antibody fragments; VHH domains are relatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigen-binding fragments thereof, and therefore

show high(er) penetration into tissues and can be administered in higher doses

than such conventional 4-chain antibodies and antigen-binding fragments thereof;

VHH domains can show so-called cavity-binding properties (inter alia due to their extended CDR3 loop, compared to conventional VH domains) and can therefore also access targets and epitopes not accessible to conventional 4-chain antibodies and antigen-binding fragments thereof.

Methods of obtaining VHH domains binding to a specific antigen or epitope have been described earlier, e.g. in WO2006/040153 and WO2006/122786. As also described therein in detail, VHH domains derived from camelids can be “humanized” by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. A humanized VHH domain can contain one or more partially or fully human framework region sequences, and, in an even more specific embodiment, can contain human framework region sequences derived from DP-29, DP-47, DP-51, or parts thereof, optionally combined with JH sequences, such as JHS.

The terms “epitope” and “antigenic determinant”, which can be used interchangeably, refer to the part of a macromolecule, such as a polypeptide, that is recognized by antigen-binding molecules, such as conventional antibodies or the polypeptides of the invention, and more particularly by the antigen-binding site of said molecules. Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an immunoglobulin.

A polypeptide (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, a polypeptide of the invention, or generally an antigen binding molecule or a fragment thereof) that can “bind to” or “specifically bind to”, that “targets” or “is targeting for” that “has affinity for” and/or that “has specificity for” a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be “against” or “directed against” said epitope, antigen or protein or is a “binding” molecule with respect to such epitope, antigen or protein, or is said to be “anti”-epitope, “anti”-antigen or “anti”-protein (e.g anti-CX3CR1).

Generally, the term “specificity” refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide of the invention) can bind. The specificity of an antigen-binding protein can be determined based on its affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD), is a measure for the binding strength between an epitope and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an epitope and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is the measure of the strength of binding between an antigen-binding molecule (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide of the invention) and the pertinent antigen. Avidity is related to both the affinity between an epitope and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule.

Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code, as generally known and agreed upon in the art. When comparing two amino acid sequences, the term “amino acid difference” refers to insertions, deletions or substitutions of the indicated number of amino acid residues at a position of the reference sequence, compared to a second sequence. In case of substitution(s), such substitution(s) will preferably be conservative amino acid substitution(s), which means that an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example from WO 98/49185, wherein conservative amino acid substitutions preferably are substitutions in which one amino acid within the following groups (i)-(v) is substituted by another amino acid residue within the same group: (i) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and GIy; (ii) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and GIn; (iii) polar, positively charged residues: His, Arg and Lys; (iv) large aliphatic, nonpolar residues: Met, Leu, Ile, VaI and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows:

Ala into GIy or into Ser;

Arg into Lys;

Asn into GIn or into His;

Asp into GIu;

Cys into Ser;

GIn into Asn;

GIu into Asp;

GIy into Ala or into Pro;

His into Asn or into GIn;

Ile into Leu or into VaI;

Leu into Ile or into VaI;

Lys into Arg, into GIn or into GIu;

Met into Leu, into Tyr or into Be;

Phe into Met, into Leu or into Tyr;

Ser into Thr;

Thr into Ser;

Trp into Tyr;

Tyr into Trp or into Phe;

VaI into Ile or into Leu.

“Sequence identity” between e.g. two immunoglobulin single variable domain sequences indicates the percentage of amino acids that are identical between these two sequences. It may be calculated or determined as described in paragraph f) on pages 49 and 50 of WO08/020079. “Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.

Target Specificity

The CX3CR1-targeting polypeptides of the invention have specificity for human CX3CR1. Thus, the imaging agents of the invention comprising CX3CR1-targeting polypeptides and a detection label preferably bind to human CX3CR1 (SEQ ID NO:230).

The CX3CR1-targeting polypeptide portion of the imaging agents described herein is comprised of VHH domains. Representative VHH domains have CDR sequences shown in Tables 1, 2, 3 (representative polypeptides of families 101, 9 and 13, respectively) and 4 (representative polypeptides of optimized variants of family 101. An optimized variant is humanized and/or optimized for stability, potency, manufacturability and/or similarity to human framework regions.

Representative sequences of VHH domains that may comprise the CX3CR1-targeting polypeptide portion of the imaging agents described herein are shown in Tables 5 and 6 below:

Representative sequences of CX3CR1-binding bivalent VHH domains that may comprise the CX3CR1-targeting polypeptide portion of the imaging agents described herein are shown in Tables 7 below. As seen in the sequences, the two VHH domains are joined by a Gly/Ser linker:

The VHH domain or bivalent VHH domains that comprise the CX3CR1-targeting portion of the imaging agent may be further modified by methods known in the art in order to enable linking to the detection label as described herein, below. For example, in order enable linking to a 99m Tc detection label by the tricarbonyl method (described below), a hexahistidine (SEQ ID NO: 230) or myc-hexahistidine tag (“hexahistidine” disclosed as SEQ ID NO: 230) may be added to the C-terminal of the desired VHH domain or bivalent VHH domains. Representative examples of such modified VHH domains, monovalent or bivalent, are shown below in Table 8.

The CX3CR1-targeting polypeptide components of the imaging agents described herein may be prepared by methods known in the art, for example, see U.S. application Ser. No. 13/775,307, incorporated herein by reference. Such methods generally comprise the steps of:

culturing host cells comprising a nucleic acid capable of encoding the desired polypeptide under conditions that allow expression of the polypeptide of the invention; and, recovering or isolating the polypeptide expressed by the host cells from the culture; and optionally further purifying and/or modifying and/or formulating the polypeptide of the invention.

In one aspect of the invention these imaging agents may be used in non-invasive imaging of atherosclerosis, for example to diagnose atherosclerotic disease. In another aspect the imaging agents of the invention would be useful as a companion diagnostic for a CX3CR1-antagonist therapeutic. That is, they may be used for patient stratification, i.e. to pre-select patients with atherosclerosis that may respond favorably to a CX3CR1-antagonist therapeutic. The imaging agents may also be used to monitor the effects of treatment with any therapeutic by evaluating the progression or regression of the atherosclerotic lesion.

In another aspect of the invention, the imaging agents may be used in non-invasive imaging to diagnose other diseases characterized by increased expression of CX3CR1. Increased CX3CR1 expression is also known to be associated with multiple inflammatory disease states or conditions including cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, pancreatitis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatory pain, and cancer.

Single variable domain polypeptides, such as VHH domains have favorable properties for use in imaging agents. They have high affinity and specificity for their target as well as good physicochemical properties such as serum stability. They have molecular weights below the renal cutoff for glomerular filtration and therefore are rapidly cleared, allowing in vivo imaging of the tissues where specific binding occurs. In one embodiment the CX3CR1-binding single variable domain comprising the imaging agent is a monovalent VHH domain. In another embodiment it is bivalent, comprising two VHH domains, which may be identical or different, covalently linked by a linker peptide. The linker peptide may be a naturally occurring sequence or a non-naturally occurring sequence, preferably non-immunogenic. Non-limiting examples of linker sequences are Gly/Ser linkers of different length such as (gly x ser y ) z linkers, including (gly 4 ser) 3 (SEQ ID NO: 231), (gly 4 ser) 4 (SEQ ID NO: 232), (gly 4 ser) (SEQ ID NO: 233), (gly 3 ser) (SEQ ID NO: 234), gly 3 , and (gly 3 ser 2 ) 3 (SEQ ID NO: 235).

For use as an imaging agent the single variable domain polypeptide is linked to a detection label. Various detection labels and linking methods are known in the art. For example, non-limiting examples of detection labels may include fluorescent, chemiluminescent, bioluminescent, phosphorescent labels, paramagnetic labels, radioisotope or radiotracer labels, microbubbles or imaging dyes. The detection label may be selected according to the desired use and imaging application.

Various imaging technologies are well known and currently in use in the art. Non-limiting examples of imaging applications or technologies that may be used include:

Single photon emission computed tomography (SPECT). Non-limiting examples of radio-isotopes that may be used in detection labels for SPECT imaging include 99m Tc, 111 In, 123 I, 201 Tl and 133 Xe.

Positron emission tomography (PET). Non-limiting examples of radio-isotopes that may be used in detection labels for PET imaging include 11 C, 64 Cu, 18 F, 68 Ga, 13 N, 15 O, 82 Rb, 124 I and 89 Zr.

Near infrared fluorescence imaging (NIR or NIRF). Non-limiting examples of imaging dyes that may be used in detection labels for NIRF include Cy5.5, Alexa680, Dylight680, Dylight800 and IRDye800CW.

Ultrasound imaging. A non-limiting example of a detection label suitable for ultrasound imaging is microbubbles.

Magnetic resonance imaging (MRI). Non-limiting examples of paramagnetic materials suitable for MRI imaging include iron oxide or carbon-coated iron-cobalt nanoparticles and gadolinium chelates.

Methods for linking detection labels to a targeting antibody fragment, for example a CX3CR1-targeting single domain polypeptide, are well known in the art. Detection labels may be linked directly or indirectly, via another linking molecule, to the targeting polypeptide. The detection label may be joined covalently, for example by formation of an amide bond with an amino acid, or non-covalently, for example by an ionic interaction with a linking, chelating molecule.

Non-limiting examples of a covalent linking method include:

99m Tc linking by a tricarbonyl method. 99m Tc-tricarbonyl is reacted with the hexahistidine tagged-targeting polypeptide (“hexahistidine” disclosed as SEQ ID NO: 230) followed by purification (for example, see V. Cortez-Retamozo, 2008 ; Curr Radiopharm 1:37).

IRDye800CW linking by NHS-ester method. IRDye800CW N-hydroxysuccinimide (NHS) ester is reacted with the targeting polypeptide followed by purification (for example, see S. Oliveira, 2012 ; Mol. Imaging, 7:254-264).

Microbubble linking by biotin-streptavidin bridge. Targeting polypeptide is biotinylated. The biotinylated targeting polypeptide is coupled to biotinylated microbubble by biotin-streptavidin bridge (for example, see S. Hernot, 2012 ; J. Control. Release 158:346-353).

A non-limiting example of a chelating linking method includes:

The targeting polypeptide is reacted with Df-Bz-NCS to form the chelating linker. The modified targeting polypeptide is then radiolabeled with 68 Ga. (for example, see M. J. W. D. Vosjan, 2011 ; Eur J. Nucl. Med. Mol. Imaging, 38:753-763).

The targeting polypeptide is conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (pSCN-Bn-NOTA) and then radiolabelled with 68 Ga. (for example, see C. Xavier, 2013 ; J. Nucl. Med., 54: 776-784).

For use in in vivo imaging, the imaging agents of the invention may be formulated as a pharmaceutical preparation comprising (i) at least one imaging agent of the invention and (ii) at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant, and/or stabilizer. By “pharmaceutically acceptable” is meant that the respective material does not show any biological or otherwise undesirable effects when administered to an individual and does not interact in a deleterious manner with any of the other components of the pharmaceutical composition (such as e.g. the imaging agent) in which it is contained. Specific examples can be found in standard handbooks, such as e.g. Remington's Pharmaceutical Sciences, 18 th Ed., Mack Publishing Company, USA (1990). For example, the imaging agents of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments and other pharmaceutically active proteins. Thus, according to a further embodiment, the invention relates to a pharmaceutical composition or preparation that contains at least one imaging agent of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and/or stabilizer.

Such a formulation may be in a form suitable for parenteral administration (such as by intravenous, intramuscular, subcutaneous, intrathecal, intracavernosal or intraperitoneal injection or intravenous infusion). Such suitable administration forms—which may be solid, semi-solid or liquid, depending on the manner of administration—as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person. The preferred formulation and route of administration would be known by one skilled in the art and would depend in part on the imaging method being used and tissue being examined.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions, emulsions, or powders which comprise the active ingredient and which are suitable, optionally after a further dissolution or dilution step, for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, sterile water and pharmaceutically acceptable aqueous buffers and solutions such as physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution; water oils; glycerol; ethanol; glycols such as propylene glycol, as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof.

For use in in vivo imaging a detectable amount of the composition containing the imaging agent is administered to a subject. The detectable amount may vary depending on a number of factors including the imaging agent, the route of administration, the imaging method and the subject and tissue being examined and can be determined by one skilled in the art.

The invention provides a method for detecting CX3CR1-containing atherosclerotic plaques in vivo comprising:

1) administering an imaging agent of the present invention to a subject; and 2) detecting the presence of the bound imaging agent in the vasculature being examined;

wherein the presence of the bound imaging agent indicates the presence of atherosclerotic plaque.

Vasculature that may be examined by in vivo imaging for detecting atherosclerotic plaque includes, for example, the carotid artery, coronary artery, femoral artery, abdominal artery and thoracic artery.

The invention also provides a method for ex vivo detection of atherosclerotic disease comprising:

1) providing a sample of tissue suspected of containing atherosclerotic plaque; 2) contacting the tissue with an imaging agent of the invention; 3) removing unbound imaging agent; and 4) detecting specifically bound imaging agent in the sample;

wherein the presence of the bound imaging agent indicates the presence of atherosclerotic plaque.

In a further aspect, the invention provides the following:

An imaging agent comprising a CX3CR1-targeting polypeptide linked to a detection label.

An imaging agent according to embodiment 1, wherein the CX3CR1-targeting polypeptide is an immunoglobulin single variable domain.

An imaging agent according to embodiment 1 or 2, wherein the CX3CR1-targeting polypeptide is a VHH domain.

An imaging agent according to any one of embodiments 1 to 3, wherein the CX3CR1-targeting polypeptide includes CDR1, CDR2 and CDR3 sequences selected from:

SEQ ID No: 141, 162 and 186, respectively; or SEQ ID No: 141, 163 and 187, respectively; or SEQ ID No: 141, 164 and 186, respectively; or SEQ ID No: 141, 166 and 186, respectively; or SEQ ID No: 141, 167 and 186, respectively; or SEQ ID No: 141, 167 and 189, respectively; or SEQ ID No: 141, 168 and 186, respectively; or SEQ ID No: 141, 168 and 187, respectively; or SEQ ID No: 141, 169 and 190, respectively; or SEQ ID No: 141, 170 and 186, respectively; or SEQ ID No: 141, 171 and 186, respectively; or SEQ ID No: 141, 174 and 186, respectively; or SEQ ID No: 141, 175 and 187, respectively; or SEQ ID No: 142, 165 and 188, respectively; or SEQ ID No: 142, 173 and 188, respectively; or SEQ ID No: 143, 164 and 186, respectively; or SEQ ID No: 144, 172 and 187, respectively; or SEQ ID No: 145, 172 and 187, respectively; or SEQ ID No: 141, 214 and 186, respectively; or SEQ ID No: 141, 215 and 186, respectively; or SEQ ID No: 141, 216 and 186, respectively; or SEQ ID No: 141, 217 and 186, respectively; or SEQ ID No: 141, 218 and 186, respectively; or SEQ ID No: 141, 219 and 186, respectively; or SEQ ID No: 141, 220 and 186, respectively; or SEQ ID No: 213, 221 and 186, respectively; or SEQ ID No: 213, 214 and 186, respectively; or SEQ ID No: 146, 176 and 191, respectively; or SEQ ID No: 146, 177 and 191, respectively; or SEQ ID No: 147, 178 and 192, respectively; or SEQ ID No: 147, 179 and 192, respectively; or SEQ ID No: 147, 179 and 194, respectively; or SEQ ID No: 148, 179 and 193, respectively; or SEQ ID No: 149, 179 and 192, respectively; or SEQ ID No: 149, 180 and 192, respectively; or SEQ ID No: 149, 181 and 192, respectively; or SEQ ID No: 149, 183 and 192, respectively; or SEQ ID No: 149, 185 and 192, respectively; or SEQ ID No: 150, 179 and 194, respectively; or SEQ ID No: 150, 182 and 194, respectively; or SEQ ID No: 151, 179 and 193, respectively; or SEQ ID No: 151, 182 and 194, respectively; or SEQ ID No: 151, 184 and 196, respectively; or SEQ ID No: 152, 179 and 195, respectively; or SEQ ID No: 153, 179 and 194, respectively; or SEQ ID No: 154, 182 and 194, respectively; or SEQ ID No: 155, 179 and 195, respectively; or SEQ ID No: 156, 181 and 192, respectively; or SEQ ID No: 157, 179 and 194, respectively; or SEQ ID No: 158, 179 and 192, respectively; or SEQ ID No: 159, 178 and 192, respectively; or SEQ ID No: 160, 179 and 194, respectively; or SEQ ID No: 161, 179 and 194, respectively.

An imaging agent according to any one of embodiments 1 to 4, wherein the CX3CR1-targeting polypeptide includes CDR1, CDR2 and CDR3 sequences selected from:

SEQ ID No: 141, 162 and 186, respectively; or SEQ ID No: 141, 214 and 186, respectively; or SEQ ID No: 141, 215 and 186, respectively; or SEQ ID No: 141, 216 and 186, respectively; or SEQ ID No: 141, 217 and 186, respectively; or SEQ ID No: 141, 218 and 186, respectively; or SEQ ID No: 141, 219 and 186, respectively; or SEQ ID No: 141, 220 and 186, respectively; or SEQ ID No: 213, 221 and 186, respectively; or SEQ ID No: 213, 214 and 186, respectively; or SEQ ID No: 147, 178 and 192, respectively; or SEQ ID No: 146, 176 and 191, respectively.

An imaging agent according to any one of embodiments 1-3, wherein the CX3CR1-targeting polypeptide is a VHH domain having a sequence selected from:

any one of SEQ ID No's: 1-140 or 197-202.

An imaging agent according to embodiment 6, wherein the CX3CR1-targeting polypeptide is a VHH domain having a sequence selected from:

any one of SEQ ID No's: 1, 11, 49, 53, 121-140 or 197-202.

An imaging agent according to embodiment 1 or 2, wherein the CX3CR1-targeting polypeptide is bivalent comprising two VHH domains, which may be identical or different, covalently linked by a linker peptide, wherein the sequence of the VHH domains are selected from:

any one of SEQ ID No's: 203-212, 222 or 223.

An imaging agent according to any one of embodiments 1, 2 or 8, wherein the sequence of the bivalent CX3CR1-targeting polypeptide is selected from:

any one of SEQ ID No's: 208, 222 or 223.

An imaging agent according to any one of embodiments 1-9, wherein the detection label is selected from a radio-isotope, an imaging dye, a paramagnetic material or a microbubble.

An imaging agent according to any one of embodiments 1-10, wherein the detection label is a radio-isotope.

An imaging agent according to any one of embodiments 1-11, wherein the detection label is selected from 99m Tc, 111 In, 123 I, 201 Tl, 133 Xe, 11 C, 64 Cu, 18 F, 68 Ga, 13 N, 15 O, 82 Rb, 124 I and 89 Zr.

An imaging agent according to any one of embodiments 1-12, wherein the detection label is selected from 99m Tc and 68 Ga.

An imaging agent according to embodiment 1, wherein the CX3CR1-targeting polypeptide is an immunoglobulin that competes for binding to CX3CR1 with a VHH domain selected from:

any one of SEQ ID No's: 224, 225, 226, 227 or 228.

An in vivo method for diagnosing a disease characterized by increased expression of CX3CR1 in a subject, comprising:

a) administering an imaging agent according to any one of embodiments 1-14; b) detecting the binding of the imaging agent;

wherein the imaging agent specifically binds to CX3CR1 in the affected tissue at a level detectably higher than in undiseased tissue and the observed binding is indicative of said disease.

An in vivo method for diagnosing a disease characterized by increased expression of CX3CR1 in a subject, the method comprising:

a) administering to a subject an imaging agent according to any of embodiments 1-14; and b) detecting a higher level of binding of the imaging agent in affected tissue in the subject as compared to undiseased tissue.

A method according to embodiment 15 or 16, wherein the disease is selected from cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, pancreatitis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatory pain, and cancer.

A method according to any one of embodiments 15-17, wherein the disease is atherosclerosis.

A method according to any one of embodiments 15-18 wherein the method for detecting the binding of the imaging agent is selected from:

a) single photon emission computed tomography; b) positron emission tomography; c) near infrared fluorescence imaging; d) ultrasound imaging; and e) magnetic resonance imaging.

A method according to any one of embodiments 15-19 wherein the method for detecting the binding of the imaging agent is positron emission tomography.

A method according to any one of embodiments 15-20, wherein the subject is a human.

An ex vivo method for diagnosing a disease characterized by increased expression of CX3CR1 in a subject, comprising:

1) contacting the tissue suspected of being affected by the disease from the subject with an imaging agent according to any of embodiments 1-14; 2) removing unbound imaging agent; and 3) detecting specifically bound imaging agent in the sample;

wherein the imaging agent specifically binds to CX3CR1 in the affected tissue at a level detectably higher than undiseased tissue and the observed binding is indicative of CX3CR1-mediated disease.

An ex vivo method for diagnosing a disease characterized by increased expression of CX3CR1 in a subject, comprising:

1) contacting a tissue sample from a subject with an imaging agent according to any of embodiments 1-14; and 2) detecting specifically bound imaging agent in the sample.

A method for identifying and treating patients suffering from a disease characterized by increased expression of CX3CR1 comprising:

a) administering an imaging agent according to any of embodiments 1-14; b) detecting the binding of the imaging agent;

wherein, if the imaging agent specifically binds to CX3CR1 in the affected tissue at a level detectably higher than undiseased tissue, treating the patient with a therapeutically effective amount of a therapeutic agent known to be effective for said disease.

A method for treating a patient having a disease characterized by increased expression of CX3CR1 comprising:

a) identifying a patient as having a higher level of binding of the imaging agent according to any of embodiments 1-14 in affected tissue compared to undiseased tissue; b) administering a therapeutic agent to the patient.

The method according to embodiment 24 or 25, wherein the disease is selected from cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, pancreatitis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatory pain, and cancer.

The method according to embodiment 25 or 26, wherein the disease is atherosclerosis.

The method according to any of embodiments 24 to 27, wherein the effective therapeutic agent is a CX3CR1 antagonist.

A method for the manufacturing of an imaging agent according to any of embodiments 1 to 14 comprising the steps of

a) production of the CX3CR1-targeting polypeptide, b) binding of the detection label and c) optionally admixing further excipients.

Use of a detection label selected from a radio-isotope, an imaging dye, a paramagnetic material or a microbubble for the manufacturing of an imaging agent according to any of embodiments 1 to 14.

Use of an imaging agent according to any of embodiments 1 to 14 for the preparation of a composition for the diagnosis of a disease characterized by increased expression of CX3CR1 in a subject.

A composition comprising an imaging agent according to any of embodiments 1 to 14 for use in a method for the diagnosis of a disease characterized by increased expression of CX3CR1 in a subject.

A kit for use in a method for the diagnosis of a disease characterized by increased expression of CX3CR1 in a subject comprising an imaging agent according to any of embodiments 1 to 14.

The kit according to embodiment 33 further comprising instructions for use in a method for the diagnosis of a disease characterized by increased expression of CX3CR1 in a subject.

A polypeptide comprising an anti-CX3CR1 immunoglobulin single variable domain, wherein said polypeptide is capable of blocking the binding of human fractalkine to human CX3CR1, wherein said anti-CX3CR1 immunoglobulin single variable domain is a VHH domain comprising the sequence set forth in any one of SEQ ID NO: 200-202.

A nucleic acid molecule encoding a polypeptide according to embodiment 35.

A pharmaceutical composition comprising (i) a polypeptide according to embodiment 35, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, excipient, adjuvant and/or stabilizer.

A method for the treatment of a CX3CR1-associated disease, disorder or condition, comprising administering a therapeutic amount of a compound according to embodiment 35 to a patient in need thereof.

The method according to embodiment 38, wherein the disease, disorder or condition is selected from cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatory pain, or cancer.

The method according to embodiment 39, wherein the disease, disorder or condition is atherosclerosis.