BR122021000901B1 - POLYPEPTIDE, ITS MANUFACTURING METHOD, BACTERIAL, YEAST OR FUNGAL HOST CELL, PHARMACEUTICAL COMPOSITION, DIAGNOSTIC KIT, AND DIAGNOSTIC METHOD - Google Patents

Abstract

The present invention relates to CX3CR1 binding polypeptides, in particular polypeptides comprising specific immunoglobulin domains. The invention also relates to nucleic acids encoding similar polypeptides; to methods for making similar polypeptides; to host cells expressing or capable of expressing similar polypeptides; to compositions comprising similar polypeptides; and to uses of similar polypeptides or similar compositions, in particular for prophylactic, therapeutic and diagnostic purposes.

Description

[001] Divided from BR112014021080-2, deposited on 02.25.2013

FIELD OF THE INVENTION

[002] The present invention relates to CX3CR1 binding polypeptides, in particular polypeptides which comprise specific immunoglobulin domains. The invention also relates to nucleic acids encoding similar polypeptides; to methods for making similar polypeptides; to host cells expressing or capable of expressing similar polypeptides; to compositions comprising similar polypeptides; and to uses of similar polypeptides or similar compositions, in particular for prophylactic, therapeutic and diagnostic purposes.

BACKGROUND OF THE INVENTION

[003] CX3CR1 is an integral membrane protein coupled to G protein, which is a chemokine receptor. It is expressed predominantly 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 upregulated in monocytes by oxidized lipids and mediates the migration of these cells into and survival within plaques. Its unique ligand fractalkine (FKN) is expressed on the surface of vascular endothelial and smooth muscle cells in lesions where it modulates leukocyte adhesion. Fractalkine is also released into the circulation by proteolytic cleavage where it functions as a chemotactic agent.

[004] In humans, a CX3CR1 variant (V249I/T280M) with reduced 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). CX3CR1 colocalized with fractalkine which showed increased immunostaining by polyclonal antibodies within atherosclerotic plaques (Wong, 2002 Cardiovasc. Path. 11:332). Fractalkine staining was not observed in non-plaque arterial regions.

[005] Several independent genetic studies in mice have demonstrated a beneficial defect of CX3CR1 deficiency on atherosclerosis. A reduction in the area of injury in the aortic arch and thoracic aorta as well as a reduction in monocyte/macrophage accumulation in plaques was seen in two strains independently derived from CX3CR1-/- apoE-/- mice fed a diet of high fat content (Combadière, 2003; Circulation, 107:1009, Lesnik, 2003; J. Clin. Invest. 111:333).

[006] This shows that CX3CR1 is involved in cardiovascular disease and that modulation of its activity may provide promising therapies. Therefore, there is a need for antagonist molecules against CX3CR1 with beneficial pharmacological properties, which can be used as therapeutic agents to treat diseases, in particular cardiovascular diseases in humans.

[007] Therefore, an object of the present invention is to provide anti-CX3CR1 antagonist molecules, in particular anti-CX3CR1 antagonist molecules, which have high binding affinity for CX3CR1.

[008] An additional object of the present invention is to provide anti-CX3CR1 antagonist molecules, which have high specificity for CX3CR1.

[009] A further object of the present invention is to provide anti-CX3CR1 antagonists, which have potent activity.

[0010] A further object of the present invention is to provide anti-CX3CR1 antagonists, which have favorable bioavailability and half-life.

[0011] A further object of the present invention is to provide anti-CX3CR1 antagonists, which have favorable biophysical properties.

[0012] Additional objects of the present invention include combinations of any of the objects stipulated above.

SUMMARY OF THE INVENTION

[0013] The invention provides polypeptides which bind to human CX3CR1 and are capable of blocking the binding of human fractalkine to human CX3CR1. In one aspect, the polypeptide is an immunoglobulin which comprises an antigenic binding domain which comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1. In a further aspect, the polypeptide comprises one or more unique anti-CX3CR1 immunoglobulin variable domains, wherein said polypeptide is capable of blocking human fractalkine binding to human CX3CR1.

[0014] In one aspect, a polypeptide of the present invention is characterized by one or more of the following properties: • Binds with high affinity to human CX3CR1; • Blocks binding of soluble fractalkine to human CX3CR1; • Inhibits fractalkine-induced chemotaxis; • Inhibits fractalkine-induced internalization of human CX3CR1 receptors;

[0015] It cross-reacts with CX3CR1 cyno within 10 times the E/IC50 for human CX3CR1 for binding and functional inhibition.

[0016] In a further aspect, a polypeptide of the present invention comprises a single anti-CX3CR1 immunoglobulin variable domain and further comprises a half-life extending moiety, e.g., an albumin-binding moiety, a polyethylene molecule glycol or an Fc domain. In a further aspect, a polypeptide of the present invention comprises two or more unique anti-CX3CR1 immunoglobulin variable domains. In one aspect, the two unique variable domains of anti-CX3CR1 immunoglobulin are covalently linked together by a linker peptide. In one aspect, the two unique anti-CX3CR1 immunoglobulin variable domains in a polypeptide of the present invention have the same amino acid sequence. In another aspect, the two unique anti-CX3CR1 immunoglobulin variable domains in a polypeptide of the present invention have different amino acid sequences. In one aspect, a polypeptide of the present invention comprises two unique anti-CX3CR1 immunoglobulin variable domains and further comprises a half-life extending moiety, e.g., an albumin-binding moiety, a polyethylene glycol molecule, or a DNA domain. Fc.

[0017] In one aspect, a polypeptide of the present invention comprises a first single anti-CX3CR1 immunoglobulin variable domain covalently linked to an albumin-binding moiety by a first linker peptide, wherein said albumin-binding moiety is further covalently linked to a second unique anti-CX3CR1 immunoglobulin variable domain by a second linker peptide.

[0018] In one aspect, a polypeptide of the present invention comprises a single anti-CX3CR1 immunoglobulin variable domain covalently linked to an Fc domain by a linker peptide. In one aspect, a similar polypeptide comprising a single anti-CX3CR1 immunoglobulin variable domain covalently linked to an Fc domain by a linker peptide is provided as a dimer, for example, via disulfide bridges.

[0019] The polypeptides of the present invention are used for the prevention, treatment, mitigation and/or diagnosis of diseases, disorders or conditions associated with CX3CR1, in particular cardiovascular diseases such as atherosclerosis.

[0020] In an additional aspect, the present invention provides:

[0021] Embodiment 1: An immunoglobulin which comprises an antigenic binding domain which comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1.

[0022] Embodiment 2: A polypeptide comprising one or more unique anti-CX3CR1 immunoglobulin variable domains, wherein said polypeptide is capable of blocking human fractalkine binding to human CX3CR1.

[0023] Embodiment 3: A polypeptide according to embodiment 2, wherein said single anti-CX3CR1 immunoglobulin variable domain consists essentially of four framework regions (FR1, FR2, FR3 and FR4) and three complementarity determining regions (CDR1, CDR2 and CDR3).

[0024] Embodiment 4: A polypeptide according to embodiment 3, wherein said anti-CX3CR1 immunoglobulin single variable domain has the structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.

[0025] Embodiment 5: A polypeptide according to any one of Embodiments 2 to 4, wherein said anti-CX3CR1 immunoglobulin single variable domain is an antibody domain.

[0026] Embodiment 6: A polypeptide according to embodiment 5, wherein said anti-CX3CR1 immunoglobulin single variable domain is a VH, a VL, a VHH, a camelized VH, or a VHH that is optimized for stability , power, manufacturability and/or similarity to human framework regions.

[0027] Embodiment 7: A polypeptide according to any one of Embodiments 1 to 6, wherein said polypeptide has an affinity for human CX3CR1 to:

[0028] an EC50 of less than or equal to 10 nM, less than or equal to 5 nM, less than or equal to 2.5 nM, or less than or equal to 1 nM, as determined by cell binding FACS; or

[0029] an IC50 of less than or equal to 10 nM, less than or equal to 5 nM, less than or equal to 2.5 nM, or less than or equal to 1 nM, as determined by competition FACS.

[0030] Embodiment 8: A polypeptide according to any one of Embodiments 1 to 7, wherein said polypeptide blocks binding of human fractalkine to human CX3CR1 at an IC50 of less than or equal to 300 nM, or less than or equal to at 100 nM, or less than or equal to 20 nM, or less than or equal to 10 nM, or less than or equal to 5 nM, or less than or equal to 2.5 nM, or less than or equal to 1 nM.

[0031] Embodiment 9: A polypeptide according to any one of Embodiments 1 to 8, wherein said polypeptide inhibits human CX3CR1-mediated fractalkine-induced chemotaxis at an IC50 of less than or equal to 500 nM, or less than or equal to 100 nM, or less than or equal to 75 nM, or less than or equal to 50 nM, or less than or equal to 10 nM, or less than or equal to 5 nM.

[0032] Embodiment 10: A polypeptide according to any one of Embodiments 1 to 9, wherein said polypeptide inhibits human CX3CR1-mediated fractalkine internalization at an IC50 of less than or equal to 10 nM, or less than or equal to 5 nM or less than or equal to 1 nM.

[0033] Embodiment 11: A polypeptide according to any one of Embodiments 3 to 10, wherein said CDR3 has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5 ( SEQ ID NO: 197), where:

[0034] - Xaa1 is Pro, Ala or Gly;

[0035] - Xaa2 is Asp or Asn;

[0036] - Xaa3 is Thr or Ser;

[0037] - Xaa4 is Arg, Lys, Ala or Gly; It is

[0038] - Xaa5 is Tyr or Phe.

[0039] Embodiment 12: A polypeptide according to any one of Embodiments 3 to 11, wherein:

[0040] a)

[0041] - Xaa1 is Pro, Ala or Gly;

[0042] - Xaa2 is Asp or Asn;

[0043] - Xaa3 is Thr;

[0044] - Xaa4 is Arg or Lys; It is

[0045] - Xaa5 is Tyr,

[0046] and/or

[0047] b) wherein said CDR3 is selected from any one of SEQ ID No's: 186 to 190.

[0048] Embodiment 13: A polypeptide according to any one of Embodiments 3 to 12, wherein said CDR3 has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyr ( SEQ ID NO: 186).

[0049] Embodiment 14: A polypeptide according to any one of Embodiments 3 to 10, wherein:

[0050] the aforementioned CDR1:

[0051] has the amino acid sequence of SEQ ID NO: 141;

[0052] has an amino acid sequence that has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 141;

[0053] has an amino acid sequence that is 2 or 1 amino acid different from the amino acid sequence of SEQ ID NO: 141, wherein

[0054] - in position 2 the S was changed to T, or G;

[0055] - in position 6 the S was changed to R;

[0056] - in position 7 the N was changed to T; and/or

[0057] - in position 9 the M was changed to K; or

[0058] d) has an amino acid sequence selected from any one of SEQ ID NO's: 141 to 145 and 213;

[0059] the aforementioned CDR2:

[0060] has the amino acid sequence of SEQ ID NO: 164;

[0061] has an amino acid sequence that has at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 164;

[0062] has an amino acid sequence that is 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 164, wherein

[0063] - in position 1 the G was changed to A, L, V or S;

[0064] - in position 3 the N was changed to D, S, Q, G or T;

[0065] - in position 4 the S was changed to T, K, G or P;

[0066] - in position 5 the V was changed to A;

[0067] - in position 6 the G was changed to D;

[0068] - in position 7 the I was changed to T, or V;

[0069] - in position 8 the T was changed to A; and/or

[0070] - in position 9 the K was changed to R; or

[0071] d) has an amino acid sequence selected from any one of SEQ ID NO's: 162 to 175 and 214 to 221; It is

[0072] the aforementioned CDR3:

[0073] has the amino acid sequence of SEQ ID NO: 186;

[0074] has an amino acid sequence that has at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 186;

[0075] has an amino acid sequence that is 3, 2, or 1 amino acid different from the amino acid sequence of SEQ ID NO: 186, wherein

[0076] - in position 2 the P was changed to A, or G;

[0077] - in position 7 the D was changed to N; and/or

[0078] - in position 9 the R was changed to K; or

[0079] d) has an amino acid sequence selected from any of SEQ ID NO's: 186 to 190.

[0080] Embodiment 15: A polypeptide according to any one of Embodiments 3 to 10, wherein

[0081] said CDR1 has the amino acid sequence of SEQ ID NO: 146;

[0082] ii) said CDR2 has an amino acid sequence that a) has at least 90% amino acid identity with the amino acid sequence of SEQ ID NO: 176 or b) has the amino acid sequence of SEQ ID NO : 176 or 177; It is

[0083] said CDR3 has the amino acid sequence of SEQ ID NO: 191.

[0084] Embodiment 16: A polypeptide according to any one of Embodiments 3 to 10, wherein

[0085] the aforementioned CDR1:

[0086] has the amino acid sequence of SEQ ID NO: 147; or

[0087] has an amino acid sequence that is 6, 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 147, wherein

[0088] - in position 1 the G was changed to K, R, or A;

[0089] - in position 2 the T was changed to I, P, S or L;

[0090] - in position 3 the I was changed to V, or T;

[0091] - in position 4 the F was changed to L;

[0092] - in position 5 the S was changed to R, or D;

[0093] - in position 6 the N was changed to S, T, or D; and/or

[0094] - in position 7 the N was changed to T, or Y; or

[0095] c) has an amino acid sequence selected from any one of SEQ ID NO's: 147 to 161;

[0096] ii) the said CDR2:

[0097] has the amino acid sequence of SEQ ID NO: 179; or

[0098] has an amino acid sequence that is 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 179, wherein

[0099] - in position 3 the S was changed to T, or G;

[00100] - in position 4 the N was changed to S, or I;

[00101] - in position 5 the S was changed to T;

[00102] - in position 6 the G was changed to Y; and/or

[00103] - in position 8 the T was changed to A; or

[00104] has an amino acid sequence selected from any one of SEQ ID NO's: 178 to 185; It is

[00105] iii) said CDR3:

[00106] has the amino acid sequence of SEQ ID NO: 192; or

[00107] has an amino acid sequence that has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 192; or

[00108] has an amino acid sequence that is 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 192, wherein

[00109] - in position 2 the A was changed to G;

[00110] - in position 8 the T was changed to S;

[00111] - in position 9 the A was changed to G; and/or

[00112] - in position 10 the Y was changed to F; or d) has an amino acid sequence selected from any of SEQ ID NO's: 192 to 196.

[00113] Embodiment 17: A polypeptide according to embodiment 3, in which the amino acid sequences of said CDR1, CDR2 and CDR3 are stipulated in:

[00114] - SEQ ID No: 141, 162 and 186, respectively; or

[00115] - SEQ ID No: 141, 163 and 187, respectively; or

[00116] - SEQ ID No: 141, 164 and 186, respectively; or

[00117] - SEQ ID No: 141, 166 and 186, respectively; or

[00118] - SEQ ID No: 141, 167 and 186, respectively; or

[00119] - SEQ ID No: 141, 167 and 189, respectively; or

[00120] - SEQ ID No: 141, 168 and 186, respectively; or

[00121] - SEQ ID No: 141, 168 and 187, respectively; or

[00122] - SEQ ID No: 141, 169 and 190, respectively; or

[00123] - SEQ ID No: 141, 170 and 186, respectively; or

[00124] - SEQ ID No: 141, 171 and 186, respectively; or

[00125] - SEQ ID No: 141, 174 and 186, respectively; or

[00126] - SEQ ID No: 141, 175 and 187, respectively; or

[00127] - SEQ ID No: 142, 165 and 188, respectively; or

[00128] - SEQ ID No: 142, 173 and 188, respectively; or

[00129] - SEQ ID No: 143, 164 and 186, respectively; or

[00130] - SEQ ID No: 144, 172 and 187, respectively; or

[00131] - SEQ ID No: 145, 172 and 187, respectively; or

[00132] - SEQ ID No: 141, 214 and 186, respectively; or

[00133] - SEQ ID No: 141, 215 and 186, respectively; or

[00134] - SEQ ID No: 141, 216 and 186, respectively; or

[00135] - SEQ ID No: 141, 217 and 186, respectively; or

[00136] - SEQ ID No: 141, 218 and 186, respectively; or

[00137] - SEQ ID No: 141, 219 and 186, respectively; or

[00138] - SEQ ID No: 141, 220 and 186, respectively; or

[00139] - SEQ ID No: 213, 221 and 186, respectively; or

[00140] - SEQ ID No: 213, 214 and 186, respectively.

[00141] Embodiment 18: A polypeptide according to embodiment 3, in which the amino acid sequences of said CDR1, CDR2 and CDR3 are stipulated in:

[00142] - SEQ ID No: 146, 176 and 191, respectively; or

[00143] - SEQ ID No: 146, 177 and 191, respectively.

[00144] Embodiment 19: A polypeptide according to embodiment 3, in which the amino acid sequences of said CDR1, CDR2 and CDR3 are stipulated in:

[00145] - SEQ ID No: 147, 178 and 192, respectively; or

[00146] - SEQ ID No: 147, 179 and 192, respectively; or

[00147] - SEQ ID No: 147, 179 and 194, respectively; or

[00148] - SEQ ID No: 148, 179 and 193, respectively; or

[00149] - SEQ ID No: 149, 179 and 192, respectively; or

[00150] - SEQ ID No: 149, 180 and 192, respectively; or

[00151] - SEQ ID No: 149, 181 and 192, respectively; or

[00152] - SEQ ID No: 149, 183 and 192, respectively; or

[00153] - SEQ ID No: 149, 185 and 192, respectively; or

[00154] - SEQ ID No: 150, 179 and 194, respectively; or

[00155] - SEQ ID No: 150, 182 and 194, respectively; or

[00156] - SEQ ID No: 151, 179 and 193, respectively; or

[00157] - SEQ ID No: 151, 182 and 194, respectively; or

[00158] - SEQ ID No: 151, 184 and 196, respectively; or

[00159] - SEQ ID No: 152, 179 and 195, respectively; or

[00160] - SEQ ID No: 153, 179 and 194, respectively; or

[00161] - SEQ ID No: 154, 182 and 194, respectively; or

[00162] - SEQ ID No: 155, 179 and 195, respectively; or

[00163] - SEQ ID No: 156, 181 and 192, respectively; or

[00164] - SEQ ID No: 157, 179 and 194, respectively; or

[00165] - SEQ ID No: 158, 179 and 192, respectively; or

[00166] - SEQ ID No: 159, 178 and 192, respectively; or

[00167] - SEQ ID No: 160, 179 and 194, respectively; or

[00168] - SEQ ID No: 161, 179 and 194, respectively.

[00169] Embodiment 20: A polypeptide according to embodiment 3, wherein the amino acid sequences of said CDR1, CDR2 and CDR3 are stipulated in: SEQ ID NO's: 141, 164 and 186, respectively, or SEQ ID NO's: 141 , 162 and 186, respectively.

[00170] Embodiment 21: A polypeptide according to embodiment 3, wherein the amino acid sequences of said CDR1, CDR2 and CDR3 are stipulated in: SEQ ID NO's: 213, 214 and 186 respectively, SEQ ID NO's: 213, 221 and 186 respectively, or SEQ ID NO's: 141, 162 and 186 respectively.

[00171] Embodiment 22: A polypeptide according to any one of Embodiments 2 to 10, wherein said anti-CX3CR1 immunoglobulin single variable domain is a VHH domain comprising the sequence stipulated in:

[00172] the amino acid sequence of SEQ ID NO: 3;

[00173] amino acid sequences that have at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 3;

[00174] amino acid sequences that have 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequences of SEQ ID NO: 3; or

[00175] an amino acid sequence of any one of SEQ ID NO: 1 to 48, 121 to 140 or 222 to 224.

[00176] Embodiment 23: A polypeptide according to any one of Embodiments 2 to 10, wherein said anti-CX3CR1 immunoglobulin single variable domain is a VHH domain comprising the sequence stipulated in:

[00177] the amino acid sequence of SEQ ID NO: 49;

[00178] an amino acid sequence that has at least 95% amino acid identity with the amino acid sequences of SEQ ID NO: 49;

[00179] an amino acid sequence having 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequences of SEQ ID NO: 49; or

[00180] an amino acid sequence of any one of SEQ ID NO: 49 to 52.

[00181] Embodiment 24: A polypeptide according to any one of Embodiments 2 to 10, wherein said anti-CX3CR1 immunoglobulin single variable domain is a VHH domain comprising the sequence stipulated in:

[00182] the amino acid sequence of SEQ ID NO: 67;

[00183] an amino acid sequence that has at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 67;

[00184] an amino acid sequence that is 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequences of SEQ ID NO: 67; or

[00185] an amino acid sequence of any one of SEQ ID NO: 53 to 120.

[00186] Embodiment 25: A polypeptide according to embodiment 2, wherein said anti-CX3CR1 immunoglobulin single variable domain comprises the sequence stipulated in SEQ ID NO: 1 or SEQ ID NO: 3.

[00187] Embodiment 26: A polypeptide according to embodiment 2, wherein said anti-CX3CR1 immunoglobulin single variable domain comprises the sequence stipulated in any one of SEQ ID NO: 121 to 140 or SEQ ID NO: 222 to 224 .

[00188] Embodiment 27: A polypeptide according to any of the above embodiments, which is humanized and/or optimized for stability, potency, manufacturability and/or similarity to human framework regions.

[00189] Embodiment 28: A polypeptide according to embodiment 27, which is humanized and/or has sequence optimization in one or more of the following positions (according to Kabat numbering): 1, 11, 14, 16 , 74, 83, 108.

[00190] Embodiment 29: A polypeptide according to embodiment 28, comprising one or more of the following mutations: E1D, S11L, A14P, E16G, A74S, K83R, Q108L.

[00191] Embodiment 30: A polypeptide according to any one of Embodiments 3 to 29, in which:

[00192] FR1 is selected from SEQ ID NO's: 198 to 204;

[00193] ii) FR2 is selected from SEQ ID NO's: 205 to 208;

[00194] iii) FR3 is selected from SEQ ID NO's: 209 to 210; and/or

[00195] FR4 is selected from SEQ ID NO's: 211 to 212.

[00196] Embodiment 31: A polypeptide according to any one of embodiments 3 to 30, which is humanized and/or has sequence optimization at one or more of the following positions (according to Kabat numbering): 52, 53.

[00197] Embodiment 32: A polypeptide according to embodiment 31, comprising one or more of the following mutations: N52S, S53T.

[00198] Embodiment 33: A polypeptide according to any one of Embodiments 3 to 32, in which CDR2 is selected from SEQ ID NO's: 214 to 221.

[00199] Embodiment 34: A polypeptide according to any one of Embodiments 2-33, wherein said anti-CX3CR1 immunoglobulin single variable domain comprises the sequence stipulated in any one of SEQ ID NO's: 138 to 140 or 222 to 224 .

[00200] Embodiment 35: A polypeptide according to any one of Embodiments 22 to 26, wherein said VHH domain consists of any one of said amino acid sequences.

[00201] Embodiment 36: A polypeptide according to any one of Embodiments 2 to 35, wherein said single immunoglobulin variable domain blocks the binding of at least one of the unique immunoglobulin variable domains of SEQ ID NO's: 1 to 120, 121 to 140 and 222 to 224 to CX3CR1.

[00202] Embodiment 37: A polypeptide according to any one of embodiments 2 to 35, wherein said single immunoglobulin variable domain is blocked from *binding to CX3CR1 by at least one of the amino acid sequences of SEQ ID NO's: 1 to 120, 121 to 140 and 222 to 224.

[00203] Embodiment 38: A polypeptide according to any one of Embodiments 2 to 37, wherein the polypeptide further comprises a half-life extending moiety.

[00204] Embodiment 39: A polypeptide according to embodiment 38, wherein said half-life extension moiety is covalently linked to said polypeptide and is selected from the group consisting of an albumin-binding moiety, such such as an anti-albumin immunoglobulin domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin domain, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of serum albumin domain, an albumin-binding peptide or an Fc domain.

[00205] Embodiment 40: A polypeptide according to embodiment 38 or 39, wherein said half-life prolonging portion consists of a single anti-albumin immunoglobulin variable domain.

[00206] Embodiment 41: A polypeptide according to embodiment 40, in which the single immunoglobulin variable domain is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, an antibody domain, a single domain antibody and/or "dAb"s.

[00207] Embodiment 42: A polypeptide according to embodiment 41, in which the single anti-albumin immunoglobulin variable domain is selected from SEQ ID NO's: 230 to 232.

[00208] Embodiment 43: A polypeptide according to any one of Embodiments 2 to 39, wherein said polypeptide is linked to an Fc portion (such as a human Fc, for example, as stipulated in SEQ ID NO: 252), optionally via a suitable linker or a hinge region.

[00209] Embodiment 44: A polypeptide according to any one of Embodiments 2 to 39, wherein said polypeptide is additionally linked to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/ to form an Fc portion), an Fc portion, and/or one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more receptors of Fc, optionally via a suitable linker or a hinge region.

[00210] Embodiment 45: A polypeptide according to any one of Embodiments 2 to 37, wherein said polypeptide further comprises a second single immunoglobulin variable domain, preferably a second single anti-CX3CR1 immunoglobulin variable domain.

[00211] Embodiment 46: A polypeptide according to embodiment 45, wherein said first and said second single immunoglobulin variable domains are covalently linked by a linker peptide.

[00212] Embodiment 47: A polypeptide according to embodiment 45 or 46, wherein said second single immunoglobulin variable domains essentially consist of four framework regions (FR1 to FR4) and three complementarity determining regions (CDR1 to CDR3) .

[00213] Embodiment 48: A polypeptide according to any one of Embodiments 45 to 47, wherein said first and said second single immunoglobulin variable domains are antibody domains.

[00214] Embodiment 49: A polypeptide according to any one of embodiments 45 to 48, wherein said first and second single immunoglobulin variable domains are a VH, a VL, a VHH, a camelized VH, or a VHH that is optimized for stability, power, manufacturability, and/or similarity to human framework regions.

[00215] Embodiment 50: A polypeptide according to any one of embodiments 45 to 49, wherein said CDR1 to CDR3 of said single immunoglobulin variable domain are stipulated in any one of embodiments 11 to 21.

[00216] Embodiment 51: A polypeptide according to any one of Embodiments 45 to 50, wherein said first and said second single immunoglobulin variable domains comprise the same CDR3.

[00217] Embodiment 52: A polypeptide according to embodiment 51, wherein said CDR3 is stipulated in any one of embodiments 11 to 13.

[00218] Embodiment 53: A polypeptide according to any one of Embodiments 45 to 53, wherein said first and said second single immunoglobulin variable domains comprise the same CDR1, CDR2 and CDR3.

[00219] Embodiment 54: A polypeptide according to embodiment 53, wherein said CDR1 to CDR3 are stipulated in any one of embodiments 11 to 21.

[00220] Embodiment 55: A polypeptide according to any one of Embodiments 45 to 54, wherein said first and said second single immunoglobulin variable domains comprise the same VHH domain.

[00221] Embodiment 56: A polypeptide according to any one of embodiments 45 to 55, wherein said VHH domain is stipulated in any one of embodiments 22 to 37.

[00222] Embodiment 57: A polypeptide comprising a first single immunoglobulin variable domain which comprises the CDR1, the CDR2 and the CDR3 stipulated in SEQ ID NO's: 141, 164 and 186 or SEQ ID NO's: 141, 162 and 186 and a second single immunoglobulin variable domain as stipulated in any of Embodiments 2 to 37.

[00223] A similar polypeptide may in particular be a polypeptide according to any one of embodiments 45 to 56.

[00224] Embodiment 58: A polypeptide according to embodiment 57, wherein said first single immunoglobulin variable domain comprises the CDR1, the CDR2 and the CDR3 stipulated in SEQ ID NO's: 213, 214 and 186, SEQ ID NO's: 213, 221 and 186 or SEQ ID NO's: 141, 162 and 186.

[00225] Embodiment 59: A polypeptide according to embodiment 57 or 58, wherein said single immunoglobulin variable domain comprises the CDR1, the CDR2 and the CDR3 stipulated in SEQ ID NO's: 141, 164 and 186 or SEQ ID NO's: 141, 162 and 186.

[00226] Embodiment 60: A polypeptide according to embodiment 57 or 58, wherein said single immunoglobulin variable domain comprises CDR1, CDR2 and CDR3 stipulated in: SEQ ID NO's: 213, 214 and 186, SEQ ID NO's: 213, 221 and 186 or SEQ ID NO's: 141, 162 and 186.

[00227] Embodiment 61: A polypeptide comprising a first single immunoglobulin variable domain, wherein said first single immunoglobulin variable domain is a VHH domain comprising the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 and a second single immunoglobulin variable domain according to any of Embodiments 2 to 37.

[00228] A similar polypeptide may in particular be a polypeptide according to any one of embodiments 45 to 60.

[00229] Embodiment 62: A polypeptide according to embodiment 61, wherein said first single immunoglobulin variable domain is a VHH domain comprising the sequence stipulated in any one of SEQ ID NO: 121 to 140 or 222 to 224 .

[00230] Embodiment 63: A polypeptide according to embodiment 61 or 62, wherein said single immunoglobulin variable domain is a VHH domain comprising the sequence stipulated in SEQ ID NO: 1 or SEQ ID NO: 3.

[00231] Embodiment 64: A polypeptide according to embodiment 63, wherein said single immunoglobulin variable domain is a VHH domain comprising the sequence stipulated in any one of SEQ ID NO: 121 to 140 or 222 to 224.

[00232] Embodiment 65: A polypeptide according to any one of Embodiments 45 to 64, wherein the polypeptide further comprises a half-life extending moiety.

[00233] Embodiment 66: A polypeptide according to embodiment 65, wherein said half-life extension moiety is covalently linked to said polypeptide and is selected from the group consisting of an albumin-binding moiety, such such as an anti-albumin immunoglobulin domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin domain, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of serum albumin domain, an albumin-binding peptide or an Fc domain.

[00234] Embodiment 67: A polypeptide according to embodiment 66, wherein said half-life prolonging portion consists of a single anti-albumin immunoglobulin variable domain.

[00235] Embodiment 68: A polypeptide according to embodiment 67, wherein the single immunoglobulin variable domain is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, an antibody domain, a single domain antibody and/or "dAb"s.

[00236] Embodiment 69: A polypeptide according to embodiment 68, in which the single anti-albumin immunoglobulin variable domain is selected from SEQ ID NO's: 230 to 232.

[00237] Embodiment 70: A polypeptide according to any one of Embodiments 45 to 64, wherein said polypeptide is linked to an Fc moiety (such as a human Fc, for example, as stipulated in SEQ ID NO: 252), optionally via a suitable linker or a hinge region.

[00238] Embodiment 71: A polypeptide according to any one of Embodiments 45 to 66, wherein said polypeptide is additionally linked to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/ to form an Fc portion), an Fc portion, and/or one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more receptors of Fc, optionally via a suitable linker or a hinge region.

[00239] Embodiment 72: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 225 to 227.

[00240] Embodiment 73: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 249 or 277 to 281.

[00241] Embodiment 74: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 257 to 262.

[00242] Embodiment 75: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 253 or 254.

[00243] Embodiment 76: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 263 or 266.

[00244] Embodiment 77: A polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 267 to 276 and 282.

[00245] Embodiment 78: A nucleic acid molecule comprising a region encoding a polypeptide according to any one of Embodiments 1 to 77.

[00246] Embodiment 79: An expression vector comprising a nucleic acid molecule according to embodiment 78.

[00247] Embodiment 80: A host cell that carries an expression vector comprising a nucleic acid molecule, said nucleic acid molecule comprising a region encoding a polypeptide according to any one of embodiments 1 to 77, wherein said cell host is capable of expressing a polypeptide according to any one of embodiments 1 to 77, and wherein said host cell is a prokaryotic cell or a eukaryotic cell.

[00248] Embodiment 81: A pharmaceutical composition comprising (i) as the active ingredient, one or more polypeptides according to any one of Embodiments 1 to 77, and (ii) a pharmaceutically acceptable carrier, and optionally (iii) a diluent, an excipient, an adjuvant and/or a stabilizer.

[00249] Embodiment 82: A method of manufacturing a polypeptide according to any one of Embodiments 1 to 77, comprising the steps of

[00250] - cultivating a host cell under conditions that allow the expression of a polypeptide according to any one of embodiments 1 to 77,

[00251] wherein said host cell carrying an expression vector comprising a nucleic acid molecule, said nucleic acid molecule comprising a region encoding a polypeptide according to any one of embodiments 1 to 77, and wherein said host cell is a prokaryotic cell or a eukaryotic cell;

[00252] - recovering said polypeptide; It is

[00253] - Purify said polypeptide.

[00254] Embodiment 83: A method of using a polypeptide according to any one of Embodiments 1 to 77 for treating, preventing or mitigating a disease, disorder or condition, in particular in a human being.

[00255] Embodiment 84: The method of embodiment 83, wherein said disease, said disorder or said condition is a disease, disorder or condition associated with CX3CR1.

[00256] Embodiment 85: The method of Embodiment 83, wherein said disease, said disorder or said condition is atherosclerosis.

[00257] Embodiment 86: An injectable pharmaceutical composition comprising the polypeptide according to any one of Embodiments 1 to 77, said composition being suitable for intravenous or subcutaneous injection into a human.

[00258] Embodiment 87: A method for preventing and/or treating a disease or a disorder that is associated with CX3CR1, wherein said method comprises administering to a subject in need thereof a pharmaceutically active amount of at least one polypeptide according with any of the modalities 1 to 77.

[00259] Embodiment 88: A method of embodiment 85, further comprising administering an additional therapeutic agent selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an antihypertensive.

[00260] Embodiment 89: A method for inhibiting CX3CR1 binding to fractalkine in a mammalian cell, comprising administering to the cell a polypeptide according to any one of embodiments 1 to 77, whereby fractalkine-mediated signaling is inhibited .

[00261] Embodiment 90: A method for detecting and/or quantifying CX3CR1 levels in a biological sample by contacting the sample with a polypeptide according to any one of Embodiments 1 to 77 and detecting binding of the polypeptide with CX3CR1.

[00262] Embodiment 91: A method for diagnosing a disorder associated with CX3CR1 or for determining whether an individual has an increased risk of developing a disorder associated with CX3CR1, wherein the method comprises contacting a biological sample from an individual with a polypeptide according with any one of embodiments 1 through 77 and detecting binding of the polypeptide to CX3CR1 to determine CX3CR1 expression or concentration.

[00263] Embodiment 92. A polypeptide according to any one of Embodiments 1 to 77 for use in treating, preventing or mitigating a disease, disorder or condition in a human.

[00264] Embodiment 93. The polypeptide for use according to embodiment 92, wherein the disease, disorder, or condition is a disease, disorder, or condition associated with CX3CR1.

[00265] Embodiment 94. The polypeptide for use according to embodiment 92, wherein the disease, disorder or condition is selected from atherosclerotic cardio- and cerebrovascular disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescent 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, lung disease such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer.

[00266] Embodiment 95. The polypeptide for use according to embodiment 92, wherein the disease, disorder, or condition is atherosclerosis.

[00267] Embodiment 96. Use of a polypeptide according to any one of Embodiments 1 to 77 for the manufacture of a medicament for the treatment, prevention or mitigation of a disease, a disorder or a condition, in a human being.

[00268] Embodiment 97. The method according to embodiment 87, in which the disease or disorder is selected from cardio- and cerebrovascular atherosclerotic disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, nephropathy by IgA, 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, lung diseases such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer.

[00269] Embodiment 98. The method according to embodiment 87, wherein the disease, disorder, or condition is atherosclerosis.

[00270] Embodiment 99. A diagnostic kit or a diagnostic method comprising a polypeptide according to any one of Embodiments 1 to 77, or the use thereof.

[00271] Modality 100. A diagnostic kit or a diagnostic method according to modality 99, for the diagnosis of at least one of cardio- and cerebrovascular atherosclerotic disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescent 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 disease demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[00272] The above and other aspects and embodiments of the invention will become evident from the description that follows here, in this patent application, in which:

[00273] Unless otherwise indicated or defined, all terms used have their usual meaning in the art which will be apparent to the skilled person. Reference is made, for example, to routine manuals such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed.), VoIs. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), as well as general background of the art cited here, in this patent application; Furthermore, unless otherwise indicated, all methods, steps, techniques and manipulations which are not specifically described in detail can be performed and have been performed in a manner known per se, as will be apparent to the person skilled in the art. Again reference is made to, for example, the routine manuals, the general background in the art referred to above and the additional references cited therein;

[00274] b) Unless otherwise indicated, the terms "immunoglobulin" and "immunoglobulin sequence" - whether used herein, in this patent application, to refer to a heavy chain antibody or an antibody of 4 conventional chains - are used as general terms to include both the full length 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, in this patent application, (e.g., in terms such as "immunoglobulin sequence", "antibody sequence", "variable domain (single) sequence", "variable domain sequence", of VHH" or "protein sequence"), will be understood generally 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;

[00275] c) The term "domain" (of a polypeptide or a protein) as used herein in this patent application refers to a folded protein structure which has the ability to retain its tertiary structure independently of the rest of the protein . In general, domains are responsible for distinct functional properties of proteins, and in many cases they can be added, removed or transferred to other proteins without loss of function of the rest of the protein and/or domain.

[00276] d) The term "immunoglobulin domain" as used herein in this patent application refers to a globular region of an antibody chain (such as, for example, a chain of a conventional 4-chain antibody or of a heavy chain antibody), or to a polypeptide that essentially consists of a similar globular region. Immunoglobulin domains are characterized by the fact that they retain the characteristic immunoglobulin fold 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 binding of conserved disulfide.

[00277] e) The term "immunoglobulin variable domain" as used herein in this patent application means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and in the parts that follow 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; the framework regions of which are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and in the parts that follow as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. They are the one or more immunoglobulin variable domains that confer specificity to an antibody for the antigen carrying the antigen-binding site.

[00278] f) The terms "single immunoglobulin variable domain" and "single variable domain" as used herein, in this patent application, mean an immunoglobulin variable domain which is capable of binding specifically to an unpaired antigen epitope. with an additional immunoglobulin variable domain. An example of single immunoglobulin variable domains within the meaning of the present invention are "domain antibodies", such as the VH and VL single immunoglobulin variable domains (VH domains and VL domains). Another example of unique immunoglobulin variable domains are camelid "VHH domains" (or simply "VHHs"), as defined in the parts that follow.

[00279] In view of the above definition, the antigenic 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 disulfide-linked Fv fragment or scFv fragment, or a diabody (all known in the art) derived from a similar conventional 4-chain antibody would normally not be considered as a single immunoglobulin variable domain, since in these cases binding to the respective epitope of an antigen would normally not occur by a (single) immunoglobulin domain but by a pair of immunoglobulin domains (in association) such as domains variable light and heavy chains, that is, by a VH-VL pair of immunoglobulin domains, which together bind to an epitope of the respective antigen.

[00280] f1) "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, were originally described as the immunoglobulin antigen-binding (variable) domain of "heavy chain antibodies" ( that is, of "antibodies devoid of light chains"; Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" was 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 "VH domains" or "VH domains" in this patent application). VH") and the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains" or "VL domains"). VHH domains can specifically bind to an epitope without an additional antigen-binding domain (unlike VH or VL domains in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain along with a VH domain) . VHH domains are small, robust and efficient antigenic recognition units formed by a single immunoglobulin domain.

[00281] In the context of the present invention, the terms VHH domain, VHH, VHH domain, VHH antibody fragment, VHH antibody, as well as "Nanobody®" and "Nanobody® domain" ("Nanobody" being a registered trademark of the company Ablynx N.V.; Ghent; Belgium) are used interchangeably and are typical of single immunoglobulin variable domains (having the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and binding specifically to an epitope without requiring the presence of an epitope). second immunoglobulin variable domain), and which are distinguished from VH domains by so-called "hallmark residues", as defined, for example, in International Patent Publication No. WO2009/109635, Fig. 1.

[00282] The amino acid residues of a VHH domain are numbered according to the general numbering for VH domains provided by Kabat et al. ("Sequence of proteins of immunological interest")"", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to the VHH domains of Camelids, as shown by for example, in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999). According to this numbering,

[00283] - FR1 comprises amino acid residues at positions 1 to 30,

[00284] - CDR1 comprises amino acid residues at positions 31 to 35,

[00285] - FR2 comprises amino acids at positions 36 to 49,

[00286] - CDR2 comprises amino acid residues at positions 50 to 65,

[00287] - FR3 comprises amino acid residues at positions 66 to 94,

[00288] - CDR3 comprises amino acid residues at positions 95 to 102, and

[00289] - FR4 comprises amino acid residues at positions 103 to 113.

[00290] However, it should be noted that - as is common knowledge in the art for VH 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. than indicated by the Kabat numbering (i.e., 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 by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[00291] Alternative methods for numbering the amino acid residues of VH 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 will be followed and applied to the VHH domains as described above, unless otherwise indicated.

[00292] The total number of amino acid residues in a VHH domain will generally be in the range from 110 to 120, often between 112 and 115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein in this patent application.

[00293] Additional structural features and functional properties of VHH domains and polypeptides containing the same can be summarized as follows:

[00294] 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 structural unit, as a domain or as a functional antigen-binding polypeptide. This distinguishes the VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suitable for practical application as single antigenic binding proteins or single immunoglobulin variable domains, but need to be combined. in some form or other so as to provide a functional antigenic binding moiety (as for example, in conventional antibody fragments such as Fab fragments; in scFv's, which consist of a VH domain covalently linked to a VL domain).

[00295] Due to 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):

[00296] - only a single domain is needed to bind an antigen with high affinity and with high selectivity, so there is no need to have two separate domains present, nor to ensure that these two domains are present in spatial conformation and in the correct configuration (ie, through the use of specially designed chainers, conforming to scFv's);

[00297] - VHH domains can be expressed from a single gene and do not require post-translational folding or modifications;

[00298] - VHH domains can be easily manipulated to multivalent and multispecific formats (as further discussed here, in this patent application);

[00299] - VHH domains are highly soluble and do not have a tendency to aggregate (as per the mouse-derived antigenic binding domains described by Ward et al., Nature 341: 544-546 (1989));

[00300] - VHH domains are highly stable to heat, pH, proteases and other conditions or denaturing agents and, therefore, can be prepared, stored or transported without the use of refrigeration equipment, transferring savings in costs, time and environmental;

[00301] - VHH domains are easy and relatively inexpensive to prepare, even at the scale required for production. For example, VHH domains and polypeptides containing them can be produced using microbial fermentation (e.g., as further described below) and do not require the use of mammalian expression systems, conforming with, e.g., conventional antibody fragments;

[00302] - VHH domains are relatively small (approximately 15 kDa, or 10 times smaller than a conventional IgG) compared to conventional 4-chain antibodies and antigenic binding fragments thereof, and therefore

[00303] -- have high (greater) penetration into tissues and

[00304] -- can be given in larger doses

[00305] than said conventional 4-chain antibodies and antigen-binding fragments thereof;

[00306] - VHH domains can present so-called cavity binding properties (among others due to their extended CDR3 loop, compared to conventional VH domains) and therefore can also access targets and epitopes not accessible to conventional 4-chain antibodies and fragments their antigenic binding.

[00307] Methods of obtaining VHH domains binding to an antigen or a specific epitope have been previously described, for example, in International Patent Publications Nos. 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 with one or more of the amino acid residues that occur at the corresponding position or in the corresponding positions in a VH domain of a conventional 4-chain antibody from a human. A humanized VHH domain may contain one or more fully human framework region sequences, and, in an even more specific embodiment, may contain human framework region sequences derived from DP-29, DP-47, DP-51, or parts thereof, optionally combined with JH sequences, such as JH5.

[00308] f2) "Domain Antibodies", also known as "Dab"s, "Domain Antibodies", and "dAbs" (the terms "Domain Antibodies" and "dAbs" being used as trademarks by the GlaxoSmithKline group of companies ) have been described, for example, in Ward, E.S., et al.: "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli"; Nature 341: 544-546 (1989); Holt, L.J. et al.: "Domain antibodies: proteins for therapy"; TRENDS in Biotechnology 21(11): 484-490 (2003); and in International Patent Publication No. WO2003/002609.

[00309] Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammals, in particular human 4-chain antibodies. In order to bind an epitope as a single antigen-binding domain, i.e. without being paired with a VL or VH domain, respectively, specific selection for similar antigenic-binding properties is required, e.g. using libraries of domain sequences Human single VH or VL.

[00310] Domain antibodies have, like VHHs, a molecular weight of about 13 to about 16 kDa and, if derived from fully human sequences, do not require humanization for, for example, therapeutic use in humans. As in the case of VHH domains, they are also well expressed in prokaryotic expression systems, providing a significant reduction in the total manufacturing cost.

[00311] Domain antibodies, as well as VHH domains, can be subjected to affinity maturation by introducing one or more changes to the amino acid sequence of one or more CDRs, which changes result in an improved affinity of the resulting single immunoglobulin variable domain by its respective antigen, in comparison with the respective parent molecule. Affinity-matured single immunoglobulin variable domain molecules of the invention can be prepared by methods known in the art, for example, as described by Marks et al., 1992, Biotechnology 10:779-783, or Barbas, et al., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3): 889 896; KS Johnson and RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[00312] f3) Furthermore, it will also be apparent to the person concerned that it is possible to "graft" one or more of the CDR's mentioned above onto other "scaffolds", including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for said CDR grafting are known in the art.

[00313] g) The terms "epitope" and "antigenic determinant", which may be used interchangeably, refer to the part of a macromolecule, such as a polypeptide, that is recognized by antigenic binding molecules, such as antibodies conventional peptides or the polypeptides of the invention, and more particularly by the antigenic binding site of said molecules. Epitopes define the minimal binding site for an immunoglobulin, and therefore represent the specificity target of an immunoglobulin.

[00314] The part of an antigenic binding molecule (such as a conventional antibody or a polypeptide of the invention) that recognizes the epitope is called a paratope.

[00315] h) The term "biparatopic" (antigenic) binding molecule or "biparatopic" polypeptide as used herein, in this patent application, shall mean a polypeptide comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as defined herein, in this patent application, wherein these two variable domains are capable of binding to two different epitopes of an antigen, which epitopes are not normally bound at the same time by a monospecific immunoglobulin, such as, for example, an antibody conventional or a single immunoglobulin variable domain. Biparatopic polypeptides may be composed of variable domains which have different epitope specificities, and do not contain pairs of mutually complementary variable domains which bind to the same epitope. The two variable domains therefore do not compete with each other for target binding.

[00316] i) A polypeptide (such as an immunoglobulin, an antibody, a single immunoglobulin variable domain, a polypeptide of the invention, or generally an antigenic binding molecule or fragment thereof) that can "bind to" or "bind specifically for", which "has affinity for" and/or which "has specificity for" a given epitope, a given antigen or a given protein (or at least a part, a fragment or an epitope thereof) it is said that is "against" or "directed against" said epitope, said antigen or said protein or is a "binding" molecule with respect to like epitope, antigen or protein, or is said to be "anti"-epitope, "anti"-antigen or "anti"-protein (eg, anti-CX3CR1).

[00317] k) Generally, the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigenic binding molecule or antigenic binding protein (such as an immunoglobulin, an antibody, a single immunoglobulin 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. Affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigenic binding protein (KD), is a measure of the strength of binding between an epitope and an antigenic binding site on the antigenic binding protein: the smaller the KD value, the greater the binding strength between an epitope and the antigenic binding molecule (alternatively, affinity can also be expressed as the affinity constant (KA), which is 1/KD). As will be evident to the person skilled in the art (for example, based on the further description herein, in this patent application), the affinity can be determined in a manner known per se, depending on the specific antigen of interest. Avidity is a measure of the strength of binding between an antigenic 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 both to the affinity between an epitope and its antigenic binding site on the antigenic binding molecule and to the number of pertinent binding sites present on the antigenic binding molecule.

[00318] l) Sedron amino acid residues indicated according to the standard three-letter or one-letter amino acid code, as is generally known and agreed 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 one position in the reference sequence, compared to a second sequence. In the case of one or more substitutions, the one or more substitutions referred to will preferably be one or more conservative amino acid substitutions, meaning that an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence about the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions referred to are well known in the art, for example in International Patent Publication No. WO 98/49185, where conservative amino acid substitutions preferably are substitutions in which an amino acid within groups (i) a (v) that follows is replaced by another amino acid residue within the same group: (i) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and GIy; (ii) polar, negatively charged residues and their amides (uncharged): Asp, Asn, GIu and GIn; (iii) polar, positively charged residues: His, Arg and Lys; (iv) large non-polar aliphatic residues: Met, Leu, Ile, VaI and Cys; and (v) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows:

[00319] Ala for GIy or Ser;

[00320] Arg for Lys;

[00321] Asn for GIn or for His;

[00322] Asp for GIu;

[00323] Cys to Ser;

[00324] GIn to Asn;

[00325] GIu to Asp;

[00326] GIy for Wing or for Pro;

[00327] His for Asn or for GIn;

[00328] Ile to Leu or to Val;

[00329] Read to Ile or to Val;

[00330] Lys for Arg, for GIn or for GIu;

[00331] Met for Leu, for Tyr or for Ile;

[00332] Phe to Met, to Leu or to Tyr;

[00333] Ser to Thr;

[00334] Thr to Ser;

[00335] Trp to Tyr;

[00336] Tyr to Trp or to Phe;

[00337] Go to Ile or Leu.

[00338] m) A polypeptide or nucleic acid molecule is considered to be "essentially isolated (in form)" - for example, when compared to its native biological source and/or the reaction medium or culture medium from the from which it was obtained - when it has been separated from at least one other component with which it is generally associated in said source or in said medium, such as another nucleic acid, other protein/polypeptide, other biological component or macromolecule or at least one contaminant, impurity or minor component. In particular, a polypeptide or nucleic acid molecule is considered "essentially isolated" when it has been purified at least 2 times, in particular at least 10 times, more in particular at least 100 times, and up to 1000 times or more. A polypeptide or nucleic acid molecule that is "in essentially isolated form" is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographic technique, such as polyacrylamide gel electrophoresis;

[00339] n) "Sequence identity" between e.g. two sequences of unique immunoglobulin variable domains indicates the percentage of amino acids that are identical between these two sequences. It can be calculated or determined as described in paragraph f) on pages 49 and 50 of International Patent Application No. WO08/020079. "Sequence similarity" indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions.

target specificity

[00340] The polypeptides of the invention have specificity for human CX3CR1. Thus, polypeptides of the invention preferentially bind to human CX3CR1 (SEQ ID NO: 255). In one aspect, the polypeptides of the present invention also bind to cynomolgus CX3CR1 (SEQ ID NO: 256).

Polypeptides of the invention

[00341] The invention provides new pharmaceutically active agents for the prevention, treatment, mitigation and/or diagnosis of diseases, disorders or conditions associated with CX3CR1, such as cardiovascular diseases. In particular, the invention provides polypeptides which bind human CX3CR1 and are capable of blocking human fractalkine binding to human CX3CR1. In one aspect, the polypeptide is an immunoglobulin which comprises an antigenic binding domain which comprises three complementarity determining regions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1. In a further aspect, the polypeptide comprises one or more unique anti-CX3CR1 immunoglobulin variable domains, wherein said polypeptide is capable of blocking human fractalkine binding to human CX3CR1.

[00342] In one aspect, a polypeptide of the present invention is characterized by one or more of the following properties: • It binds with high affinity to human CX3CR1, for example, at an EC50 of less than or equal to 10 nM, less than or equal to at 5 nM, less than or equal to 2.5 nM or less than or equal to 1 nM, as determined by cell binding FACS; • Blocks binding of human fractalkine to human CX3CR1, for example, at an IC50 of less than or equal to 300 nM, or less than or equal to 100 nM, or less than or equal to 20 nM, or less than or equal to 10 nM, or less than or equal to 5 nM, or less than or equal to 2.5 nM or less than or equal to 1 nM; • Inhibits fractalkine-induced chemotaxis mediated by human CX3CR1, for example, at an IC50 of less than or equal to 500 nM, or less than or equal to 100 nM, or less than or equal to 75 nM, or less than or equal to 50 nM, or less than or equal to 10 nM or less than or equal to 5 nM; the inhibition effectiveness obtained is greater than or equal to 15%, or greater than or equal to 50%, or greater than or equal to 80%, or greater than or equal to 95%; • Inhibits fractalkine-induced internalization mediated by human CX3CR1, eg, at an IC50 of less than or equal to 10 nM or less than or equal to 5 nM; • Cross-reacts with cynomolgus CX3CR1, eg, within 10 fold of the E/IC50 for human CX3CR1 for binding and functional inhibition.

[00341] In a further aspect, a polypeptide of the present invention further comprises a half-life extending moiety, for example, an albumin-binding moiety, a polyethylene glycol molecule or an Fc domain. In a further aspect, a polypeptide of the present invention comprises two or more unique anti-CX3CR1 immunoglobulin variable domains. In one aspect, the two unique variable domains of anti-CX3CR1 immunoglobulin are covalently linked together by a linker peptide. In one aspect, the two unique anti-CX3CR1 immunoglobulin variable domains in a polypeptide of the present invention have the same amino acid sequence. In another aspect, the two unique anti-CX3CR1 immunoglobulin variable domains in a polypeptide of the present invention have different amino acid sequences. In one aspect, a polypeptide of the present invention comprises two unique anti-CX3CR1 immunoglobulin variable domains and further comprises a half-life extending moiety, e.g., an albumin-binding moiety, a polyethylene glycol molecule, or a DNA domain. Fc.

[00342] In one aspect, a polypeptide of the present invention comprises a first single anti-CX3CR1 immunoglobulin variable domain covalently linked to an albumin-binding moiety by a first linker peptide, wherein said albumin-binding moiety is further covalently linked to a second unique anti-CX3CR1 immunoglobulin variable domain by a second linker peptide.

[00343] In one aspect, a polypeptide of the present invention comprises a single anti-CX3CR1 immunoglobulin variable domain covalently linked to an Fc domain by a linker peptide. In one aspect, a similar polypeptide comprising a single anti-CX3CR1 immunoglobulin variable domain covalently linked to an Fc domain by a linker peptide is provided as a dimer, for example, via disulfide bridges.

[00344] Polypeptides according to the present invention are obtained as described in the following parts. In summary, unique variable domains of the present invention were identified from a library expressing unique variable domains (VHH) derived from a llama immunized with DNA encoding human CX3CR1. The phage library was panned over hCX3CR1 viral lipoparticles and binding phage were screened for their ability to compete for receptor binding with Alexa-fluor-tagged fractalkine (AF-FKN). Typical unique variable domains of the present invention are described here, in this patent application, in further detail.

[00345] In one aspect, a single immunoglobulin variable domain of the present invention essentially consists of four framework regions (FR1, FR2, FR3 and FR4) and three complementarity determining regions (CDR1, CDR2 and CDR3). In particular, the single immunoglobulin variable domain has the structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. In one aspect, the single immunoglobulin variable domain is an antibody domain.

[00346] In one aspect, the CDR3 of a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4- Xaa5 as stipulated in SEQ ID NO: 197, where:

[00347] - Xaa1 is Pro, Ala or Gly;

[00348] - Xaa2 is Asp or Asn;

[00349] - Xaa3 is Thr or Ser;

[00350] - Xaa4 is Arg, Lys, Ala or Gly; It is

[00351] - Xaa5 is Tyr or Phe.

[00352] In one aspect, the CDR3 of a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4 -Xaa5 as stipulated in SEQ ID NO: 197, where:

[00353] - Xaa1 is Pro, Ala or Gly;

[00354] - Xaa2 is Asp or Asn;

[00355] - Xaa3 is Thr;

[00356] - Xaa4 is Arg or Lys; It is

[00357] - Xaa5 is Tyr.

[00358] In one aspect, the CDR3 of a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg -Tyr as stipulated in SEQ ID NO: 186.

[00359] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the following CDR1, CDR2 and CDR3:

[00360] - CDR1:

[00361] a) has the amino acid sequence of GSIFSSNAMA (SEQ ID NO: 141); or

[00362] b) has an amino acid sequence that has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 141; or

[00363] c) has an amino acid sequence that is 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 141, wherein

[00364] - in position 2 the S was changed to T, or G;

[00365] - in position 6 the S was changed to R;

[00366] - in position 7 the N was changed to T; and/or

[00367] - in position 9 the M was changed to K; or

[00368] d) has an amino acid sequence selected from any one of SEQ ID NO's: 141 to 145 and 213;

[00369] - CDR2:

[00370] a) has the amino acid sequence of GINSVGITK (SEQ ID NO: 164); or

[00371] b) has an amino acid sequence that has at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 164; or

[00372] c) has an amino acid sequence that is 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 164, wherein

[00373] - in position 1 the G was changed to A, L, V or S;

[00374] - in position 3 the N was changed to D, S, Q, G or T;

[00375] - in position 4 the S was changed to T, K, G or P;

[00376] - in position 5 the V was changed to A;

[00377] - in position 6 the G was changed to D;

[00378] - in position 7 the I was changed to T, or V;

[00379] - in position 8 the T was changed to A; and/or

[00380] - in position 9 K was changed to R; or

[00381] d) has an amino acid sequence selected from any one of SEQ ID NO's: 162 to 175 and 214 to 221; It is

[00382] - CDR3:

[00383] a) has the amino acid sequence of DPRRGWDTRY (SEQ ID NO: 186); or

[00384] b) has an amino acid sequence that has at least 70% amino acid identity with the amino acid sequence of SEQ ID NO: 186; or

[00385] c) has an amino acid sequence that is 3, 2, or 1 amino acid different from the amino acid sequences of SEQ ID NO: 186, wherein

[00386] - in position 2 the P was changed to A, or G;

[00387] - in position 7 the D was changed to N; and/or

[00388] - in position 9 the R was changed to K; or

[00389] d) has an amino acid sequence selected from any of SEQ ID NO's: 186 to 190.

[00390] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the following CDR1, CDR2 and CDR3, wherein:

[00391] - said CDR1 has the amino acid sequence of GRTFSSYAMG (SEQ ID NO: 146);

[00392] - said CDR2 has an amino acid sequence that a) has at least 90% amino acid identity with the amino acid sequence of GISGSASRKY (SEQ ID NO: 176) or b) has the amino acid sequence of SEQ ID NO : 176 or 177; It is

[00393] - said CDR3 has the amino acid sequence of SNSYPKVQFDY (SEQ ID NO: 191).

[00394] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the following CDR1, CDR2 and CDR3:

[00395] - the aforementioned CDR1:

[00396] a) has the amino acid sequence of GTIFSNNAMG (SEQ ID NO: 147); or

[00397] b) has an amino acid sequence that is 6, 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 147, wherein

[00398] - in position 1 the G was changed to K, R, or A;

[00399] - in position 2 the T was changed to I, P, S or L;

[00400] - in position 3 the I was changed to V, or T;

[00401] - in position 4 the F was changed to L;

[00402] - in position 5 the S was changed to R, or D;

[00403] - in position 6 the N was changed to S, T, or D; and/or

[00404] - in position 7 the N was changed to T, or Y; or

[00405] c) has an amino acid sequence selected from any of SEQ ID NO's: 147 to 161;

[00406] - the aforementioned CDR2:

[00407] a) has the amino acid sequence of SISNSGSTN (SEQ ID NO: 179); or

[00408] b) has an amino acid sequence that is 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 179, wherein

[00409] - in position 3 the S was changed to T, or G;

[00410] - in position 4 the N was changed to S, or I;

[00411] - in position 5 the S was changed to T;

[00412] - in position 6 the G was changed to Y; and/or

[00413] - in position 8 the T was changed to A; or

[00414] c) has an amino acid sequence selected from any of SEQ ID NO's: 178 to 185; It is

[00415] - the aforementioned CDR3:

[00416] a) has the amino acid sequence of DARRGWNTAY (SEQ ID NO: 192); or

[00417] b) has an amino acid sequence that has at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 192; or

[00418] c) has an amino acid sequence that is 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 192, wherein

[00419] - in position 2 the A was changed to G;

[00420] - in position 8 the T was changed to S;

[00421] - in position 9 the A was changed to G; and/or

[00422] - in position 10 the Y was changed to F; or

[00423] d) has an amino acid sequence selected from any of SEQ ID NO's: 192 to 196.

[00424] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the following CDR1, CDR2 and CDR3:

[00425] - SEQ ID No: 141, 162 and 186, respectively or

[00426] - SEQ ID No: 141, 163 and 187, respectively; or

[00427] - SEQ ID No: 141, 164 and 186, respectively or

[00428] - SEQ ID No: 141, 166 and 186, respectively or

[00429] - SEQ ID No: 141, 167 and 186, respectively or

[00430] - SEQ ID No: 141, 167 and 189, respectively or

[00431] - SEQ ID No: 141, 168 and 186, respectively; or

[00432] - SEQ ID No: 141, 168 and 187, respectively or

[00433] - SEQ ID No: 141, 169 and 190, respectively or

[00434] - SEQ ID No: 141, 170 and 186, respectively or

[00435] - SEQ ID No: 141, 171 and 186, respectively or

[00436] - SEQ ID No: 141, 174 and 186, respectively or

[00437] - SEQ ID No: 141, 175 and 187, respectively or

[00438] - SEQ ID No: 142, 165 and 188, respectively or

[00439] - SEQ ID No: 142, 173 and 188, respectively or

[00440] - SEQ ID No: 143, 164 and 186, respectively or

[00441] - SEQ ID No: 144, 172 and 187, respectively or

[00442] - SEQ ID No: 145, 172 and 187, respectively or

[00443] - SEQ ID No: 141, 214 and 186, respectively or

[00444] - SEQ ID No: 141, 215 and 186, respectively or

[00445] - SEQ ID No: 141, 216 and 186, respectively or

[00446] - SEQ ID No: 141, 217 and 186, respectively or

[00447] - SEQ ID No: 141, 218 and 186, respectively; or

[00448] - SEQ ID No: 141, 219 and 186, respectively or

[00449] - SEQ ID No: 141, 220 and 186, respectively or

[00450] - SEQ ID No: - SEQ ID No: 213, 221 and 186, respectively or

[00451] - SEQ ID No: 213, 214 and 186, respectively.

[00452] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the following CDR1, CDR2 and CDR3:

[00453] - SEQ ID No: 146, 176 and 191, respectively; or

[00454] - SEQ ID No: 146, 177 and 191, respectively.

[00455] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the following CDR1, CDR2 and CDR3:

[00456] - SEQ ID No: 147, 178 and 192, respectively or

[00457] - SEQ ID No: 147, 179 and 192, respectively or

[00458] - SEQ ID No: 147, 179 and 194, respectively; or

[00459] - SEQ ID No: 148, 179 and 193, respectively or

[00460] - SEQ ID No: 149, 179 and 192, respectively or

[00461] - SEQ ID No: 149, 180 and 192, respectively or

[00462] - SEQ ID No: 149, 181 and 192, respectively or

[00463] - SEQ ID No: 149, 183 and 192, respectively; or

[00464] - SEQ ID No: 149, 185 and 192, respectively or

[00465] - SEQ ID No: 150, 179 and 194, respectively or

[00466] - SEQ ID No: 150, 182 and 194, respectively or

[00467] - SEQ ID No: 151, 179 and 193, respectively or

[00468] - SEQ ID No: 151, 182 and 194, respectively; or

[00469] - SEQ ID No: 151, 184 and 196, respectively or

[00470] - SEQ ID No: 152, 179 and 195, respectively or

[00471] - SEQ ID No: 153, 179 and 194, respectively or

[00472] - SEQ ID No: 154, 182 and 194, respectively or

[00473] - SEQ ID No: 155, 179 and 195, respectively or

[00474] - SEQ ID No: 156, 181 and 192, respectively or

[00475] - SEQ ID No: 157, 179 and 194, respectively or

[00476] - SEQ ID No: 158, 179 and 192, respectively or

[00477] - SEQ ID No: 159, 178 and 192, respectively or

[00478] - SEQ ID No: - SEQ ID No: 160, 179 and 194, respectively or

[00479] - SEQ ID No: 161, 179 and 194, respectively.

[00480] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the CDR1, the CDR2 and the CDR3 stipulated in:

[00481] - SEQ ID NO's: 141, 164 and 186; or

[00482] - SEQ ID NO's: 141, 162 and 186.

[00483] In a further aspect, a polypeptide of the present invention, in particular an immunoglobulin single domain of the present invention, has the CDR1, the CDR2 and the CDR3 stipulated in:

[00484] - SEQ ID NO's: 213, 214 and 186; or

[00485] - SEQ ID NO's: 213, 221 and 186; or

[00486] - SEQ ID NO's: 141, 162 and 186.

[00487] Typical polypeptides of the present invention having the CDRs described above are shown in Tables 1, 2, 3 (typical polypeptides of families 101, 9 and 13, respectively) and 4 (typical polypeptides of optimized variants of family 101. TABLE 1: FAMILY 101

[00488] *CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Sequence numbers in the Table (SEQ) refer to the sequences in the listing sequences of this application. TABLE 2: FAMILY 9

[00489] *CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Sequence numbers in the Table (SEQ) refer to the sequences in the listing sequences of this application. TABLE 3: FAMILY 13

[00490] *CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Sequence numbers in the Table (SEQ) refer to the sequences in the listing sequences of this application. TABLE 4: OPTIMIZED VARIANTS

[00491] *CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010. Sequence numbers in the Table (SEQ) refer to the sequences in the listing sequences of this application.

[00492] In a further aspect, the present invention provides polypeptides having one or more VHH domains.

[00493] In one aspect, a VHH domain of the present invention comprises or essentially consists of the sequence stipulated in:

[00494] a) the amino acid sequence of SEQ ID NO: 3; or

[00495] b) amino acid sequences that have at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 3; or

[00496] c) amino acid sequences that have 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequences of SEQ ID NO: 3 or

[00497] d) an amino acid sequence of any one of SEQ ID NO: 1 to 48, or SEQ ID NO: 121 to 140, or SEQ ID NO: 222 to 224.

[00498] In a further aspect, a VHH domain of the present invention comprises or essentially consists of the sequence stipulated in:

[00499] a) the amino acid sequence of SEQ ID NO: 49; or

[00500] b) an amino acid sequence that has at least 95% amino acid identity with the amino acid sequences of SEQ ID NO: 49; or

[00501] c) an amino acid sequence that has 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequences of SEQ ID NO: 49; or

[00502] d) an amino acid sequence of any one of SEQ ID NO: 49 to 52.

[00503] In an additional aspect, a VHH domain of the present invention comprises or essentially consists of the sequence stipulated in:

[00504] a) the amino acid sequence of SEQ ID NO: 67; or

[00505] b) an amino acid sequence that has at least 90% amino acid identity with the amino acid sequences of SEQ ID NO: 67; or

[00506] c) an amino acid sequence having 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference with the amino acid sequences of SEQ ID NO: 67; or

[00507] d) an amino acid sequence of any one of SEQ ID NO: 53 to 120.

[00508] In a further aspect, a VHH domain of the present invention comprises or essentially consists of the amino acid sequence stipulated in either one of SEQ ID NO: 121 to 140, or SEQ ID NO: 222 to 224.

[00509] In a further aspect, a VHH domain of the present invention comprises or essentially consists of the amino acid sequence stipulated in any one of SEQ ID NO: 138 to 140.

[00510] In a further aspect, a VHH domain of the present invention comprises or essentially consists of the amino acid sequence stipulated in any one of SEQ ID NO: 222 to 224.

[00511] Typical VHH domains of the present invention are shown in Table 5 and optimized VHH domains typical of the present invention are shown in Table 6 below: TABLE 5: VHH DOMAINS

[00512] SEQ ID NO: 1 to 48 are VHH family 101 domains. SEQ ID NO: 49 to 52 are VHH family 9 domains. SEQ ID NO: 53 to 120 are VHH family 13 domains. TABLE 6: OPTIMIZED VHH DOMAINS

[00513] In a further aspect, a polypeptide according to the present invention, in particular a single immunoglobulin variable domain of the present invention, is humanized and/or optimized for stability, potency, manufacturability and/or similarity to regions of framework. For example, the polypeptide is humanized and/or has sequence optimization at one or more of the following positions (in accordance with Kabat numbering): 1, 11, 14, 16, 74, 83, 108. In one aspect, the polypeptide comprises one or more of the following mutations: E1D, S11L, A14P, E16G, A74S, K83R, Q108L.

[00514] In one aspect, one or more framework regions of a polypeptide according to the present invention, in particular a single immunoglobulin variable domain of the present invention, are humanized and/or have sequence optimisation. In one aspect, a polypeptide according to the present invention, in particular a single immunoglobulin variable domain of the present invention, comprises framework regions (FR) for example, as stipulated below:

[00515] i) FR1 is selected from any one of SEQ ID NO's: 198 to 204;

[00516] ii) FR2 is selected from any of SEQ ID NO's: 205 to 208;

[00517] iii) FR3 is selected from any one of SEQ ID NO's: 209 to 210; and/or

[00518] iv) FR4 is selected from any of SEQ ID NO's: 211 to 212.

[00519] Human immunoglobulin framework region (FR) sequences that can also be used as framework region sequences for the unique immunoglobulin variable domains as described above are known in the art. Furthermore, methods for humanizing framework regions of single variable domains of immunoglobulin derived from different human species are known in the art.

[00520] In a further aspect, one or more CDR regions of a polypeptide according to the present invention, in particular a single immunoglobulin variable domain of the present invention, are humanized and/or have sequence optimisation. In one aspect, a polypeptide according to the present invention, in particular a single immunoglobulin variable domain of the present invention, is humanized and/or has sequence optimization at one or more of the following positions (according to numbering Kabbat): 52, 53.

[00521] In a further aspect, a polypeptide according to the present invention, in particular a single immunoglobulin variable domain of the present invention, comprises one or more of the following mutations: N52S, S53T.

[00522] In a further aspect, a polypeptide according to the present invention, in particular a single immunoglobulin variable domain of the present invention, comprises a CDR2 selected from any one of SEQ ID NO's: 214 to 221.

[00523] Typical humanized and/or optimized sequences of the present invention are shown in Table 4 and 6 in the following parts and in Table 7 below here in this patent application. TABLE 7: OPTIMIZED SEQUENCE VARIANTS

[00524] Table 7a shows the FR1-CDR1-FR2-CRD2 of the sequence-optimized variants, Table 7b shows the FR3-CDR3-FR4-CDR4 of said variants. The sequence numbers in the tables (SEQ) refer to the sequences in the sequence listing of the present application. Table 7a: Optimized sequence variants (FR1-CDR1-FR2-CDR2) Table 7b: Optimized sequence variants (FR3-CDR3-FR4)

[00525] In one aspect of the present invention, a polypeptide of the invention may additionally contain modifications such as glycosyl residues, modified amino acid side chains, and the like.

[00526] It will be apparent to the skilled person that for pharmaceutical uses in humans, the polypeptides of the invention are preferably directed against human CX3CR1, whereas for veterinary purposes, the polypeptides of the invention are preferably directed against CX3CR1 of the species to be treated.

[00527] Furthermore it will be apparent to the skilled person that when used as a therapeutic agent in humans, the single immunoglobulin variable domains comprised in the polypeptides according to the invention are preferably humanized single immunoglobulin variable domains.

[00528] According to the invention, a single immunoglobulin variable domain can be an antibody domain, i.e., VL or VH antibody, and/or VHH domains as described above, and/or any other type of single variable domain of immunoglobulin, for example, camelized VH, provided that these single immunoglobulin variable domains are anti-CX3CR1 single immunoglobulin variable domains.

[00529] In one aspect of the invention, the single immunoglobulin variable domain essentially consists of either an antibody domain sequence or a VHH domain sequence as described above. In particular, the single immunoglobulin variable domain essentially consists of a VHH domain sequence.

[00530] In a further aspect, a polypeptide of the present invention comprises two or more unique anti-CX3CR1 immunoglobulin variable domains. In a further aspect, a polypeptide of the present invention comprises two unique anti-CX3CR1 immunoglobulin variable domains, e.g., anti-CX3CR1 VHHs. In one aspect, the two unique anti-CX3CR1 immunoglobulin variable domains in a polypeptide of the present invention have the same amino acid sequence. In another aspect, the two unique anti-CX3CR1 immunoglobulin variable domains in a polypeptide of the present invention have different amino acid sequences.

[00531] According to another embodiment of the invention, the at least two unique immunoglobulin variable domains present in a polypeptide of the invention can be linked to each other directly (that is, without using a linker) or through a linker - deador. The linker is preferably a linker peptide and, according to the invention, will be selected so as to allow the binding of at least two unique immunoglobulin variable domains to CX3CR1, either within one and the same CX3CR1 molecule, or within of two different molecules.

[00532] Suitable chains among others will depend on the epitopes, and specifically the distance between the epitopes on CX3CR1 to which the unique immunoglobulin variable domains bind, and will be apparent to the skilled person based on the description herein, in this patent application , optionally after some limited degree of routine experimentation.

[00533] Furthermore, when the two or more single variable domains of anti-CX3CR1 immunoglobulin are domain antibodies or VHH domains, they may also be linked to each other via a third domain antibody or VHH domain (in which the two or more single immunoglobulin variable domains can be linked directly to the third domain antibody or VHH domain or via suitable linkers). A third domain antibody or VHH-like domain can be, for example, a domain antibody or a VHH domain that provides an increased half-life, as further described herein in this patent application. For example, the latter domain antibody or VHH domain can be a domain antibody or a VHH domain that is capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin, as further described here in this patent application.

[00534] Alternatively, the two or more single variable domains of anti-CX3CR1 immunoglobulin can be linked in series (either directly or via a suitable linker) and the third (single) antibody domain or VHH domain (which may provide half -increased life, as described above) can be connected directly or via a linker to one of these two or more immunoglobulin sequences mentioned above.

[00535] Suitable linkers are described herein, in this patent application, in connection with specific polypeptides of the invention and may comprise - for example, and without limitation - an amino acid sequence, which amino acid sequence preferably has a length of 5 or more amino acids , 7 or more amino acids, 9 or more amino acids, 11 or more amino acids, 15 or more amino acids, or at least 17 amino acids, such as about 20 to 40 amino acids. However, the upper limit is not crucial but is chosen for reasons of convenience considering, for example, the biopharmaceutical production of similar polypeptides.

[00536] The chainer sequence can be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutic purposes, the trigger is preferably non-immunogenic in the subject to which the polypeptide of the invention is administered.

[00537] A useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in International Patent Publication Nos. WO 96/34103 and WO 94/04678.

[00538] Other examples are polyalanine linker sequences such as Ala-Ala-Ala.

[00539] Additional preferred examples of chainer sequences are Gly/Ser chainers of different length such as (glyxsery)z chainers, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3 , and (gly3ser2)3.

[00540] If the polypeptide of the invention is modified by the attachment of a polymer, for example, a polyethylene glycol (PEG) moiety, the linker sequence preferably includes an amino acid residue, such as a cysteine or a lysine, allowing said modification, for example, PEGilation, in the chainer region.

EXAMPLES OF CHAINERS ARE:

[00541] GGGGS (5 GS chainer, SEQ ID NO: 233)

[00542] SGGSGGS (7GS chainer, SEQ ID NO: 234)

[00543] GGGGCGGGS (8GS chainer, SEQ ID NO: 235)

[00544] GGGGSGGGS (9GS chainer, SEQ ID NO: 236)

[00545] GGGGSGGGGS (10GS chainer, SEQ ID NO: 237)

[00546] GGGGSGGGGGSGGGGS (15GS chainer, SEQ ID NO: 238)

[00547] GGGGSGGGGSGGGGGGGS (18GS chainer, SEQ ID NO: 239)

[00548] GGGGSGGGGSGGGGSGGGGS (20GS chainer, SEQ ID NO: 240)

[00549] GGGGSGGGGSGGGGSGGGGSGGGGS (25GS chainer, SEQ ID NO: 241)

[00550] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (30GS chainer, SEQ ID NO: 242)

[00551] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (35GS chainer, SEQ ID NO: 243)

[00552] EPKSCDKTHTCPPCP (G1 link chain, SEQ ID NO: 244)

[00553] GGGGSGGGSEPKSCDKTHTCPPCP (G1 9GS-joint chainer, SEQ ID NO: 245)

[00554] EPKTPKPQPAAA (Llama upper long joint region, SEQ ID NO: 246)

[00555] ELKTPL- GDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSC DTPPPCPRCP (G3 joint, SEQ ID NO: 247)

[00556] AAA (Wing chainer, SEQ ID NO: 248)

[00557] Furthermore, the linker can also be a poly(ethylene glycol) moiety, as shown for example in International Patent Publication No. WO04/081026.

[00558] Non-limiting examples of polypeptides which comprise or consist of two or more single anti-CX3CR1 immunoglobulin variable domains are given in Table 8a. TABLE 8A: BIVALENT ANTI-CX3CR1 POLYPEPTIDES

[00559] In another embodiment, the at least two unique immunoglobulin variable domains of the polypeptide of the invention are linked to each other through another moiety (optionally through one or two linkers), such as another polypeptide which, in one preferred but non-limiting embodiment may be an additional single immunoglobulin variable domain as already described above. Said moiety can either be essentially inactive or can have a biological effect such as enhancing the desired properties of the polypeptide or can confer one or more additional desired properties on the polypeptide. For example, and without limitation, the moiety can increase the half-life of the protein or polypeptide, and/or can reduce its immunogenicity or enhance any other desired property.

[00560] In one aspect, a polypeptide of the invention includes, especially when used as a therapeutic agent, a portion which prolongs the half-life of the polypeptide of the invention in the serum or other body fluids of a patient. The term "half-life" means the time it takes for the serum concentration of the (modified) polypeptide to decrease by 50%, in vivo, for example, due to degradation of the polypeptide and/or clearance and/or sequestration by natural mechanisms. .

[00561] According to a further embodiment of the invention, the two single immunoglobulin variable domains can be fused to a serum albumin molecule, as described for example in International Patent Publications Nos. WO01/79271 and No. WO03/59934.

[00562] Alternatively, said half-life extending moiety may be covalently linked or fused to said polypeptide and may be, without limitation, an Fc moiety, an albumin moiety, a fragment of an albumin moiety, an albumin moiety, an albumin binding, such as a single anti-albumin immunoglobulin variable domain, a transferrin binding moiety, such as a single anti-transferrin immunoglobulin variable domain, a polyoxyalkylene molecule, such as a polyethylene glycol molecule, col, an albumin-binding peptide, or hydroxyethyl starch (HES) derivatives.

[00563] In another aspect, the polypeptide of the invention comprises a portion which binds to an antigen found in the blood, such as serum albumin, serum immunoglobulins, thyroxine binding protein, fibrinogen or transferrin, thereby conferring an increased in vivo half-life of the resulting polypeptide of the invention. According to one embodiment, said portion is an albumin-binding immunoglobulin, and in particular, a single albumin-binding immunoglobulin variable domain such as an albumin-binding VHH domain.

[00564] In another embodiment, the polypeptide of the invention comprises a portion which binds serum albumin, wherein said portion is an albumin-binding peptide, as described for example in International Patent Publications Nos. WO2008/068280 and WO2009/127691.

[00565] If intended for use in humans, said albumin-binding immunoglobulin single variable domain (also called anti-albumin immunoglobulin single variable domain) will preferably bind to human serum albumin and will preferably be a VHH binding domain of humanized albumin.

[00566] Single immunoglobulin variable domains binding to human serum albumin are known in the art and are described in further detail, for example, in International Patent Publication No. WO2006/122786. A specifically useful VHH albumin-binding domain consists of or contains the amino acid sequence as stipulated in any one of SEQ ID NO: 230 to 232: TABLE 8B

[00567] According to one embodiment, a polypeptide of the invention can be linked to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or can confer the ability to bind to one or more Fc receptors. For example, to this end, and without being limited thereto, antibody parts may be or may comprise CH2 and/or CH3 domains from an antibody, such as from a heavy chain antibody (as described above) and more preferably from a conventional human 4-chain antibody; specifically, the polypeptide of the invention can be linked to an Fc region, for example, of human IgG, human IgE or other human Ig. For example, International Patent Publication No. WO 94/04678 describes heavy chain antibodies comprising a Camelidae VHH domain or a humanized derivative thereof, in which the Camelidae CH2 and/or CH3 domain has been replaced by CH2 and/or Camelidae domains. or human CH3, so as to provide an immunoglobulin consisting of 2 heavy chains, each comprising a VHH domain - optionally humanized - and human CH2 and CH3 domains (but not CH1 domain), which immunoglobulin has the effector function provided by the CH2 domains and CH3, can function without the presence of any light chains, and has an increased half-life compared to the corresponding VHH domains without said modification.

[00568] In one aspect, a polypeptide of the present invention comprises two anti-CX3CR1 VHHs and one VHH capable of binding serum albumin. In one aspect, the VHHs are fused using linker peptides. Typical examples of similar polypeptides of the present invention are shown in the following parts.

[00569] In one aspect, a polypeptide of the present invention comprises a first anti-CX3CR1 VHH fused to a first linker peptide, which is itself fused to a VHH capable of binding serum albumin, which is itself fused to a second linker peptide, which is itself fused to a second anti-CX3CR1 VHH. In one aspect, the first or second peptide linker is a 9GS linker, in one aspect, the first and second peptide linker is a 9GS linker. In one aspect, the VHH capable of binding serum albumin is capable of binding human serum albumin. In one aspect, the VHH capable of binding serum albumin has the amino acid sequence set forth in SEQ ID NO: 231. In one aspect, the first and second anti-CX3CR1 VHH have the same amino acid sequence. In one aspect, the first or second anti-CX3CR1 VHH has the CDR1, CDR2 and CDR3 stipulated in:

[00570] - SEQ ID NO's: 213, 214 and 186; or

[00571] - SEQ ID NO's: 213, 221 and 186; or

[00572] - SEQ ID NO's: 141, 162 and 186.

[00573] In one aspect, the first and second anti-CX3CR1 VHH have the CDR1, CDR2 and CDR3 stipulated in:

[00574] - SEQ ID NO's: 213, 214 and 186; or

[00575] - SEQ ID NO's: 213, 221 and 186; or

[00576] - SEQ ID NO's: 141, 162 and 186.

[00577] In one aspect, the first or second anti-CX3CR1 VHH has the amino acid sequence set forth in either SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224. In one aspect, the first and second second anti-CX3CR1 VHH have the same amino acid sequence, wherein said amino acid sequence is the sequence stipulated in either SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224.

[00578] Non-limiting examples of polypeptides of the present invention are polypeptides of any one of SEQ ID NO: 225 to 227, 249 or 277 to 281. TABLE 9

[00579] In another aspect, a polypeptide of the present invention comprises an anti-CX3CR1 VHH and an Fc domain. In one aspect, a polypeptide of the present invention comprises an anti-CX3CR1 VHH fused to a linker peptide, which is itself fused to an Fc domain. In one aspect, the linker peptide is a 15GS linker. In one aspect, the Fc domain has the amino acid sequence set forth in SEQ ID NO: 250 or 252. In one aspect, the VHH has the CDR1, CDR2 and CDR3 set forth in:

[00580] - SEQ ID NO's: 213, 214 and 186; or

[00581] - SEQ ID NO's: 213, 221 and 186; or

[00582] - SEQ ID NO's: 141, 162 and 186.

[00583] In one aspect, the VHH has the amino acid sequence set forth in either SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224. In one aspect the polypeptide is in the form of a dimer, for example, where the dimer is formed by one or more disulfide bridges.

[00584] Non-limiting examples of polypeptides of the present invention are the polypeptides of SEQ ID NO: 251, 253 or 254. TABLE 10

[00585] A polypeptide of the invention can be modified to improve its properties. In one aspect, a polypeptide of the present invention can be modified to increase its stability after storage. In one aspect, a polypeptide of the present invention can be modified to facilitate its expression in a particular host system. For example, the first codon of a polypeptide of the present invention can be modified. In one aspect, a polypeptide of the present invention starts with a glutamic acid (glu) as its first amino acid. In another aspect, a polypeptide of the present invention starts with an aspartic acid (asp) as its first amino acid, for example, to reduce pyroglutamate formation at the N-terminus during storage and thereby increase product stability. In another aspect, a polypeptide of the present invention starts with an alanine (ala) or a valine (val) as its first amino acid, for example, to facilitate expression of the polypeptide in a prokaryotic expression system, such as Escherichia coli. . Said modification of a polypeptide according to the present invention is done using techniques known in the art.

[00586] Typical examples of polypeptides according to the present invention with a modified first codon are stipulated in any one of SEQ ID NO: 257 to 262 and 263-266 and are shown in Tables 11 and 12 below: TABLE 11 TABLE 12

[00587] In an additional aspect, a polypeptide of the present invention is characterized by one or more of the following properties: • Binds with high affinity to human CX3CR1; • Inhibits binding of soluble fractalkine to human CX3CR1; • Inhibits fractalkine-induced chemotaxis; • Inhibits fractalkine-induced internalization of human CX3CR1 receptors; • Cross-reacts with cyno CX3CR1 within 10 fold of the E/IC50 for human CX3CR1 for binding and functional inhibition.

[00588] Accordingly, in one aspect, a polypeptide of the present invention has an affinity for human CX3CR1 at an IC50 less than or equal to 10 nM, or less than or equal to 5 nM, or less than or equal to 2.5 nM or less than or equal to 1 nM as determined by competition FACS.

[00589] In a further aspect, a polypeptide of the present invention has an affinity for human CX3CR1 at an EC50 of less than or equal to 10 nM, or less than or equal to 5 nM, or less than or equal to 2.5 nM or less than or equal to 1 nM, as determined by cell binding FACS.

[00590] In a further aspect, a polypeptide of the present invention blocks binding of human CX3CR1 to human fractalkine at or above 50%, or at or above 60%, or at or above 70%, or at or above 80%, or at or above 90%, or at or above 95% as determined by human fractalkine competition FACS.

[00591] In a further aspect, a polypeptide of the present invention blocks binding of human fractalkine to human CX3CR1 at an IC50 of less than or equal to 300 nM, or less than or equal to 100 nM, or less than or equal to 20 nM, or less than or equal to 10 nM, less than or equal to 5 nM, less than or equal to 2.5 nM, or less than or equal to 1 nM as determined by FACS competition with human fractalkine.

[00592] In a further aspect, a polypeptide of the present invention inhibits fractalkine-induced chemotaxis mediated by human CX3CR1 at or above 10%, or at or above 30%, or at or above 40%, or at or above of 50%, or at or above 60%, or at or above 70%, or at or above 80%, or at or above 90%.

[00593] In a further aspect, a polypeptide of the present invention inhibits fractalkine-induced chemotaxis mediated by human CX3CR1 at an IC50 of less than or equal to 500 nM, or less than or equal to 100 nM, or less than or equal to at 75 nM, or less than or equal to 50 nM, or less than or equal to 10 nM, or less than or equal to 5 nM.

[00594] In a further aspect, a polypeptide of the present invention inhibits fractalkine-induced human CX3CR1 receptor internalization at an IC50 of less than or equal to 10 nM, or less than or equal to 5 nM or or less than or equal to to 1 nM.

[00595] According to yet another embodiment, a half-life prolonging modification of a polypeptide of the invention (said modification also reducing the immunogenicity of the polypeptide) comprises linking a suitable pharmacologically acceptable polymer, such as poly( straight or branched chain ethylene glycol) (PEG) or derivatives thereof (such as methoxypoly(ethylene glycol) or mPEG). In general, any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to domain antibodies and scFv's); reference is made, for example, to: Chapman, Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Disc. 2 (2003); International Patent Publication No. WO 04/060965; and US Patent No. US6,875,841.

[00596] Various reagents for PEGylation of polypeptides are also commercially available, for example, from Nektar Therapeutics, USA, or from NOF Corporation, Japan, such as the EA Series, SH Series, MA Series, CA Series, and ME Series from Sunbright ®, such as the Sunbright® ME-100MA, Sunbright® ME-200MA, and Sunbright® ME-400MA.

[00597] Preferably, site-directed PEGylation is used, in particular via a cysteine residue (see, for example, Yang et al., Protein Engineering 16, 761-770 (2003)). For example, to this end, PEG can be linked to a naturally occurring cysteine residue in a polypeptide of the invention, a polypeptide of the invention can be modified so as to suitably introduce one or more cysteine residues for PEG binding, or a sequence of amino acids comprising one or more cysteine residues for PEG binding can be fused to the N- and/or C-terminus and/or PEG can be linked to a linker region that bridges between two or more functional domains of a polypeptide of the invention, all using protein engineering techniques known per se to the skilled person.

[00598] Preferably, for the polypeptides of the invention, a PEG having a molecular weight of more than 5 kDa, such as more than 10 kDa and less than 200 kDa, such as less than 100 kDa, is used; for example, in the range of 20 kDa to 80 kDa.

[00599] With respect to PEGylation, it should be noted that, generally speaking, the invention also encompasses any polypeptide of the invention that has been PEGylated at one or more amino acid positions, preferably in such a way that said PEGylation or ( 1) increases in vivo half-life; (2) reduces immunogenicity; (3) provides one or more additional beneficial properties known per se for PEGylation; (4) does not essentially affect the affinity of the polypeptide for CX3CR1 (e.g., does not reduce said affinity by more than 50%, and more preferably not by more than 10%, as determined by a suitable test, such as those described in the Examples below); and/or (4) does not affect any of the other desired properties of the polypeptides of the invention. Suitable PEG moieties and methods for linking the same, whether specifically or non-specifically, will be apparent to the skilled person.

[00600] According to a specifically preferred embodiment of the invention, a PEGylated polypeptide of the invention includes a PEG portion of linear PEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEG portion is linked to the polypeptide in a linker region and specifically at a Cys residue, for example at position 5 of a GS8 linker peptide as shown in SEQ ID NO: 235.

[00601] Preferred examples of PEGylated polypeptides of the invention are PEGylated preferably with one of the PEG reagents as mentioned above, such as "Sunbright® ME-400MA" as shown in the following chemical formula:

[00602] which has an average molecular weight of 40 kDa.

therapeutic uses

[00603] In one aspect, the present invention provides a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for use as a medicament.

[00604] In one aspect, the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerotic cardio- and cerebrovascular disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescent glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neural disorders - rodegenerative and demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer.

[00605] In another aspect, the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis.

[00606] In another aspect, the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis by preventing and/or reducing the formation of new lesions or atherosclerotic plaques and /or by preventing or slowing the progression of existing lesions and plaques.

[00607] In another aspect, the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis by modifying the composition of the plaques in order to reduce the risk of rupture of plaques and atherothrombotic events.

[00608] In one aspect, the present invention also provides a method of treating, or reducing the risk of, cardio- and cerebrovascular atherosclerotic disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescent glomerulonephritis, nephropathy by IgA, 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, lung diseases such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer, in a person suffering from, or at risk of, said disease or condition, in which the method comprises administering to the person a therapeutically effective amount of a polypeptide according to the present invention or a pharmaceutical composition comprising said polypeptide.

[00609] In one aspect, the present invention also provides a method of treating, or reducing the risk of atherosclerosis in a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective form of polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide.

[00610] In one aspect, the present invention also provides a method of treating or reducing the risk of atherosclerosis by preventing and/or reducing the formation of new lesions or atherosclerotic plaques and/or by preventing or decreasing the progression of lesions and plaques existing in a person suffering from, or at risk of, said disease or condition, the method comprising administering to the person a therapeutically effective amount of polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide.

[00611] In one aspect, the present invention also provides a method of treating, or reducing the risk of, atherosclerosis by modifying plaque composition so as to reduce the risk of plaque rupture and atherothrombotic events in a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the subject a therapeutically effective amount of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide.

[00612] In one aspect, a polypeptide of the present invention is indicated for use in the treatment or prophylaxis of a disease or a disorder that is associated with CX3CR1.

[00613] In one aspect, a polypeptide of the present invention is indicated for use in the treatment or prophylaxis of diseases or conditions in which modulation of activity at the CX3CR1 receptor is desirable. In one aspect, the present invention also provides a method of treating, or reducing the risk of, diseases or conditions in which CX3CR1 receptor antagonism is beneficial which comprises administering to a person suffering from, or at risk of, said disease or condition, a polypeptide of the present invention.

[00614] Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered from a previous episode of, or are otherwise considered to be at increased risk of, the disease or condition in question. People at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening as being particularly susceptible to developing the disease or condition.

[00615] In the context of the present invention, the term "prevention, treatment and/or mitigation" not only comprises prevention and/or treatment and/or mitigation of the disease, but also generally comprises prevention of the onset of the disease, decrease or reversing the progress of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or duration of the disease, and/or of any symptoms associated with the same and/or prevention of a further increase in the severity of the disease and/or of any symptoms associated with the same, prevention, reduction or reversal of any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

[00616] The individual to be treated will be a mammal, and more particularly a human being. As will be apparent to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases, disorders or conditions mentioned herein in this patent application.

[00617] Furthermore, it will be apparent to the skilled person that the above methods of treating a disease include preparing a medicament for treating said disease. Furthermore, it is evident that the polypeptides of the invention can be used as an active ingredient in a medicament or in a pharmaceutical composition intended for the treatment of the above diseases. Accordingly, the invention also relates to the use of a polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention, treatment and/or mitigation of any of the diseases, disorders or conditions mentioned above. The invention further relates to a polypeptide of the invention for therapeutic or prophylactic use, and specifically for the prevention, treatment and/or mitigation of any of the diseases, disorders or conditions mentioned above. The invention additionally relates to a pharmaceutical composition for the prevention, treatment and/or mitigation of the diseases, disorders or conditions mentioned above, said composition comprising at least one polypeptide of the invention.

[00618] The polypeptides of the invention and/or the compositions comprising the same can be administered to a patient in need thereof in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, the polypeptides of the invention and/or compositions comprising the same can be administered, for example, intravenously, subcutaneously, intramuscularly, intraperitoneally, transdermally, orally, sublingually (for example, in the form of a sublingual tablet, spray or drop placed under the tongue and adsorbed through the mucous membranes into the network of capillaries under the tongue), (intra-)nasally (by e.g. in the form of a nasal spray and/or as an aerosol), topically, via a suppository, by inhalation, intravitreally (esp. for the treatment of dry macular degeneration (in English, dry AMD) or glaucoma), or any other suitable manner in an effective amount or dose.

[00619] The polypeptides of the invention and/or the compositions comprising the same are administered according to a treatment regimen that is suitable for preventing, treating and/or mitigating the disease, disorder or condition to be prevented, treated or mitigated. The physician will generally be able to determine an appropriate treatment regimen depending on factors such as the disease, disorder or condition to be prevented, treated or alleviated, the severity of the disease, the severity of the symptoms of the disease, the polypeptide specific invention to be used, the specific route of administration and the pharmaceutical formulation or composition to be used, the age, sex, weight, diet, general condition of the patient, and similar factors known to the physician. In general, the treatment regimen will comprise administration of one or more polypeptides of the invention, or one or more compositions comprising the same, in therapeutically and/or prophylactically effective amounts or doses.

[00620] In general, for the prevention, treatment and/or mitigation of the diseases, disorders and conditions mentioned here, in this patent application, and depending on the disease, disorder or specific condition to be treated, the potency of the specific polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the polypeptides of the invention will generally be administered in an amount between 0.005 and 20.0 mg per kilogram of body weight and dose , preferably between 0.05 and 10.0 mg/kg/dose, and most preferably between 0.5 and 10 mg/kg/dose, either continuously (e.g., by infusion) or as single doses (such as by daily, weekly, or monthly doses; cf. below), but they can vary significantly, especially depending on the above-mentioned parameters.

[00621] For prophylactic applications, compositions containing the polypeptides of the invention can also be administered in similar or slightly lower dosages. The dosage can also be adjusted by the individual's physician in case of any complications.

[00622] Depending on the specific polypeptide of the invention and its specific pharmacokinetics and other properties, it may be administered daily, every two, third, four, five or six days, weekly, monthly, and the like. An administration regimen may include weekly, long-term treatment. By "long term" is meant a minimum of two weeks and preferably months or years of duration.

[00623] The effectiveness of the polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro test, cellular test, in vivo test and/or animal model known per se, or any combination thereof, depending on the specific disease involved. Suitable animal tests and models will be apparent to the skilled person, and for example include the animal tests and models used in the Examples below.

[00624] For pharmaceutical use, the polypeptides of the invention can be formulated as a pharmaceutical preparation comprising (i) at least one polypeptide of the invention and (ii) at least one pharmaceutical vehicle, diluent, excipient, adjuvant, and/or stabilizer - clinically acceptable, and (iii) optionally one or more polypeptides and/or additional pharmaceutically active compounds. By "pharmaceutically acceptable" is meant that the respective material does not exhibit any biological or otherwise undesirable effects when administered to an individual and does not interact in a harmful manner with any of the other components of the pharmaceutical composition (such as, for example, the pharmaceutically active ingredient) in which it is contained. Specific examples can be found in routine manuals, such as, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, USA (1990). For example, the polypeptides of the invention can be formulated and administered in any manner known per se for conventional antibodies and antibody fragments and other pharmaceutically active proteins. Therefore, according to a further embodiment, the invention relates to a pharmaceutical composition or preparation that contains at least one polypeptide of the invention and at least one pharmaceutically acceptable vehicle, diluent, excipient, adjuvant and/or stabilizer, and optionally a or more additional pharmaceutically active substances.

[00625] By way of non-limiting examples, a similar formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular, subcutaneous, intrathecal, intracavernous or intraperitoneal injection or by intravenous infusion ), for topical administration, for sublingual administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, by transdermal, nasal, intravitreal, rectal or vaginal administration, and the like. The suitable forms of administration referred to - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as the methods and vehicles for use in preparing the same, will be apparent to the person skilled in the art.

[00626] Pharmaceutical preparations for parenteral administration, such as intravenous, intramuscular, subcutaneous injection or intravenous infusion may be, for example, sterile solutions, suspensions, dispersions, emulsions, or powders which comprise the active ingredient and which are suitable after an additional dissolution or dilution step, for infusion or injection. Suitable carriers or diluents for the foregoing 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 blends thereof.

[00627] Solutions of the active compound or its salts may also contain a preservative to prevent the growth of microorganisms, such as antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal (thiomersal), and the like . In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Proper fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions or by using surfactants. Other agents delaying absorption, for example aluminum monostearate and gelatin, can also be added.

[00628] In all cases, the final dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as needed, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization) techniques, which produce a powder of the active ingredient plus any additional desired ingredients present in the solutions. previously filtered sterile.

[00629] Generally, aqueous solutions or suspensions will be preferred. In general, suitable formulations for therapeutic proteins such as the polypeptides of the invention are buffered protein solutions, such as solutions including the protein in a suitable concentration (such as from 0.001 to 400 mg/mL, preferably 0.005 to 200 mg/mL, more preferably 0.01 to 200 mg/mL, most preferably 1.0 to 100 mg/mL, such as 1.0 mg/mL (i.v. administration) or 100 mg/mL (i.v. administration) s.c.) and an aqueous buffer such as:

[00630] - phosphate buffered saline, pH 7.4,

[00631] - other phosphate buffers, pH 6.2 to 8.2,

[00632] - histidine buffers, pH 5.5 to 7.0,

[00633] - succinate buffers, pH 3.2 to 6.6, and

[00634] - citrate buffers, pH 2.1 to 6.2,

[00635] and, optionally, salts (eg NaCl) and/or sugars or polyalcohols (such as trehalose, mannitol, or glycerol) to provide isotonicity of the solution.

[00636] Preferred buffered protein solutions are solutions including about 0.05 mg/mL of the polypeptide of the invention dissolved in 25 mM phosphate buffer, pH 6.5, adjusted to isotonicity by adding 220 mM trehalose. In addition, other agents such as a detergent, for example 0.02% Tween-20 or Tween-80, can be included in similar solutions. Formulations for subcutaneous application can include significantly higher concentrations of the polypeptide of the invention, such as up to 100 mg/ml or even above 100 mg/ml. However, it will be apparent to the person skilled in the art that the ingredients and amounts thereof as provided above only represent a preferred option. Alternatives and variations thereof will be immediately apparent to the skilled person, or can easily be devised starting from the above description.

[00637] The polypeptides of the invention may also be administered using suitable depot, slow-release or gradual-release formulations, for example, suitable for injection, using controlled-release devices for implantation under the skin, and/or using a pump dosing or other devices known per se for the administration of pharmaceutically active substances or principles. Furthermore, the polypeptides of the invention can be formulated in the form of a gel, cream, spray, drop, plaster or film which, if placed on the skin, passes through the skin.

[00638] Furthermore, compared with conventional antibodies or antibody fragments, an important advantage of using the polypeptides of the invention is that they can also be easily administered through routes other than parenteral administration and can be easily formulated for similar administration . For example, as described in International Patent Application No. WO2004/041867, similar polypeptides can be formulated for oral, intranasal, intrapulmonary and transdermal administration.

[00639] According to another embodiment of the invention, a pharmaceutical combination is provided comprising at least one polypeptide of the invention as described herein, in this patent application, and at least one other therapeutic agent selected from the group consisting of statins, antiplatelet agents, anticoagulants, antidiabetics and antihypertensives.

[00640] The pharmaceutical combination mentioned optionally and additionally may comprise a diluent, an excipient, an adjuvant and/or a stabilizer.

[00641] When two or more substances or principles are to be used as part of a combined treatment regimen, they may be administered via the same route of administration or via different routes of administration, essentially at the same time or at different times (e.g. , essentially simultaneously, consecutively, or according to an alternating regimen). When the substances or principles must be administered simultaneously through the same route of administration, they can be administered as different formulations or pharmaceutical compositions or as part of a combined formulation or pharmaceutical composition. Furthermore, when two or more substances or active principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used. is used alone, and said combined use may or may not lead to a synergistic effect. However, when the combined use of two or more substances or active principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This can be useful, for example, to avoid, limit or reduce any unwanted side effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, whilst still achieving the desired pharmaceutical or therapeutic effect.

[00642] Yet, an additional embodiment of the invention is a method of treating the diseases and disorders as set forth above, comprising administering to a subject, simultaneously, separately or sequentially, an effective amount of at least one polypeptide of the invention and at the at least one agent selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic, and an antihypertensive.

[00643] According to a further aspect of the invention, the polypeptide of the invention is prepared to be administered in combination with other drugs used for the treatment of the diseases and disorders stipulated above, the other drugs referred to being selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic, and an antihypertensive.

[00644] and according to yet another aspect of the invention, drugs used for the treatment of the diseases and disorders stipulated above, said drugs being selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an anti- hypertensive are prepared to be administered in combination with the polypeptide of the invention.

[00645] According to a further aspect of the invention, the polypeptide of the invention is used in combination with a device useful for administering the polypeptide, such as a syringe, injector pen, or miscellaneous device.

[00646] According to yet another embodiment of the invention, there is provided a method of diagnosing a disease, disorder or condition mediated by dysfunction of CX3CR1 comprising the steps of:

[00647] a) obtain a sample from an individual, and

[00648] b) contacting, in vitro, the sample with a polypeptide of the invention as defined above, and

[00649] c) detecting the binding of said polypeptide with said sample, and

[00650] d) comparing the binding detected in step (c) with a standard, wherein a difference in binding relative to said sample is diagnostic of a disease, disorder, or condition characterized by dysfunction of CX3CR1.

[00651] According to another embodiment of the invention, there is provided a method of diagnosing a disease, disorder or condition mediated by dysfunction of CX3CR1 comprising the steps of:

[00652] a) obtain a sample from an individual, and

[00653] b) contacting the sample with a polypeptide of the invention as defined above;

[00654] c) determine the amount of CX3CR1 in the sample; It is

[00655] d) comparing the amount determined in step (c) with a standard, wherein a difference in the amount relative to said sample is diagnostic of a disease, a disorder or a condition characterized by dysfunction of CX3CR1.

[00656] The above diagnostic methods can also be used to monitor the effectiveness of an individual's therapeutic treatment.

[00657] According to another embodiment of the invention, a kit is provided for diagnosing a disease, a disorder or a condition mediated by dysfunction of CX3CR1, for use in a method as defined above, said kit comprising at least one polypeptide of the invention and, optionally, one or more means, detection means and/or in vitro or in vivo imaging agents, and, additionally and optionally, instructions for use. Suitable in vivo imaging agents include 99mTc, 111Indium, 123Iodine, and, for study by magnetic resonance imaging, paramagnetic compounds.

[00658] The invention additionally provides a kit comprising at least one polypeptide of the invention and, additionally, one or more miscellaneous components selected from the group consisting of other drugs used for the treatment of diseases and disorders as described above, and devices as described above.

[00659] The invention additionally provides methods of manufacturing a polypeptide of the invention, said methods generally comprising the steps of:

[00660] - cultivating host cells comprising a nucleic acid capable of encoding a polypeptide of the invention (in the following parts: "nucleic acid of the invention") under conditions that allow expression of the polypeptide of the invention; It is,

[00661] - recovering or isolating the polypeptide expressed by the host cells from the culture; It is

[00662] - optionally further purifying and/or modifying and/or formulating the polypeptide of the invention.

[00663] A nucleic acid of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or the intended host organism). According to an embodiment of the invention, the nucleic acid of the invention is in essentially isolated form, as defined above.

[00664] The nucleic acid of the invention can also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, a cosmid or a YAC, which again can be in essentially isolated. The vector can especially be an expression vector, i.e., a vector that can provide for expression of the polypeptide in vitro and/or in vivo (e.g., in a suitable host cell, in a suitable host organism, and/or in a suitable environment). appropriate expression system). Said expression vector generally comprises at least one nucleic acid of the invention which is operably linked to one or more suitable regulatory elements, such as one or more promoters, one or more enhancers, one or more terminators, and the like. Specific examples of similar regulatory elements and other elements, such as one or more integrating factors, one or more selectable markers, one or more signal or leader sequences, one or more reporter genes, and the like, useful or necessary for expressing the polypeptides of the invention are described, for example, at pp. 131 to 133 of International Patent Publication No. WO2006/040153.

[00665] The nucleic acids of the invention can be prepared or obtained in a manner known per se (for example, by automated DNA synthesis and/or by recombinant DNA technology), based on the information on the amino acid sequences for the poly - peptides of the invention provided herein, in this patent application, and/or can be isolated from a suitable natural source.

[00666] According to another embodiment, the invention relates to a host or a host cell that expresses or is capable of expressing a polypeptide of the invention; and/or which contains a nucleic acid encoding a polypeptide of the invention. According to a particularly preferred embodiment, said host cells are bacterial cells, yeast cells, fungal cells or mammalian cells.

[00667] Suitable bacterial cells include cells from gram-negative bacterial strains such as Escherichia coli, Protes, and Pseudomonas strains, and gram-positive bacterial strains such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains. Suitable fungal cells include cells from Trichoderma, Neurospora, and Aspergillus species. Suitable yeast cells include cells from Saccharomyces species (for example, Saccharomyces cerevisiae), Schizosaccharomyces (for example, Schizosaccharomyces pombe), Pichia (for example, Pichia pastoris and Pichia methanolica), and Hansenula.

[00668] Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, NS0 cells, HEK cells, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins can also be used.

[00669] For industrial scale production, preferred heterologous hosts for (industrial) production of immunoglobulin single variable domain polypeptides and protein therapeutics containing the same include strains of E. coli, Pichia pastoris, and S. cerevisiae which are suitable for large-scale expression, production and fermentation, and in particular for (bio-)pharmaceutical expression, large-scale production and fermentation.

[00670] The choice of the specific expression system will depend in part on the need for high post-translational modifications, more specifically glycosylation. Production of a polypeptide of the invention for which glycosylation is desired or required will necessitate the use of mammalian expression hosts that have the capacity to glycosylate the expressed protein. In this regard, it will be apparent to the skilled person that the glycosylation pattern obtained (i.e. the type, number and position of appended residues) will depend on the cell or cell line that is used for expression.

[00671] Polypeptides of the invention produced in a cell as stipulated above can be produced either intracellularly (for example, in the cytosol, in the periplasm or in inclusion bodies) and then isolated from host cells and optionally further purified; or they can be produced extracellularly (secreted into the medium in which the host cells are grown) and then isolated from the culture medium and optionally further purified.

[00672] Additional methods and reagents used for the recombinant production of polypeptides, such as appropriate expression vectors, transformation or transfection methods, selection markers, methods of inducing protein expression, culture conditions, and the like, are known in the art. Similarly, protein isolation and purification techniques useful in a method of manufacturing a polypeptide of the invention are well known to the skilled person.

[00673] Production of the polypeptides of the invention by fermentation in convenient recombinant host organisms such as E. coli and yeast is cost-effective compared to conventional antibodies which also require expensive mammalian cell culture facilities. Furthermore, the obtainable expression levels are high and the yields of the polypeptides of the invention are in the range of 1 to 10 g/l (E. coli) and up to 10 g/l (yeast) and more.

EXAMPLES

[00674] Production of CHO, Baf/3, Caki and HEK293 cell lines overexpressing human CX3CR1 or cynomolgus CX3CR1

[00675] CHO and Baf/3 cells overexpressing human or cynomolgus CX3CR1 were generated using techniques known in the art. Cells expressing human CCR2 or CCR5 were also generated using techniques known in the art.

[00676] The cDNA was cloned into pCDNA3.1(+)-neo for human CX3CR1 whereas pcDNA-DEST40-neo was used for mouse CX3CR1.

[00677] The amino acid sequences of human CX3CR1 and cynomolgus CX3CR1 are depicted in SEQ ID NO: 255 and 256, respectively.

[00678] In order to establish camel kidney cells (Caki, Camel Kidney) overexpressing human CX3CR1 or mouse CX3CR1, parental Caki cells were electroporated with pCD-NA3.1(+)-neo-hCX3CR1 or pcDNA-DEST40 -neo-mCX3CR1, respectively. For all conditions, transfectants were selected by adding 1 mg/mL of geneticin (Invitrogen, Carlsbad, CA, USA).

[00679] Human embryonic kidney (HEK293, Human Embyonic Kidney) cells overexpressing human CX3CR1 or cynomolgus CX3CR1 were generated by lipid-mediated transfection with Fugene (Roche) of plasmids pCDNA3.1(+)-neo-hCX3CR1 or cyCX3CR1 , respectively, in the parental cell line HEK293. These cells were used as transient transfectants and thus were not put under selection. Briefly, 2*10E6 cells were seeded by T75 and incubated overnight prior to transfection. After removal from the culture medium, the cells were transfected with the respective plasmids (9 μg) and Fugene (27 μl) according to the manufacturer's instructions. 48 hours after transfection, cells were harvested and frozen for later use.

EXAMPLE 1: IMMUNIZATION WITH CX3CR1 INDUCES A HUMORAL IMMUNE REACTION IN LLAMA 1.1. Immunizations

[00680] After approval by the Ethics Committee (University Antwerp, Belgium, UA2008A1, 2008/096, 2007/068), 9 llamas (designated No. 368, 369, 370, 381, 382, 384, 312, 313 and 314 ) were immunized.

[00681] Six llamas (312, 313, 314, 381, 382, and 384) were immunized with 4 intramuscular injections (2 mg/dose at weekly or biweekly intervals) of plasmid vector pVAX1-huCX3CR1 (Invitrogen, Carlsbad, CA , USA ). Three llamas (381, 382 and 384) then received 4 subcutaneous injections of human CX3CR1 overexpressing Caki cells which were established as described above. Cells were resuspended in D-PBS and kept on ice before injection.

[00682] Three additional llamas (designated No. 368, 369 and 370) were immunized according to routine protocols with 4 subcutaneous injections of human CX3CR1 overexpressing Caki cells which were established as described above. Cells were resuspended in D-PBS and kept on ice before injection. Then, these llamas were given two injections with recombinant CX3CR1 NT/EC3 fragment coupled to BSA (TABLE 13). Peptides were ordered in NeoMPS (PolyPeptide Group, Strasbourg, France) and coupled to BSA according to routine protocols. TABLE 13 SEQUENCE OF PEPTIDE FRAGMENTS USED FOR IMMUNIZATION BOOST

[00683] The first injection was formulated in Complete Freund's Adjuvant (Difco, Detroit, MI, USA), while the subsequent injection was formulated in Incomplete Freund's Adjuvant (Difco, Detroit, MI, USA).

1.2. Evaluation of llama-induced immune reactions

[00684] In order to evaluate the induction of immune reactions in animals against human CX3CR1 by ELISA or FACS, sera were collected from llamas 312, 313 and 314 on day 0 (pre-immune), and different time points in the immunization schedule (peripheral blood lymphocyte [PBL] collection time).

[00685] In summary, Neutravidin (2 μg/mL) was immobilized overnight at 4°C in a 96-well Maxisorb plate (Nunc, Wiesbaden, Germany). The wells were blocked with a solution of casein (1%) in PBS. Then biotinylated recombinant NT fragment (Polypeptide, Strasbourg, France) or biotinylated EC3 fragments of CX3CR1 (Polypeptide, Strasbourg, France) were captured at 2 μg/mL. After the addition of serum dilutions, specifically bound immunoglobulins were detected using horseradish peroxidase (HRP) conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc., Montgomery, TX, USA) and a subsequent enzymatic reaction in the presence of TMB One substrate (3,3',5,5'-tetramentylbenzidine) (Promega, Mannheim, Germany), showing that a significant antibody-dependent immune reaction against CX3CR1 was induced after immunizations with the peptides.

[00686] Additionally, serum titers of animals immunized with cells were confirmed by FACS analysis on actively growing human CX3CR1 overexpressing CHO cells. CX3CR1 serum titre responses for llamas 368, 369, and 370 were determined with serum sampled after 4 cell immunizations (day 49), 4 cell immunizations and 1 peptide boost (day 77), and 4 cell immunizations and 2 peptide boosts. peptide (day 81). Cells were harvested and washed before incubation with serum dilutions. Detection was performed with goat anti-llama IgG (Bethyl, Montgomery, TX, USA) followed by PE-coupled donkey anti-goat (Jackson Laboratories, Suffolk, UK) and reading by FACSArray analysis (BD Biosciences). A summary of serum responses obtained as determined by either ELISA or FACS is shown in TABLE 14 and TABLE 15. TABLE 14 ANALYSIS OF SERUM TITERS FOR ANIMALS IMMUNIZED WITH CELLS/PEPTIDES TABLE 15 ANALYSIS OF SERUM TITERS FOR ANIMALS IMMUNIZED WITH DNA/CELLS

[00687] For llamas immunized with DNA only (312, 313 and 314) no serum titers were determined.

EXAMPLE 2: CLONING OF THE HEAVY CHAIN ANTIBODY FRAGMENT REPERTORIES ONLY AND PHAGE PREPARATION

[00688] After the final immunogen injection of each subgroup, immune tissues as the source of B cells that produce the heavy chain antibodies were collected from the immunized llamas. For llama 312, 313 and 314, two 150 mL blood samples collected 4 and 8 days after the last antigen injection were collected per animal. For llamas 368, 369 and 370 four 150 ml blood samples were collected 5 and 7 days after the last cellular immunization and additionally 4 and 8 days after the last peptide immunization. Next to these, two lymph node biopsies were taken, 12 days after the last cellular immunization and 12 days after the last peptide immunization. For llamas 381, 382 and 384 five blood samples of 150 mL were collected 8 days after the last DNA immunization and additionally 4 days after the first cell booster, 8 and 11 days after the second cell booster and 8 days after the last cellular immunization. Next to these, a lymph node biopsy was taken 8 days after the second cellular immunization.

[00689] From the blood samples, peripheral blood lymphocytes (PBLs) were prepared using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). From PBLs and lymph node (LN) biopsy, total RNA was extracted, which was used as starting material for RT-PCR in order to amplify the DNA segments encoding VHH.

[00690] For each immunized llama, libraries were built by grouping the total RNA isolated from samples originating from a certain subgroup of the immunization scheme, that is, after a type of immunization antigen, and for some llamas samples from different animals were gathered in a library (TABLE 16). TABLE 16 GROUPING OF DIFFERENT SAMPLES FOR LIBRARY CONSTRUCTION

[00691] In summary, the PCR-amplified VHH repertoire was cloned through specific restriction sites into a vector designed to facilitate phage display of the VHH library. The vector was derived from pUC119 and contains the LacZ promoter, an M13 phage gIII protein coding sequence, an ampicillin or carbenicillin resistance gene, a multiple cloning site, and a gIII-pelB hybrid leader sequence (pAX050) . In frame with the VHH coding sequence, the vector encodes a C-terminal c-myc tag and a His6 tag. Phages were prepared according to routine protocols and stored after filter sterilization at 4°C or -80°C in 20% glycerol for later use.

EXAMPLE 3: SELECTION OF SPECIFIC VHHS FOR CX3CR1 VIA PHAGE DISPLAY

[00692] VHH repertoires obtained from all llamas and cloned as a phage library were used in different selection strategies, applying a multiplicity of selection conditions. Variables include i) the form of presentation of the CX3CR1 protein (on different cellular backgrounds or on liposomes/VLPs), ii) the method of antigen presentation (in solution when using cells or coated on plates when using VLPs), iii) the antigen concentration iv) the ortholog used (human or cynomolgus) v) the number of selection rounds and vi) different elutriation methods (non-specific through trypsin or specific through Fractalcine ligand). All coated solid phase selections were made in 96-well Maxisorp plates (Nunc, Wiesbaden, Germany).

[00693] Selections were performed as follows: CX3CR1 antigen preparations for solid and solution phase selection formats were presented as described above in multiple concentrations. After 2 hours of incubation with the phage libraries followed by extensive washing, the bound facts were eluted with trypsin (1 mg/ml) for 15 minutes. When trypsin was used for phage elutriation, protease activity was immediately neutralized by applying 0.8 mM ABSF protease inhibitor. As a control, selections without antigen were performed in parallel.

[00694] The phage yields were used to infect E. coli which were then in turn used to prepare phage for the next round of selection (phage rescue). After the second round of selection, the phage yields were used to infect E. coli which were then plated onto agar plates (LB+carb+glucose2%) for analysis of individual VHH clones. In order to screen a selection yield for specific ligands, single colonies were selected from among the agar plates and grown in 1 ml deep 96-well plates. LacZ-controlled VHH expression was induced by adding IPTG (1 mM final) in the absence of glucose. Periplasmic extracts (in a volume of ~80 uL) were prepared according to routine protocols.

EXAMPLE 4: SCREENING OF PERIPLASMIC EXTRACTS IN CX3CR1-FRACTALKIN COMPETITION FACS TEST

[00695] Periplasmic extracts were screened in a human FACS CX3CR1/human Fractalcine competition test in order to assess the ability to block the expressed VHHs. Human CX3CR1 was present on CHO cells overexpressing CX3CR1. Both a setup using cells harvested from an actively growing culture and a setup using frozen cells were used. As a detection reagent labeled fractalcine (R&D Systems, Minneapolis, MN, USA) labeled with alexa647 (A647-Fractalkine) at a labeling grade of 1 was used. In order to set up the test, a series of titrations of the labeled fractalkine on CHO-huCX3CR1 cells in order to determine the EC50 value for binding. Initially, screening was performed at a higher concentration of fractalcine (3 nM) in order to increase the robustness of the test. In order to increase the screening sensitivity to a maximum, the concentration at EC30 (1 nM) was chosen for subsequent screening. Briefly, 50 μl of periplasmic extract was added to 6 nM labeled fractalkine (50 μl) and 200,000 CHO-huCX3CR1 cells. After one hour of incubation at 4°C, the cells were washed three times before being read on a FACS Array (Becton Dickinson). First a barrier (gate) was set over the intact cells as determined from the diffusion profile. Then, dead cells were excluded (gated out) by their fluorescence profile from the PI strain (Sigma, St Louis, EU). The alexa647 tag fluorescence profile was determined for each sample and was used for blocking capacity calculation. As controls, conditions were taken together where there was no VHH present in the peri-extract or a known irrelevant VHH and samples were included where excess cold fractalcine was included. For each sample the percent blockage was determined using the control samples to determine the test window.

[00696] From this screening, VHHs were selected and sequence analysis revealed 120 unique VHHs belonging to 3 different B cell lineages. The total number of variants found for each B cell lineage is represented in TABLE 17. TABLE 17SELECTION PARAMETERS USED FOR THE IDENTIFICATION OF HUMAN CX3CR1-SPECIFIC VHH B CELL LINEAGES

[00697] An overview of the selection procedure and performance during initial screening is given for all VHHs in Table 18. TABLE 18SELECTION CONDITIONS AND PRIMARY SCREENING RESULTS FOR THE SPECIFIC VHH FOR HUCX3CR1

[00698] The amino acid sequences of all the unique VHHs obtained are shown in the Sequence Listing and above (CDRs and framework regions have been indicated).

EXAMPLE 5: CHARACTERIZATION OF PURIFIED VHHS

[00699] Inhibitory anti-CX3CR1 VHHs selected from the screening described in Example 4 were further purified and characterized. Selected VHHs were expressed in E. coli TG1 as c-myc-tag proteins, His6. Expression was induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37°C. After centrifuging the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets. These extracts were used as starting material and VHHs were purified by IMAC and size exclusion chromatography (SEC) resulting in 95% purity as judged by SDS-PAGE.

Inhibition by anti-CX3CR1 VHHs of human Fractalkin binding to human CX3CR1 expressed on BA/F3 cells

[00700] The blocking capacity for the fractalkine ligand of the VHHs was evaluated in a human CX3CR1 competition FACS as summarized in Example 4. Either CHO-huCX3CR1 cells, BA/F3-huCX3CR1 cells or transiently transfected HEK293T cells were used . The amount of labeled binder used in the different competition settings was also varied. IC50 values for VHHs blocking the interaction of human fractalkine with human CX3CR1 are depicted in TABLE 19. TABLE 19VHH POTENCY AND EFFECTIVENESS IN A BINDERS COMPETITION FACS

Inhibition by anti-CX3CR1 VHHs of human Fractalkine-induced chemotaxis of BA/F3 cells overexpressing human CX3CR1

[00701] In order to evaluate the inhibition of Fractalkine-induced chemotaxis, a chemotaxis test was set up using the disposable ChemoTx chamber with pore size of 5 µm (Neuroprobe, Gaithersburg, EU). Cells were harvested from an actively growing culture and were washed prior to use in test medium, RPMI (Gibco, Carlsbad, US) supplemented with 0.1% BSA. The bottom chamber was filled with 320 pM of human Fractalcine in a total volume of 300 µl. After membrane application, 0.13E6 cells were deposited on top of the membrane in a total volume of 70 µl. Chemotaxis was allowed for 3 hours at 37°C in a CO2 humidified chamber. After this incubation period, the membrane was removed and the cells in the bottom chamber were resuspended. The amount of ATP present in the wells was determined using the CellTiter-Glo kit (Promega, Madison WI, EU). Reading was performed on an Envision (Perkin Elmer, Massachusetts, US) with routine settings for luminescence reading. Series of titrations were performed in triplicate and each plate contained control samples also in triplicate. As a control, a sample without VHH was included as well as a sample where no human Fractalcin was added to the bottom chamber. A summary of the results is shown in TABLE 20. TABLE 20 POTENCY AND EFFECTIVENESS OF VHH IN BLOCKING FRACTALKIN-INDUCED CHEMOTAXIS

Evaluation of cross-reactivity of anti-CX3CR1 VHHs against cynomolgus CX3CR1

[00702] Initially, a FACS-based binding setup was used to assess cross-reactivity with cynomolgus. For this, the VHHs were incubated with the respective cells for 30 minutes at 4°C followed by three washing steps and subsequently incubated with the detection reagents. As detection, a mouse anti-cmyc antibody (Serotec, MCA2200) was used followed by a PE-coupled goat anti-mouse antibody (Jackson 115-116-071), each incubation for 30 minutes at 4°C, followed by three washing steps. The test results are shown in TABLE 21. TABLE 21 EC50 VALUE FOR RESPECTIVE VHH BINDING ON HUMAN CX3CR1 OR CYNOMOLGUS CX3CR1

[00703] For subsequently identified VHHs, a human Fractalkine competition FACS was set up using human or cynomolgus CX3CR1 expressed in HEK293T cells. Both the human receptor and the cynomolgus receptor were transiently transfected into HEK293T cells and the transfections were combined by binding the labeled ligand, human fractalkine. Competition was evaluated using the EC30 concentration of fractalkine and thus the IC50 values obtained are a good estimate of the Ki value, a measure for affinity (TABLE 22). The experiment was performed as described in Example 4. The ratio of IC50 values in cynomolgus monkey and human CX3CR1 was used to assess potential differences in affinity for CX3CR1 in both species. TABLE 22 EFFECTIVENESS AND POTENCY OF VHHS IN COMPETITION FACS OF LIGAND FOR HUMAN AND CYNOMOLGUS CX3CR1

Bindings of anti-human CX3CR1 VHHs to human CCR2, human CCR5, or mouse CX3CR1

[00704] Receptor specificity of huCX3CR1 was evaluated by performing a FACS binding experiment on parental CHO-K1 cells or CHO cells expressing huCCR2, huCCR5 or msCX3CR1. The VHHs were incubated with the respective cell lines for 30 minutes at 4°C followed by three washing steps and then incubated with the detection reagents. As detection, a mouse anti-cmyc antibody (Serotec, MCA2200) was used followed by a PE-coupled goat anti-mouse antibody (Jackson 115-116-071), each incubation for 30 minutes at 4°C, followed by three washing steps. For each cell line, a quality control with specific receptor antibody was included. Furthermore, the highest concentration of each VHH was also incubated with CHO cells expressing huCX3CR1 as a positive control. No binding to msCX3CR1, huCCR2 or huCCR5 could be observed.

Bin epitope determination

[00705] A competitive binding experiment was set up in order to determine whether VHHs bind overlapping epitopes on CX3CR1. For this, VHH 66B02 tagged with alexa647 was used in a competition FACS on BA/F3 cells expressing huCX3CR1. Typical VHHs from the three functional families were used as competitors for labeled 66B02 binding. The IC50 values obtained are shown in TABLE 23. TABLE 23 EPITOP BINNING BASED ON COMPETITION FACS

[00706] As a complete inhibition of 66B02 binding can be obtained by all typical VHHs of the different ligand blocking families, it can be concluded that all functional families bind in close enough proximity to each other such that they compete with the binding of 66B02.

EXAMPLE 6: VHHS FORMATTING FOR BIVALENCE Construction of bivalents

[00707] In order to increase the potency and/or effectiveness of a selection of the obtained VHHs, bivalent molecules were constructed by genetic engineering. Two VHHs were genetically linked together with a 35GS linker between the two building blocks and subsequently expressed in E.coli as described above for the monovalent VHHs. Different bivalent constructs were produced as listed in TABLE 24. TABLE 24 TYPICAL BIVALENT FORMATS

Inhibition by anti-CX3CR1 VHHs of human Fractalkin binding to human CX3CR1 expressed on BA/F3 cells

[00708] Inhibition of ligand binding to human CX3CR1 was investigated for the different formats as described in Example 4. For this characterization, the BA/F3-huCX3CR1 cell line showing stable expression of the human CX3CR1 receptor was used. The alexa647-labeled fractalkine linker was used at its EC30 concentration and thus the IC50 values obtained are reflective of the Ki values. An overview of the data obtained is shown in table 25. TABLE 25 POWER OF BIVALENT FORMATS IN BINDER COMPETITION

Inhibition by anti-CX3CR1 VHHs of human Fractalkine-induced chemotaxis of BA/F3 cells overexpressing human CX3CR1

[00709] Similar to what was described for the monovalent anti-CX3CR1 VHHs, the inhibition of chemotaxis induced by fractalkine on BA/F3-huCX3CR1 cells was evaluated for the bivalent constructs. An identical test setup as described above was used and the results obtained are summarized in Table 26. TABLE 26 INHIBITION OF FRAC-TALCIN-INDUCED CHEMOTAXIS BY BIVALENT VHH CONSTRUCTS

Evaluation of cross-reactivity of anti-CX3CR1 VHHs against cynomolgus CX3CR1

[00710] Also for the bivalent constructs the cross-reactivity for CX3CR1 from cynomolgus was evaluated and compared with the human reactivity. As described earlier, either a binding configuration (Table 27) or a ligand competition configuration (TABLE 28) were applied using transiently transfected HEK293T cells. Pools of transiently transfected cells were matched by their level of receptor expression. TABLE 27 BINDING OF BIVALENT CONSTRUCTS TO HUMAN OR CYNOMOLGUS CX3CR1 TABLE 28 BIVALENT CONSTRUCTS BINDER COMPETITION OVER HUMAN OR CYNOMOLGUS CX3CR1

EXAMPLE 7: LENGTH EXPLORATION OF THE LINEAGER AND HALF-LIFE EXTENSION Evaluation of the chain length and positioning of the VHH alb11

[00711] As the length of the chain used in a bivalent format can drastically impact the power obtained, different chain lengths were evaluated.

[00712] Furthermore, Alb11, a Nanobody binding to human serum albumin was included in order to increase the in vivo half-life of the formatted molecules (International Patent Publication No. WO 06/122787). Different formats were produced including variations on the lengths of the chain used, but also the positioning of the different VHHs in the composition. A summary of the formats explored is shown in TABLE 29. TABLE 29 EXPLORATION OF HALF-LIFE AND LENGTH EXPLORATION OF CHAINER LENGTH

[00713] Coding sequences for the formatted VHH were cloned into a plasmid constructed internally (in-house) allowing expression in Pichia pastoris and secretion in the culture medium. The expression vector was derived from pPICZa (Invitrogen) and contained the AOX1 promoter for tightly regulated methanol-induced expression, a Zeocin™ resistance gene, a multiclonatem site, and the α-factor secretion signal. After transformation, expression cultures were grown and VHH expression was induced by addition of methanol and allowed to continue for 48 hours at 30°C.

[00714] The potency of these different formats was assessed using the ligand competition test as described above. Seeing that the binder concentration used is below the EC50 value, the IC50 values obtained are equivalent to the Ki values. The Ki obtained for the different formats is summarized in Table 30. TABLE 30 POTENCY OF EXTENDED HALF-LIFE FORMATS IN LINDOR COMPETITION

Impact of human serum albumin on potency

[00715] Binding of human serum albumin (HSA) to alb11 VHH can impact on formate potency and therefore ligand competition was repeated in the presence of HSA. In summary, in order to allow binding of HSA to alb11 VHH, the constructs under evaluation and fractalkine were pre-incubated with HSA for 30 minutes prior to addition to the cells. In addition cells were resuspended in FACS buffer supplemented with HSA. The final concentration of HSA used was a 50-fold excess over the highest concentration of VHH used. Then competition was allowed for 2 hours and further processing was as described in Example 4.

[00716] The potential interference of HSA was also evaluated in an adapted chemotaxis setup by including HSA in the different test compartments. The HSA concentration used was again a 50-fold excess over the highest construct concentration used and the constructs were loaded with HSA for 30 minutes before the start of the test. The test buffer was also supplemented with HSA such that HSA is present throughout the entire length of the experiment. As described above, the disposable ChemoTx chamber with a pore size of 5 µm (Neuroprobe, Gaithersburg, MD, USA) was used. Cells were harvested from an actively growing culture and were washed prior to use in test medium, RPMI (Gibco, Carlsbad, EU) supplemented with 0.1% BSA and 62.5 µM HSA (Sigma, A8763) . The bottom chamber was filled with 320 pM human Fractalcine in a total volume of 300 μl. After membrane application, 0.13E6 cells were deposited on top of the membrane in a total volume of 70 µl. Chemotaxis was allowed for 3 hours at 37°C in a CO2 humidified chamber. After this incubation period, the membrane was removed and the cells in the bottom chamber were resuspended. The amount of ATP present in the well was determined using the Cell-Titer-Glo kit (Promega, Madison WI, USA). Reading was performed on an Envision (Perkin Elmer, Waltham, MA, USA) with routine factory settings for luminescence reading. Series of titrations were performed in triplicate and each plate contained control samples also in triplicate. As a control, a sample without VHH was included as well as a sample where no human Fractalcin was added to the bottom chamber. The IC50 values obtained are listed in TABLE 31. TABLE 31 FRACTALKIN-INDUCED CHEMOTAXIS IN THE PRESENCE OF HSA

Inhibition of Fractalkine internalization by extended half-life bivalent formated polypeptides

[00717] Additional functional tests were performed to demonstrate the antagonist activity of bivalent polypeptides with extended half-life. The polypeptides were evaluated for their ability to inhibit the internalization of A647-Fractalkine in CHO huCX3CR1 cells. Briefly, 1E4 cells/well were plated onto 96-well dark black bottom clear plates (BD, Franklin Lakes, NJ, USA) and grown overnight. Cells were washed once and then equilibrated in assay buffer (Calcium Magnesium HBSS (Gibco) supplemented with 10 mM HEPES and 0.1% BSA). The formatted polypeptide constructs were added and the plates were incubated for 15 minutes at 37°C. A647-Fractalcine was then added to a final concentration of 8 nM and the cells were incubated for 60 minutes at 37°C. The medium was removed and cells were fixed for 10 minutes with 3.7% formaldehyde solution (Polysciences, Warrington, PA, USA). Cells were rinsed once with PBS and nuclei were stained with Hoechst dye (Life Technologies, Grand Island, NY, USA). In order to quantify internalized labeled Fractalkine, cells were imaged using the BD Pathway bioimaging system. Image segmentation was performed by identifying the marked cell nucleus and drawing a 3-pixel ring around that mask. The average intensity of A647 was measured in the cytoplasmic ring. The formatted polypeptides potently inhibited Fractalkine internalization as summarized in Table 32: TABLE 32 INHIBITION OF A647-FRACTALKIN INTERNALIZATION

An anti-CX3CR1 formatted bivalent extended half-life polypeptide is devoid of agonist activity

[00718] In order to confirm that a bivalent anti-CX3CR1 extended half-life polypeptide had no agonist activity, CX3CR1BII036 was evaluated for induction of calcium influx into huCX3CR1 CHO cells. Fractalkine mediated increases in cytosolic calcium levels in these cells in a CX3CR1-dependent manner, and CX3CR1BII036 inhibited this response.

[00719] CHO huCX3CR1 cells were plated at 5E4 cells/well in clear black bottom (BD) 96-well plates and grown overnight. Cells were incubated with Calcium-4 dye /2 mM probenicid (Molecular Devices, Sunnyvale, CA, USA) in HBSS supplemented with 20 mM HEPES for 60 minutes at 37oC. To demonstrate the antagonism of the polypeptides, CX3CR1BII036 was pre-incubated with the cells for 15 minutes of addition of Fractalkine at its EC80 value. Calcium mobilization was monitored on a FLIPR Tetra system (Molecular Devices) according to the manufacturer's instructions. To determine agonism, there was no pre-incubation with the polypeptide and instead, CX3CR1BII036 was used instead of Fractalkine stimulation. While CX3CR1BII036 inhibited Fractalkine-mediated calcium influx with an IC50 of 1.3 nM, no increases in cytosolic calcium levels were observed when the isolated polypeptide was added at concentrations up to 1 µM.

EXAMPLE 8: EXPLORING HALF-LIFE EXTENSION FORMATS USING MOUSE FC

[00720] In order to investigate alternative half-life extension modalities, the 66B02 VHH domain was produced as a fusion protein with a mouse IgG2 Fc domain (66B02-mFc). An aspartic acid to alanine mutation (D265A) was incorporated into the CH2 domain to abrogate potential Fc-mediated effector function in this construct ( Baudino, J. Immunol., 181, 6664-6669 (2008 )). 66B02-mFc was expressed in HEK293T cells or NS0 cells and purified by Protein A affinity chromatography followed by ion exchange chromatography. This molecule was tested for activity using the test formats described in Example 7. The results are summarized in Table 33: TABLE 33 66B02-MFC ACTIVITY

[00721] Although 66B02-mFc potently inhibited Fractalkine-mediated activation of CX3CR1, it did not show agonist activity. No increase in cytosolic calcium levels was observed with treatment with up to 1 μM of this molecule.

EXAMPLE 9: INHIBITION OF PLAQUE PROGRESSION IN AN EXTENDED HALF-LIFE DIVALENT POLYPEPTIDE MOUSE MODEL OF ATHEROSCLEROSIS Production of human CX3CR1 knock-in in Apo E -/- mice

[00722] Given the lack of cross-reactivity of the identified VHHs to mouse CX3CR1 (Example 5), a human CX3CR1 knock-in mouse strain (hu CX3CR1 KI) was generated at TaconicArtemis (Koeln, Germany) to allow experiments on these molecules in mouse disease models. A strategy was employed that allowed expression of the human chemokine receptor under the control of the corresponding mouse promoter while disrupting expression of the endogenous mouse protein. In summary, a targeting vector was constructed where the mouse CX3CR1 coding region in exon 2 was replaced with the complete human CX3CR1 open reading frame and flanked by selection markers and loxP sites. The targeting vector was introduced into mouse ES cells and clones that had successfully undergone homologous recombination were used to generate chimeric mice. These mice were bred to highly efficient Flp-deleter mice in order to obtain selection marker removal and germline transmission. The resulting CX3CR1 KI hu mice in a C57BL/6 background were then crossed with Apo E -/- mice (The Jackson Laboratory, Bar Harbor, Maine, USA) to generate CX3CR1 KI Apo E -/- hu mice . The Apo E -/- mouse model provides a robust method for inducing extensive atherosclerotic plaque formation that is broadly similar to human disease with respect to the site-specific localization of plaque formation, histologic composition, and risk factors. known risks (cholesterol, inflammation, hypertension, etc).

Evaluation of bivalent anti-CX3CR1 polypeptides with extended half-life in the mouse model of atherosclerosis Apo E-/

[00723] Female hu CX3CR1KI ApoE -/- mice were fed a high-fat/high-cholesterol diet containing 1.5% cholesterol for 16 weeks starting at four weeks of age. After 10 weeks, the animals were administered by injection ip vehicle (20 mM Na Citrate pH 6.0, 115 mM NaCl), 10 mg/kg 66B02-mFc once or twice a week or 30 mg/kg of CX3CR1BII036 twice a week for 6 weeks. Animals were anesthetized by gas anesthesia and perfused with 0.9% saline. The descending aorta to the iliac bifurcation was carefully removed and fixed in formalin. It was then opened longitudinally, and stained with Sudan IV for 15 minutes, followed by 70% methanol for 2 minutes. The pots were washed under running water and covered with PBS. Tissues were photographed with a digital camera using SPOT Advanced software (SPOT Imaging Solutions, Sterling Heights, MI, USA). The percentage of lipid staining was determined with image analysis software (Image-Pro Plus, MediaCybernetics, Rockville, MD, USA) and expressed as a percentage of positive vessel staining. The results of this study are summarized in Table 34: TABLE 34 QUANTIFICATION OF PLAQUE SIZE IN DESCENDING AORTA IN FEMALE HU CX3CR1 KI APO E -/- MICE

[00724] Both 66B02-mFc and CX3CR1BII036 significantly inhibited plaque progression when dosed twice weekly. This correlated with coverage as plasma levels of these molecules could be confirmed to be maintained throughout the study. For once-weekly dosing of 66B02-mFc, detectable plasma levels were not maintained and this correlated with the lack of significant efficacy observed after 6 weeks of treatment. No molecule significantly affected plasma levels of cholesterol or triglycerides.

EXAMPLE 10: PARENTAL VHH SEQUENCE OPTIMIZATION

[00725] In general, during VHH sequence optimization, parental wild-type VHH sequences are mutated to produce VHH sequences that are more identical to human VH3-JH germline consensus sequences. Specific amino acids in the framework regions that differ between the VHH and the human VH3-JH germline consensus are changed to the human equivalent such that the structure, activity, and stability of the protein are kept intact. To investigate this, all sequence optimization variants were compared to the parental VHH in three different tests: (i) determination of the melting temperature (Tm) in a Thermal Shift Assay (TSA), (ii) in vitro potency analysis in fractalkine competition FACS, and for some constructs (iii) in vitro potency analysis in the fractalkine-induced chemotaxis test.

Mutation of framework residues

[00726] For sequence optimization, the following mutations were investigated: E1D, S11L, A14P, E16G, R44Q, D46E, A74S, K83R and Q108L. The individual mutants that were generated in the parental sequence of CX3CR1BII66B02 are represented in Table 35: TABLE 35 MUTATIONS INVESTIGATED DURING SEQUENCE OPTIMIZATION OF 66B02

[00727] All constructs were cloned into an E. coli expression vector, and expressed in E. coli as myc/His-tagged proteins in a culture volume of 0.25 L to 0.5 L of TB medium. Expression was induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37°C and 250 rpm. Cells were pelleted, and periplasmic extracts were prepared by freeze-thaw and resuspension in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using Histrap FF (GE heal-thcare) raw columns. Nanobodies were eluted from the column with 250 mM imidazole and then desalted into dPBS. The purity and integrity of the Nanobodies was verified by reducing SDS-PAGE.

[00728] As summarized in Table 36, the mutations A14P, A74S, K83R and Q108L had no clear effect on potency as determined from the competition FACS. Similarly, the additional E1D, S11L and E16G mutations did not affect potency. On the other hand, introduction of either R44Q or D46E resulted in a significant drop in potency that was even more pronounced if both mutations were introduced. TABLE 36 POWER OF SEQUENCE OPTIMIZATION CONSTRUCTS DETERMINED BY BINDER COMPETITION FACS

[00729] In addition, the melting temperature, predictive for the stability of the VHH, was evaluated. Most of the individual mutations had limited or no effect with the exception of the D46E mutation, which increased the melting temperature by approximately 6°C. The introduction of the combined mutations also increased thermal stability, cfr 057 and 060.

[00730] Due to the important effects on potency in the FACS of ligand competition, the mutations R44Q and D46E were not included in the final sequence.

CDR residue mutation

[00731] Based on the in silico analysis of the parental sequence, a glycosylation site was predicted at position 52. Therefore, two libraries were constructed; one for position 52 and one for position 53, which was designed to include all possible amino acids at the respective position. Libraries were screened as periplasmic extracts in a ligand competition FACS. First, a dilution series was made of periplasmic material from the parental sequence and three dilutions were selected for further screening. A first dilution point (two-fold) was chosen to provide complete blockage of the ligand interaction whereas the other two dilution points (128 and 512-fold) will result in 70% and 40% blockade respectively. After producing the periplasmic extracts from the library, all samples were divided into two and one of them was subjected to a heat treatment. Both untreated and heat-treated samples were then analyzed in the binder competition FACS at the three dilution points. The impact of the mutation can be estimated by comparing the obtained block with the parental sequence block. Analysis of heat-treated samples provides a measure for a potential impact on mutation stability.

[00732] Based on the initial screening results, seven mutations were selected for further characterization. The potency obtained in the ligand competition FACS is shown in Table 37. TABLE 37 GLYCOSYLATION SITE REMOVAL AT POSITION 52

[00733] From this analysis, sequence alignment with the human reference sequence and based on an in silico T cell epitope recognition prediction program, it was decided to include the N52S and S53T mutations in the sequence.

[00734] For stability concerns, an additional library was produced for position 32. The ligand competition screen was set up in a similar fashion as described above. Again, three dilutions of the periplasmic extracts were screened and the % blocking obtained was compared with the % blocking obtained for the parental sequence. After analysis of the various mutants, the N32T replacement was chosen and included in the final optimized sequence variant.

EXAMPLE 11: ANALYSIS OF OPTIMIZED VARIANTS

[00735] In a final characterization round, the constructs listed in TABLE 38 were characterized. TABLE 38 OPTIMIZED SEQUENCE VARIANTS OF VHH 66B02 LIDER

[00736] A competition FACS experiment was performed as described above as well as a melting temperature determination. The values obtained are represented in Table 39. TABLE 39 COMPETITION FACS AND MELTING TEMPERATURE (TM) OF OPTIMIZED SEQUENCE VARIANTS

[00737] These constructs were also characterized in fractalkine-induced chemotaxis as described above (Table 40). TABLE 40 LIGAND-INDUCED CHEMOTAXIS WITH OPTIMIZED SEQUENCE VARIANTS

[00738] Selected constructs were evaluated for inhibition of A647-Fractalkine induced internalization in CHO huCX3CR1 cells. The results are summarized in Table 41: TABLE 41 LIGAND INDUCED INTERNALIZATION WITH OPTIMIZED SEQUENCE VARIANTS

An extended half-life anti-CX3CR1 sequence-optimized polypeptide is devoid of agonist activity

[00739] In order to confirm that the sequence-optimized anti-CX3CR1 polypeptide with extended half-life does not have agonist activity, CX3CR1BII00313 was evaluated for induction of calcium influx in huCX3CR1 CHO cells. While preincubation with CX3CR1BII00313 inhibited Fractalkine-mediated calcium influx with an IC50 of 1.3 nM, no increase in cytosolic calcium levels was observed when isolated polypeptide was added at concentrations up to 1 µM.

EXAMPLE 12: EXPLORING HALF-LIFE EXTENSION FORMATS USING HUMAN FC

[00740] In order to investigate additional half-life extension modalities, the sequence-optimized VHH domains CX3CR1BII00306 and CX3CR1BII00307 were produced as fusion proteins with a human IgG1 Fc domain (306D-hFc and 307D-hFc). Two mutations were incorporated into the CH2 domain to abrogate potential Fc-mediated effector function in this construct. 306D-hFc and 307D-hFc were expressed in HEK293T cells or NS0 cells and purified by Protein A affinity chromatography followed by ion exchange chromatography. These molecules were tested for functional activity using the test formats described in Example 7. The results are summarized in Table 42: TABLE 42 HFC FUSION PROTEIN ACTIVITY

[00741] Although these molecules potently inhibited Fractalkine-mediated activation of CX3CR1, they did not exhibit agonist activity. No increases in cytosolic calcium levels were observed with treatment with up to 1 μM of these isolated Nanobodies.

EXAMPLE 13: INHIBITION OF PLAQUE PROGRESSION IN A MOUSE MODEL OF ATHEROSCLEROSIS BY A SEQUENCE-OPTIMIZED ANTI-CX3CR1 NA-NOBODY

[00742] Female hu CX3CR1KI ApoE -/- mice were fed a high-fat/high-cholesterol diet containing 1.5% cholesterol for 16 weeks starting at four weeks of age. After 10 weeks, the animals were administered by injection ip vehicle (20 mM Na Citrate pH 6.0, 115 mM NaCl), 30 mg/kg of CX3CR1BII00313 once or twice a week or 30 mg/kg of CX3CR1BII036 twice a week for 6 weeks. The animals were sacrificed and the percentage of plaque area in the descending aorta was quantified as described above. The results of this study are summarized in Table 43: TABLE 43 QUANTIFICATION OF PLAQUE SIZE IN DESCENDING AORTA IN FEMALE HU CX3CR1 KI APO E -/- MICE

[00743] Both CX3CR1BII00313 and CX3CR1BII036 significantly inhibited plaque progression when dosed twice weekly. This correlated with coverage as plasma levels of these molecules could be confirmed to be maintained throughout the study. For once-weekly dosing of CX3CR1BII00313, no detectable plasma levels were maintained and this correlated with the lack of significant efficacy observed after 6 weeks of treatment. No molecule significantly affected plasma levels of cholesterol or triglycerides.

EXAMPLE 14: BINDING OF NANOBODY TO HUMAN AND MONKEY CYNOMOLGUS PRIMARY CD14+ CELLS IN WHOLE BLOOD Competition FACS with Formatted Sequence Optimized Anti-CX3CR1 Nanobody

[00744] In order to confirm the binding of the formatted sequence-optimized anti-CX3CR1 Nanobody to primary human cells, CX3CR1BII00313 was shown to compete for the binding of A647-labeled CX3CR1BII018 (A647-018) to CD14+ cells in a competition FACS test at the whole blood. Briefly, an eFluor 450-conjugated mouse anti-human CD14 antibody (eBioscience, San Diego, CA, USA) was diluted 1:10 in EDTA-treated whole blood from a healthy human donor. 40 µl/well was added to a 96-well polystyrene round bottom plate followed by 10 µl/well of CX3CR1BII00313 diluted in BSA Stain Buffer (BD Pharmingen) at a final concentration ranging from 100 nM to 0.002 pM and the samples were incubated for 20 minutes at room temperature. 10 µl/well of A647-018 in Stain Buffer was then added to yield a final concentration of 1 nM (the EC80 of A647-018 binding) and the samples were incubated for an additional 20 minutes at room temperature. 220 µl/well of 1-Step Fix/Lyse solution (eBioscience) was then added. After an incubation at room temperature for 10 minutes, the cells were pelleted, washed twice in Stain buffer and resuspended in this buffer. Samples were analyzed on a BD LSR II flow cytometer. The mean fluorescence intensity for AlexaFluor 647 was quantified to gate the CD14 positive cell population. CX3CR1BII00313 potently inhibited binding of A647-018 to CD14 positive cells in human blood with an IC50 of 0.35 nM (n=8).

[00745] In order to confirm the binding of sequence-optimized anti-CX3CR1 Nanobody formatted to primary cynomolgus monkey cells, it was demonstrated that CX3CR1BII00313 competes for the binding of A647-labeled CX3CR1BII018 (A647-018) to CD14+ cells in a test FACS competition in cynomolgus monkey whole blood. The method used was analogous to the method summarized above with the exception that the final concentration of A647-018 was 3 nM (the EC80 of A647-018 binding) and ACK lysis buffer (Life Technologies) was used instead of the solution 1-Step Fix/Lyse. Cells were resuspended in Stain buffer supplemented with 1% formaldehyde before analysis. CX3CR1BII00313 potently inhibited binding of A647-018 to CD14 positive cells in cynomolgus monkey blood with an IC50 of 0.43 nM (n=4).

EXAMPLE 15: PHARMACOKINETICS (PK) IN CYNOMO-LGUS MONKEYS

[00746] A pharmacokinetic study was conducted in naïve male cynomolgus monkeys (Macaca fascicularis) aged 2 to 5 years with a body weight range between 2.4 to 3.5 kg. The monkeys were divided into four treatment groups. Group 1 (n=3) received 0.2 mg/kg CX3CR1BII00313 i.v.; Group 2 (n=3) received 2 mg/kg of CX3CR1BII00313 i.v.; Group 3 (n=3) received 2 mg/kg CX3CR1BII00313 s.c. and Group 4 (n=3) received 5 mg/kg CX3CR1BII00313 i.v. CX3CR1BII00313 was administered as a 2 mg/ml solution in citrate buffer (20 mM sodium citrate/115 mM sodium chloride, pH 6.0). Blood samples were collected over 6 weeks from a peripheral vein into serum separator tubes for pharmacokinetic analysis.

[00747] Serum samples were analyzed using an MSD (Meso Scale Discovery) format. Briefly, a biotinylated anti-Nanobody antibody was bound to a standard MSD streptavidin plate (Meso Scale Discovery, Rockville, MD, USA). Plates were washed with 0.05% Tween 20 in phosphate buffered saline and blocked with 5% wt/vol SeraCare BSA (SeraCare Life Sciences, Milford, MA, USA) prior to incubation with the serum samples. CX3CR1BII00313 was detected using a sulfo-labeled anti-Nanobody Nanobody and the plates were analyzed on a Sector Imager 2400 (Meso Scale Discovery). Varying concentrations of CX3CR1BII0313 from 5000 to 0.5 ng/ml in 5% monkey serum were used as standards. Target involvement was assessed by monitoring free CX3CR1 levels on gated CD14+ monocytes. This test was analogous to the competition FACS test summarized in Example 14 with the exception that no additional CX3CR1BII00313 was added. Serum samples were also monitored for the presence of primate anti-human antibodies (PAHA) as these may impact the assessment of pharmacokinetics and free CX3CR1.

[00748] ForteBio RED96 was used for PAHA detection. In summary, biotinylated CX3CR1BII0313 was captured on streptavidin sensors. Pooled naive monkey serum was then used as a negative control to calculate the cut-off value (defined as twice the average binding signal of naive sera). All serum samples were diluted 20-fold in buffer and the PAHA response was determined to be positive if the binding signal was greater than the cut-off value.

[00749] Data for time points after PAHA detection were excluded from PK/PD analysis. Pharmacokinetic data are summarized in Table 44 below. TABLE 44 PHARMACOKINETIC PARAMETERS OF CX3CR1BII00313

[00750] **Insufficient data to characterize the terminal phase

[00751] The clearance and half-life at 2.0 mg/kg i.v. were 9.4 mL/d/kg and 9.6 days, respectively. At 0.2 mg/kg i.v., clearance was substantially higher (113 mL/d/kg) consistent with saturable target-mediated disposition (TMD) pharmacokinetics. The dose-adjusted AUC(0-14d) was comparable between the 2 and 5 mg/kg i.v. doses suggesting TMD saturation at the 2 mg/kg dose. Exposure at 2 weeks after Nanobody administration either i.v. or s.c. was >70 nM and bioavailability after s.c. administration was 54%. Receptor-free traced exposure with greater than 90% average coverage maintained at exposures > 10 nM.

Claims (22)

1. Polypeptide, characterized in that it comprises a unique variable domain of anti-CX3CR1 immunoglobulin (anti-CX3 chemokine receptor 1), said polypeptide capable of blocking the binding of human fractalkine to human CX3CR1, wherein said polypeptide comprises a CDR1, CDR2 and CDR3 having the amino acid sequences defined in: - SEQ ID No: 146, 176 and 191, respectively; or - SEQ ID No: 146, 177 and 191, respectively. 2. Polypeptide, according to claim 1, characterized by the fact that the unique variable domain of the anti-CX3CR1 immunoglobulin essentially consists of four framework regions (FR1, FR2, FR3 and FR4) and three complementary determining regions (CDR1, CDR2 and CDR3). 3. Polypeptide according to claim 1 or 2, characterized in that the unique variable domain of the anti-CX3CR1 immunoglobulin is a VH, VL, VHH, camelized VH or VHH that is optimized for stability, potency, manufacturability and/or similarity to human framework regions. 4. Polypeptide according to any one of claims 1 to 3, characterized in that said single variable domain of anti-CX3CR1 immunoglobulin is a VHH domain comprising the defined sequence: a) in the amino acid sequence of SEQ ID NO: 49; or b) an amino acid sequence of any one of SEQ ID NO: 49-52. 5. Polypeptide according to any one of claims 1 to 4, characterized in that the polypeptide additionally comprises a half-life extension moiety. 6. Polypeptide according to claim 5, characterized in that said half-life extension moiety is covalently linked to said polypeptide and is selected from the group consisting of an albumin-binding moiety, such as a anti-albumin immunoglobulin domain, a transferrin binding moiety such as an anti-transferrin immunoglobulin domain, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, a peptide binding of albumin or an Fc domain. 7. Polypeptide, according to claim 5, characterized by the fact that said half-life extension portion consists of a single variable domain of anti-albumin immunoglobulin. 8. Polypeptide according to claim 7, characterized in that the unique variable domain of anti-albumin immunoglobulin comprises a sequence selected from any one of SEQ ID NO's: 230 to 232. 9. Polypeptide according to any one of claims 1 to 8, characterized in that the single immunoglobulin variable domain is selected from a VHH domain, a humanized VHH domain, a camelized VH domain, an antibody domain, an antibody single domain names and/or "dAb"s. 10. Bacterial, yeast or fungal host cell, characterized in that it comprises a nucleic acid molecule encoding a polypeptide, as defined in any one of claims 1 to 9, wherein said host cell is capable of expressing said polypeptide . 11. Pharmaceutical composition, characterized in that it comprises (i) a polypeptide, as defined in any one of claims 1 to 9, and (ii) a pharmaceutically acceptable vehicle, and optionally (iii) a diluent, an excipient, an adjuvant and/or a stabilizer. 12. Pharmaceutical composition according to claim 11, characterized in that said pharmaceutical composition is suitable for intravenous or subcutaneous injection into a human being. 13. Method of manufacturing a polypeptide, as defined in any one of claims 1 to 9, characterized in that it comprises the steps of - cultivating a host cell under conditions that allow the expression of a polypeptide, as defined in any one of claims 1 to 9, wherein said host cell is carrying an expression vector comprising a nucleic acid molecule, said nucleic acid molecule comprising a region encoding a polypeptide as defined in any one of claims 1 to 9 , and wherein said host cell is a prokaryotic cell or a eukaryotic cell. 14. Method according to claim 13, characterized in that it additionally comprises the steps of - recovering said polypeptide; and - purifying said polypeptide. 15. Polypeptide, according to any one of claims 1 to 9, characterized in that it is for use in the treatment, prevention or mitigation of a disease, disorder or condition in a human being. 16. Polypeptide according to claim 15, characterized in that the disease, disorder or condition is a disease, disorder or condition associated with CX3CR1. 17. Polypeptide, according to claim 16, characterized by the fact that the disease, disorder or condition is selected from cardio- and cerebrovascular atherosclerotic disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis , human crescent 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, sclerosis multiplex (MS), Alzheimer's disease, lung diseases such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer. 18. Polypeptide for use according to claim 17, characterized in that the disease, disorder or condition is atherosclerosis. 19. Diagnostic kit, characterized in that it comprises a polypeptide, as defined in any one of claims 1 to 9, and one or more means, detection means and/or imaging agents, and optionally, instructions for use; wherein said kit is suitable for diagnosing a disease, disorder or condition defined by CX3CR1 dysfunction. 20. Diagnostic kit, according to claim 19, characterized in that it is for the diagnosis of at least one of cardio- and cerebrovascular atherosclerotic disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescent 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, lung diseases such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer. 21. Diagnostic method, characterized in that it comprises, from a sample of an individual, the steps of: a) contacting the sample with a polypeptide as defined in any one of claims 1 to 9; b) determine the amount of CX3CR1 in the sample; and c) comparing the amount determined in step (b) to a standard, wherein a difference in amount from said sample is diagnostic of a disease, disorder or condition characterized by dysfunction of CX3CR1. 22. Diagnostic method, according to claim 21, characterized in that it is for the diagnosis of at least one of atherosclerotic cardio- and cerebrovascular disorders, peripheral arterial disease, restenosis, diabetic nephropathy, glomerulonephritis, glomerulonephritis human crescent, 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 ( in English, MS), Alzheimer's disease, lung diseases such as chronic obstructive pulmonary disease, asthma, neuropathic pain, inflammatory pain, or cancer.