CX3CR1 binding compounds, methods and uses thereof

Abstract

The present technology relates to polypeptides comprising at least three immunoglobulin single variable domains (ISVDs). In particular, the present technology relates to a polypeptide comprising an optimized ISVD that binds to the fractal chemokine receptor CX3CR1 and an optimized ISVD that binds to albumin. The present technology also relates to compositions of polypeptides; a polynucleotide encoding the polypeptide; a host or host cell comprising the polynucleotide; methods for producing polypeptides or compositions; and polypeptides, polynucleotides or compositions for use in the prevention, treatment and diagnosis of a disease or disorder.

Description

1. Technical Field

[0001] The present invention relates to a polypeptide comprising an immunoglobulin single variable domain (ISVD) that binds to CX3CR1. In particular, the present invention relates to a polypeptide comprising an optimized ISVD that binds to the fractal chemokine receptor CX3CR1 and an optimized ISVD that binds to albumin. The present invention also relates to a composition of polypeptides; a polynucleotide encoding the polypeptide; a host or host cell comprising the polynucleotide; a method for producing a polypeptide or a composition; and a polypeptide, polynucleotide or composition for use in the prevention, treatment and diagnosis of a disease or disorder. 2. Background technology

[0002] C-X3-C motif chemokine receptor 1 (CX3CR1) (also known as fractal chemokine receptor or G protein coupled receptor 13 (GPR13)) is a transmembrane protein and receptor for the chemokine fractal chemokine (also known as CX3CL1). CX3CR1-CX3CL1 signaling plays different functions in different tissue compartments, such as immune response, inflammation, cell adhesion and chemotaxis (see, e.g., Imai et al. 1997, Cell 91:521-30). CX3CR1-CX3CL1 signaling is involved in the pathogenesis of various clinical disease states or processes (such as atherosclerosis, glomerulonephritis, cardiac allograft rejection and rheumatoid arthritis) (see, e.g., Umehara et al. 2004, Arterioscler. Thromb. Vasc. Biol. 24:34-40).

[0003] In addition, polymorphisms of CX3CR1 have been shown to have clinical significance. For example, functional CX3CR1 analysis shows that fractal chemokine binding is reduced in patients homozygous for CX3CR1 variants (V249I / T280M) (variant haplotypes affecting two amino acids (isoleucine 249 and methionine 280)). CX3CR1 variants (V249I / T280M) are associated with differences in susceptibility to HIV infection (see, e.g., Faure et al. 2000, Science 287:2274-7), atherosclerotic disease and stroke (see, e.g., McDermott et al. 2001, Circ. Res. 89:401-7; Moatti et al. 2001, Blood 97:1925-8). Although HIV-infected patients homozygous for the CX3CR1 variant (V249I / T280M) progress to AIDS faster than patients with other haplotypes (see, e.g., Faure et al. 2000, Science 287:2274-7), the CX3CR1 variant (V249I / T280M) has also been shown to be associated with a lower risk of cardiovascular disease (see, e.g., McDermott et al. 2001, Circ. Res. 89:401-7; McDermott et al. 2003, J. Clin. Invest. 111:1241; Ghilardi et al. 2004, Stroke 35:1276).

[0004] Therefore, modulation of CX3CR1 activity may provide a promising therapy.For example, several independent mouse genetic studies have shown beneficial effects of CX3CR1 deficiency on atherosclerosis (Combadière et al. 2003, Circulation 107:1009; Lesnik et al. 2003, J. Clin. Invest. 111:333).

[0005] Eisai Co. developed a small molecule modulator of CX3CR1 activity, E-6130 (e.g., Wakita et al. 2017, Mol. Pharmacol. 92:502), and the small molecule modulator entered clinical trials in 2016. However, in 2018, Eisai announced the discontinuation of these clinical studies. KAND-145 and KAND-567 are other small molecule antagonists of CX3CR1 in different diseases developed by Kancera.

[0006] Eisai has developed a humanized anti-fractional chemokine (CX3CL1) monoclonal antibody, quemolimab (also known as E6011) (Tanaka et al. 2021, Modern Rheumatol. 31: 783). Clinical development of this biologic has also been discontinued.

[0007] Anti-CX3CR1 VHH is described in WO 2013 / 130381. On this basis, BI 655088 (named A041600087 in this specification) was developed as a therapeutic compound for treating atherosclerosis (see https: / / adisinsight.springer.com / drugs / 800044864; Low et al. 2020, Mabs, 12: 1709322). However, to date, there are no available and / or developing therapeutic biologics that bind to CX3CR1 for the treatment of diseases associated with fractal chemokine receptor CX3CR1 or single nucleotide polymorphism (SNP) variants of CX3CR1.

[0008] Disadvantages of available CX3CR1 modulators (eg, anti-CX3CR1 antibodies) include their manufacturing process and storage (eg, low production efficiency, low product quality) and their therapeutic applications (eg, low half-life of CX3CR1 modulators, high immunogenicity, off-target binding).

[0009] Therefore, there is a need in the art for improved CX3CR1 binding compounds to modulate CX3CR1 activity to prevent, treat and / or diagnose diseases. 3. Summary of the invention

[0010] The inventors of the present invention have developed polypeptides targeting CX3CR1 and comprising a half-life extension portion, which exhibit increased efficacy and / or efficacy of regulating CX3CR1 activity compared to available anti-CX3CR1 therapeutic compounds. The polypeptide can be effectively produced (e.g., high expression yield in a microbial host; better recovery after purification), and exhibits improved (long-term) stability under storage conditions (e.g., measured by turbidity or opalescence of a polypeptide at a concentration of 100 mg / mL). In addition, the polypeptide shows limited reactivity to pre-existing antibodies in a subject to be treated (i.e., antibodies present in a subject before the first treatment with the polypeptide). In certain embodiments, such polypeptides exhibit a sufficiently long half-life in a subject in need that can conveniently space consecutive treatments (e.g., to prevent, treat, or diagnose a disease or disorder associated with a fractal chemokine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1). Furthermore, following production and / or further purification, such polypeptides may be shown to have improved product quality (eg, reduced amounts of low molecular weight species and / or reduced amounts of high molecular weight species and / or aggregation).

[0011] In some embodiments, the technology of the present invention provides a polypeptide comprising or consisting of: at least two immunoglobulin single variable domains (ISVDs) that specifically bind to CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and a half-life extension portion that specifically binds to a serum protein such as albumin. In other embodiments, the polypeptide comprises or consists of: two ISVDs that specifically bind to a single nucleotide polymorphism (SNP) variant of CX3CR1 and / or CX3CR1 and one ISVD that specifically binds to albumin, wherein the three ISVDs are optionally directly linked or linked via a peptide linker.

[0012] In another aspect, polynucleotides capable of expressing the polypeptides of the present invention, vectors comprising the polynucleotides, and compositions comprising the polypeptides, the polynucleotides, or the vectors are provided. In some embodiments, the composition is a pharmaceutical composition.

[0013] Also provided is a host or host cell comprising the polynucleotide or a vector encoding a polypeptide according to the technology of the present invention. Also provided is a method for producing the polypeptide and the use of the polypeptide as a drug in preventing, treating and diagnosing a disease or disorder (e.g., inflammatory disease, atherosclerotic disease or stroke).

[0014] The present invention meets or solves at least some of the above needs and aims to solve the above problems in the art by providing improved products and methods defined by the independent claims. Specific embodiments are set forth in the various dependent claims and in the specific embodiments described below. The present invention provides:

[0015] Item 1. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), wherein each of the at least three ISVDs has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions and CDR1 to CDR3 refer to complementarity determining regions; and wherein the polypeptide comprises

[0016] (a) a first ISVD that specifically binds to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and comprises (according to AbM numbering)

[0017] CDR1 as shown in SEQ ID NO: 10;

[0018] CDR2 as shown in SEQ ID NO: 12; and

[0019] CDR3 as shown in SEQ ID NO: 14; and

[0020] (b) a second ISVD that specifically binds to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and comprises (according to AbM numbering)

[0021] CDR1 as shown in SEQ ID NO: 10;

[0022] CDR2 as shown in SEQ ID NO: 12; and

[0023] CDR3 as shown in SEQ ID NO: 14; and

[0024] (c) a third ISVD that specifically binds albumin and (according to AbM numbering) comprises a CDR1 as shown in SEQ ID NO: 53 or having two or one amino acid differences from SEQ ID NO: 53;

[0025] a CDR2 as shown in SEQ ID NO:55, or having two or one amino acid differences from SEQ ID NO:55; and a CDR3 as shown in SEQ ID NO:57, or having two or one amino acid differences from SEQ ID NO:57;

[0026] and

[0027] Wherein (a), (b) and (c) are directly linked or linked via a peptide linker.

[0028] Item 2. A polypeptide according to the preceding item, wherein the C-terminal ISVD of the polypeptide carries a C-terminal extension of FR4 having a length of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0029] Item 3. The polypeptide according to any one of the preceding items, wherein the order of (a), (b) and (c) indicates their relative positions within the polypeptide from the N-terminus to the C-terminus of the polypeptide.

[0030] Item 4. The polypeptide according to any one of the preceding items, wherein the ISVD in (a), (b) and (c) is selected from a VH domain and a VHH domain.

[0031] Item 5. The polypeptide according to the preceding item, wherein the ISVD in (a), (b) and (c) is selected from the group consisting of a camelized VH domain, a humanized VHH domain, a domain antibody and a dAb.

[0032] Item 6. The polypeptide according to the preceding item, wherein the ISVD in (a), (b) and (c) is a humanized VHH domain.

[0033] Item 7. A polypeptide according to any of the preceding items, wherein in (a) and / or in (b), the ISVD comprises one or more of the following: aspartic acid (D) at amino acid position 1 according to Kabat numbering, proline (P) at amino acid position 14 according to Kabat numbering, glycine (G) at amino acid position 16 according to Kabat numbering, arginine (R) at amino acid position 83 according to Kabat numbering, leucine (L) at amino acid position 89 according to Kabat numbering, and leucine (L) at amino acid position 108 according to Kabat numbering.

[0034] Item 8. A polypeptide according to any of the preceding items, wherein in (a) and / or in (b), the ISVD comprises one or more of serine (S) at amino acid position 11 according to Kabat numbering and glutamine (Q) at position 44 according to Kabat numbering.

[0035] Item 9. A polypeptide according to any of the preceding items, wherein in (a) and / or in (b), the ISVD comprises one or more of a threonine (T) at amino acid position 32 according to Kabat numbering, a serine (S) at amino acid position 52 according to Kabat numbering, and a threonine (T) at amino acid position 53 according to Kabat numbering.

[0036] Item 10. A polypeptide according to any of the preceding items, wherein in (a) and / or in (b), the ISVD comprises one or more of valine (V) at amino acid position 50 according to Kabat numbering, isoleucine (I) at amino acid position 56 according to Kabat numbering, and alanine (A) at amino acid position 96 according to Kabat numbering.

[0037] Item 11. The polypeptide according to any one of the preceding items, wherein in (a) and / or (b), the framework regions FR1 to FR4 comprise or consist of:

[0038] FR1 as shown in SEQ ID NO:9;

[0039] FR2 as shown in SEQ ID NO: 11;

[0040] FR3 as shown in SEQ ID NO: 13; and

[0041] FR4 as shown in SEQ ID NO:15.

[0042] Item 12. A polypeptide according to any of the preceding items, wherein in (a) and / or in (b), the ISVD comprises an amino acid sequence having a sequence identity of greater than 90% or greater than 95% to SEQ ID NO: 2, wherein for the purpose of determining the % sequence identity, the amino acid sequence of the CDR region is ignored.

[0043] Item 13. A polypeptide according to any of the preceding items, wherein in (a) and / or in (b), the ISVD comprises an amino acid sequence having a sequence identity of greater than 90% or greater than 95% to SEQ ID NO: 2.

[0044] Item 14. A polypeptide according to any of the preceding items, wherein in (a), the ISVD consists of an amino acid sequence as shown in SEQ ID NO:3 or amino acids having at least 90% sequence identity with SEQ ID NO:3, and in (b), the ISVD consists of an amino acid sequence as shown in SEQ ID NO:2 or amino acids having at least 90% sequence identity with SEQ ID NO:2, wherein for the purpose of determining the % sequence identity, the amino acid sequence of the CDR region is ignored.

[0045] Item 15. A polypeptide according to any of the preceding items, wherein in (a), the ISVD consists of an amino acid sequence as shown in SEQ ID NO:3 or an amino acid having at least 90% sequence identity with SEQ ID NO:3, and in (b), the ISVD consists of an amino acid sequence as shown in SEQ ID NO:2 or an amino acid having at least 90% sequence identity with SEQ ID NO:2.

[0046] Item 16. A polypeptide according to any of the preceding items, wherein in (a), the ISVD consists of an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid differences with SEQ ID NO: 2, and in (b), the ISVD consists of an amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid differences with SEQ ID NO: 2.

[0047] Item 17. The polypeptide according to any one of the preceding items, wherein in (a) and / or in (b), the ISVD comprises the amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0048] Item 18. The polypeptide according to any one of the preceding items, wherein in (a), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 3, and in (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 2.

[0049] Item 19. A polypeptide according to any one of the preceding items, wherein in (a), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 3, and in (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 2, and wherein the order of (a), (b) and (c) indicates their relative positions within the polypeptide from the N-terminus to the C-terminus of the polypeptide.

[0050] Item 20. The polypeptide according to any one of the preceding items, wherein in (c), the ISVD specifically binds to human serum albumin.

[0051] Item 21. A polypeptide according to any one of the preceding items, wherein in (c), the framework regions FR1 to FR4 comprise

[0052] FR1 as shown in SEQ ID NO:52;

[0053] FR2 as shown in SEQ ID NO:54;

[0054] FR3 as shown in SEQ ID NO:56; and

[0055] FR4 as shown in SEQ ID NO:58.

[0056] Item 22. A polypeptide according to any of the preceding items, wherein in (c), the ISVD comprises or consists of: an amino acid sequence as shown in SEQ ID NO: 37 or an amino acid sequence having a sequence identity of greater than 90% or greater than 95% identity to SEQ ID NO: 37, wherein for the purpose of determining the % sequence identity, the amino acid sequence of the CDR region is ignored.

[0057] Item 23. A polypeptide according to any one of the preceding items, wherein in (c), the ISVD comprises or consists of an amino acid sequence as shown in SEQ ID NO: 37 or an amino acid sequence having a sequence identity of greater than 90% or greater than 95% to SEQ ID NO: 37.

[0058] Item 24. The polypeptide according to any one of the preceding items, wherein in (c), the ISVD comprises or consists of the amino acid sequence as shown in SEQ ID NO: 37.

[0059] Item 25. A polypeptide according to any of the preceding items, wherein in (a) and (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 2, and in (c), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 37.

[0060] Item 26. A polypeptide according to any one of the preceding items, wherein in (a), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:3, in (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:2, and in (c), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:37.

[0061] Item 27. A polypeptide according to any one of the preceding items, wherein the ISVD located at the C-terminus of the polypeptide carries the C-terminal extension on the C-terminus of FR4, wherein the C-terminus of FR4 is SEQ ID NO: 135 and the C-terminal extension of FR4 comprises 1, 2, 3, 4 or 5 amino acids.

[0062] Item 28. The polypeptide according to the preceding item, wherein the C-terminal extension of FR4 comprises or consists of: one, two or three alanine (Ala) residues.

[0063] Item 29. The polypeptide according to the preceding item, wherein the C-terminal extension of FR4 is a single Ala residue.

[0064] Item 30. The polypeptide according to any one of the preceding items, wherein the peptide linker comprises 3 to 62 amino acids.

[0065] Item 31. The polypeptide according to the preceding item, wherein the amino acid in the peptide linker is selected from alanine (A), glycine (G) and serine (S).

[0066] Item 32. The polypeptide according to the preceding item, wherein the peptide linker is selected from a 9GS linker, a 20GS linker and a 35GS linker.

[0067] Item 33. The polypeptide according to the preceding item, wherein the peptide linker comprises or consists of a 9GS linker.

[0068] Item 34. The polypeptide according to any one of the preceding items, comprising two peptide linkers, wherein each peptide linker is located between two ISVDs within the polypeptide;

[0069] Wherein the two peptide linkers comprise or consist of the amino acid sequence shown in SEQ ID NO: 69.

[0070] Item 35. A polypeptide according to any one of the preceding items, wherein the polypeptide comprises an amino acid sequence having a sequence identity of greater than 90% or greater than 95% to SEQ ID NO: 82.

[0071] Item 36. A polypeptide according to any of the preceding items, wherein the polypeptide comprises an amino acid sequence having a sequence identity of greater than 90% or greater than 95% with SEQ ID NO: 82, wherein the CDR regions are ignored for the purpose of determining the sequence identity.

[0072] Item 37. The polypeptide according to the preceding item, wherein the polypeptide comprises the amino acid sequence as shown in SEQ ID NO:82.

[0073] Item 38. The polypeptide according to the preceding item, wherein the polypeptide consists of the amino acid sequence shown in SEQ ID NO:82.

[0074] Item 39. A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 82

[0075] Item 40. A polypeptide according to any one of the preceding items, wherein the single nucleotide polymorphism (SNP) variant of CX3CR1 comprises at least one SNP selected from SNP rs938203, rs2669849, rs1050592, rs3732379 and rs3732378.

[0076] Item 41. The polypeptide according to any of the preceding items, wherein the first ISVD in (a) and / or the second ISVD in (b) is characterized by reduced antigenicity compared to an ISVD consisting of the amino acid sequence as shown in SEQ ID NO: 1;

[0077] Optionally, wherein said antigenicity is assessed by binding at a human leukocyte antigen DR isotype (HLA-DR) allele.

[0078] Item 42. The polypeptide according to any one of the preceding items, wherein the polypeptide is characterized in that the yield is increased after expression in the yeast Pichia pastoris compared to a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83.

[0079] Item 43. The polypeptide according to any of the preceding items, wherein the polypeptide is characterized in that at least one biophysical property is improved compared to a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83;

[0080] wherein the biophysical property is selected from

[0081] i. improved stability after purification of the polypeptide, for example as measured by turbidity / opalescence of the polypeptide at a concentration of 100 mg / mL;

[0082] ii. after expressing the polypeptide in a suitable host (e.g., after expressing the polypeptide in Pichia pastoris), the amount of low molecular weight substances is reduced, for example, as measured by the pre-peak on reverse phase chromatography (e.g., RP-UPLC);

[0083] iii. the amount of low molecular weight species reduced after purification of the polypeptide, for example as measured by capillary gel electrophoresis (CGE);

[0084] iv. a reduction in the amount of high molecular weight species after expressing the polypeptide in a suitable host (e.g., after expressing the polypeptide in Pichia pastoris), for example as measured by size exclusion high performance liquid chromatography (SE-HPLC);

[0085] v. the amount of high molecular weight species reduced after purification of the polypeptide, for example as measured by size exclusion high performance liquid chromatography (SE-HPLC); and

[0086] vi. A combination of two or more of the biophysical properties described.

[0087] Item 44. The polypeptide according to any of the preceding items, wherein the polypeptide is characterized in that at least one biophysical property is improved compared to a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83;

[0088] wherein the biophysical property is selected from

[0089] i. improved stability after purification of the polypeptide, as measured by turbidity / opalescence of the polypeptide at a concentration of 100 mg / mL;

[0090] ii. reduced amount of low molecular weight species after expression of the polypeptide in Pichia pastoris, as measured by peak front on reverse phase chromatography (e.g., RP-UPLC);

[0091] iii. reduced amount of low molecular weight species after purification of the polypeptide as measured by capillary gel electrophoresis (CGE);

[0092] iv. reduced amount of high molecular weight species after expression of the polypeptide in Pichia pastoris, as measured by size exclusion high performance liquid chromatography (SE-HPLC);

[0093] v. the amount of high molecular weight species reduced after purification of the polypeptide, as measured by size exclusion high performance liquid chromatography (SE-HPLC); and

[0094] vi. A combination of two or more of the biophysical properties described.

[0095] Item 45. A polypeptide according to any one of the preceding items, wherein the polypeptide is characterized in that the recovery rate after the purification step is higher than the recovery rate of a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83, wherein the purification step is selected from:

[0096] i. Capture step on resin;

[0097] ii. a pH adjustment step after the capture step on the resin;

[0098] iii. Fine purification step;

[0099] iv. an ultrafiltration / diafiltration / ultrafiltration step; and

[0100] v. A combination of two or more of the purification steps described.

[0101] Item 46. A polypeptide according to the preceding item, wherein the capture step (i) is a purification step on Protein A chromatography.

[0102] Item 47. The polypeptide according to any one of the preceding items, wherein the polishing step (iii) is an ion exchange chromatography step.

[0103] Item 48. A polypeptide according to any one of the preceding items, wherein the polypeptide has a reduced amount of missing disulfide bridges after the polypeptide is expressed in a suitable host compared to a reference polypeptide consisting of an amino acid sequence as shown in SEQ ID NO:83, wherein the amount of the polypeptide having a reduced amount of missing disulfide bridges is measured by reverse phase high performance liquid chromatography (RP-HPLC).

[0104] Item 49. The polypeptide according to the preceding item, wherein the host is Pichia pastoris.

[0105] Item 50. A polypeptide according to any one of the preceding items, wherein the polypeptide binds to the cell surface glycoprotein MUC18 with lower affinity than the binding of a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83.

[0106] Item 51. A polynucleotide encoding the polypeptide according to any one of the preceding items.

[0107] Item 52. A (non-human) host or host cell, comprising the polynucleotide according to the preceding item.

[0108] Item 53. A composition comprising a polypeptide or polynucleotide according to any one of the preceding items; optionally, wherein the composition is a pharmaceutical composition; and further optionally, wherein the composition comprises at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant.

[0109] Item 54. A method for producing a polypeptide or composition according to any one of the preceding items, the method comprising the following steps:

[0110] - expressing the polypeptide in a suitable host cell or host organism or in another suitable expression system;

[0111] and further optionally

[0112] - isolating and / or purifying said polypeptide.

[0113] Item 55. The polypeptide, polynucleotide or composition according to any one of the preceding items, which is used to inhibit the binding of CX3CR1 to fractal chemokines in mammalian cells.

[0114] Item 56. The polypeptide, polynucleotide or composition according to any one of the preceding items, for use as a medicament.

[0115] Item 57. A polypeptide, polynucleotide or composition according to any one of the preceding items, which is used for preventing, treating and / or diagnosing a disease or disorder.

[0116] Item 58. The polypeptide, polynucleotide or composition for use according to the preceding item, wherein the disease or disorder is a disease or disorder associated with the fractal chemokine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1.

[0117] Item 59. A polypeptide, polynucleotide or composition for the use described in the preceding item, wherein the disease or disorder is selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allogeneic transplant rejection, systemic sclerosis, neurodegenerative disorders and demyelinating diseases of the nervous system, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain and cancers including ovarian cancer.

[0118] Item 60. A method for inhibiting the binding of CX3CR1 to a fractal chemokine in a mammalian cell, the method comprising administering to the cell a polypeptide according to any one of the preceding items, thereby inhibiting signal transduction mediated by the fractal chemokine.

[0119] Item 61. A method for treating a disease or disorder, wherein the disease or disorder is selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allogeneic transplant rejection, systemic sclerosis, neurodegenerative disorders and demyelinating diseases of the nervous system, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain and cancer including ovarian cancer, and wherein the method comprises administering a pharmaceutically active amount of a polypeptide, polynucleotide or composition according to any one of the preceding items to a subject in need thereof.

[0120] Item 62. Use of the polypeptide, polynucleotide or composition according to any one of the preceding items in the preparation of a pharmaceutical composition for inhibiting the binding of CX3CR1 to fractal chemokines in mammalian cells.

[0121] Item 63. Use of a polypeptide, polynucleotide or composition according to any one of the preceding items in the preparation of a pharmaceutical composition for treating a disease or disorder.

[0122] Item 64. Use of a polypeptide, polynucleotide or composition according to any one of the preceding items in the preparation of a pharmaceutical composition for treating a disease or disorder, wherein the disease or disorder is a disease or disorder associated with the fractal chemokine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1.

[0123] Item 65. Use of a polypeptide, polynucleotide or composition according to any one of the preceding items in the preparation of a pharmaceutical composition for treating a disease or disorder selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allogeneic transplant rejection, systemic sclerosis, neurodegenerative disorders and demyelinating diseases of the nervous system, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain and cancer including ovarian cancer. 4. Description of the drawings

[0124] Figure 1 .Alignment of the sequences of the ISVD building blocks described in WO 2013 / 130381 (66B02, 54A12 and 307) with the sequences of exemplary ISVD building blocks of the present technology (66B02_SO and 54A12_SO).

[0125] Figure 2 Expression profiling of exemplary multivalent ISVD constructs as described in Example 4. The amount of expressed protein was quantified using the ProtA biosensor on OctetRED384.

[0126] FIG. 3 (AB). Binding of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) to CHO K1 cells expressing (A) human CX3CR1 and (B) cynomolgus monkey CX3CR1.

[0127] FIG. 4 (AB). CX3CL1 competition assay of huCX3CL1-AF647 with exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) and a negative control (IRR00163; VHH reference) on CHO K1 cells expressing (A) human CX3CR1 and (B) cynomolgus monkey CX3CR1.

[0128] Figure 5 . Inhibition of CX3CL1 -induced chemotaxis by exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) as measured by luminescence (RLU).

[0129] Fig. 6 (AD). Binding of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) (see upper panels) and human CX3CL1 / Fractalchemokine (full length)-Alexa647 (see lower panels) to HEK293T cells transfected with different human CX3CR1 variants: (A) human CX3CR1 wild type (WT); (B) human CX3CR1 V249I; (C) human CX3CR1 T280M; (D) human CX3CR1 V249I and T280M.

[0130] Figure 7 Binding of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) (see upper panels) and purified anti-human CCR2 (see lower panels) to CHO K1 human CCR2.

[0131] Figure 8 Binding of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) (see upper panels) and purified mouse anti-human CCR5 (see lower panels) to CHOK1 human CCR5.

[0132] Fig. 9Levels of high molecular weight products (HMW) of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) during large-scale purification, after capture (loading polishing), after polishing (loading UF / DF / UF), and in formulated products (formulations) as determined by size exclusion high performance liquid chromatography (SE-HPLC).

[0133] Fig.10 Levels of low molecular weight products (LMW) of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) during large-scale purification, after capture (load polishing), after polishing (load UF / DF / UF) and in formulated products (formulations) as determined by capillary gel electrophoresis (GCE).

[0134] Fig.11 . Visual inspection of exemplary multivalent ISVD constructs (upper panels: A041600085, A041600034, A041600035; lower panels: A041600025, A041600041) by direct observation of the final formulated product. For ISVD constructs with three flask batches: left flask, buffer; middle flask, low concentration batch (50 mg / mL); and right flask, high concentration batch (100 mg / mL). For ISVD constructs with two flask batches: left flask: low concentration batch (50 mg / mL); right flask: high concentration batch (100 mg / mL). At the high concentration of 100 mg / mL, four of the five multivalent ISVD constructs were found to be "slightly turbid", and A041600025 was observed to have significant turbidity and / or opalescence and was therefore classified as "turbid".

[0135] Fig.12 Turbidity measurements of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) by OD500 measurement of the final formulated product.

[0136] Fig.13 Characterization of exemplary multivalent ISVD constructs (A041600034, A041600035, A041600085) for blocking human CX3CL1-induced phosphorylation of ERK in human CX3CR1-expressing BA / F3 cells as measured by the ratio of pERK signals as described in Example 8.1.

[0137] Figure 14 (AD). Characterization of an exemplary multivalent ISVD construct (A041600035) and anti-mouse CX3CR1 for inhibition of CX3CL1-induced ERK phosphorylation in CHO K1 cells expressing CX3CR1, as measured by the ratio of pERK signals, as described in Example 8.1. A: Human CX3CL1-induced ERK phosphorylation in CHO K1 cells expressing human CX3CR1. B: Mouse CX3CL1-induced ERK phosphorylation in CHO K1 cells expressing human CX3CR1. C: Human CX3CL1-induced ERK phosphorylation in CHO K1 cells expressing mouse CX3CR1. D: Mouse CX3CL1-induced ERK phosphorylation in CHO K1 cells expressing mouse CX3CR1.

[0138] FIG. 15 (AC). Monocyte chemotaxis assay of exemplary multivalent ISVD constructs (A041600035, A014600087) and negative controls (negative control 1: IRR00164, an ISVD that does not bind CX3CR1; negative control 2: no ISVD / antibody) in trans-well cultures from healthy donors (n=3), as measured by arbitrary migration index (fold change relative to medium control wells) of (A) classical monocytes, (B) intermediate monocytes, and (C) non-classical monocytes.

[0139] Fig.16 Monocyte chemotaxis assay in trans-well cultures from healthy donors (n=5; upper panels) and lupus nephritis (LN) donors (n=6; lower panels), as measured by arbitrary migration index of total monocytes (left), CD16+ monocytes (middle), and CD16- monocytes (right). *=p value < 0.05; ***=p value < 0.001; ns=p value > 0.05.

[0140] Figure 17 (AB). Evaluation of exemplary multivalent ISVD constructs (A) A041600035 and (B) A041600087 for binding in the human membrane protein array (MPA) to analyze specificity and target selectivity. Binding of a protein library (including carrier, DCC, DSC1, MCAM, FCGR2B, CX3CR1) and a positive control (Protein A) was measured on the Intellicyt iQue using fluorescently labeled secondary antibodies on unfixed cells.

[0141] Fig.18. Pharmacokinetic (PK) profiles of exemplary multivalent ISVD constructs A041600035 and A041600087 in cynomolgus monkeys for normalized single IV doses of 0.1 mg / kg and 1 mg / kg.

[0142] Fig.19 .Design of the 7-day non-accelerated NTN mouse model.

[0143] Figure 20. Representative illustration of the altered F4 / 80 (A), CD11b / CD4 (B), and Mac-2 / Galectin-3 (C) distribution patterns on the renal cortex of an hCX3CR1KI mouse treated with IRR00163 compared to a naive mouse at day 7 after NTS mono injection.

[0144] Fig.21 .Design of the 21-day non-accelerated NTN mouse model.

[0145] Figure 22. Representative illustration of the altered F4 / 80 (A), CD11b-CD4 (B) and Mac-2 / Galectin-3-CD8 (C) distribution patterns on the renal cortex of hCX3CR1KI mice treated with NTS and IRR00163 compared to naive mice at day 21 after NTS exclusive injection. 5. Specific implementation methods

[0146] 5.1 Definitions

[0147] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0148] According to the present technology, the term "comprising" can be optionally replaced by the term "consisting of..." each time it occurs. Unless otherwise clearly indicated by contrast, the articles "a / an" and "the" used herein refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one element or more than one element. Unless the context clearly indicates otherwise, the term "or" is used herein to mean the term "and / or" and can be used interchangeably with the term "and / or".

[0149] As used herein, the term "about" refers to a deviation of ±10% from the stated value. When the term "about" is used herein to refer to a number, it should be understood that yet another embodiment includes the number not modified by the presence of the term "about". In the absence of the term "about", and unless the context dictates otherwise, generally accepted rounding rules apply to the specified value.

[0150] Unless otherwise indicated or defined, all terms used have their ordinary meanings in the art, which will be clear to the skilled person. For example, reference is made to standard manuals such as Sambrook et al. 1989 (Molecular Cloning: A Laboratory Manual, 2nd edition, volumes 1-3, Cold Spring Harbor Laboratory Press), Ausubel et al. 1987 (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York), Lewin 1985 (Genes II, John Wiley & Sons, New York, NY), Old et al. 1981 (Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd edition, University of California Press, Berkeley, CA), Roitt et al. 2001 (Immunology, 6th edition, Mosby / Elsevier, Edinburgh), Roitt et al. 2001 (Roitt's Essential Immunology, 10th edition, Blackwell Publishing, UK), and Janeway et al. 2005 (Immunobiology, 6th edition, Garland Science Publishing / Churchill Livingstone, New York), and the general background art cited herein.

[0151] Unless otherwise indicated, as will be clear to the skilled person, all methods, steps, techniques and operations not specifically described in detail can be carried out and have been carried out in a manner known per se. For example, reference is again made to the standard manuals and general background art mentioned herein and to the further references cited therein; and to reviews such as Presta 2006 (Adv. Drug Deliv. Rev. 58: 640), Levin and Weiss 2006 (Mol. Biosyst. 2: 49), Irving et al. 2001 (J. Immunol. Methods 248: 31), Schmitz et al. 2000 (Placenta 21 Suppl. A: S106), Gonzales et al. 2005 (Tumour Biol. 26: 31), which describe techniques for protein engineering such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0152] The terms "polypeptide", "protein", or "peptide" are used to refer to and each refers to a linear polymer composed of covalently linked amino acids (also referred to as an "amino acid sequence"), which may be composed of natural L amino acids (commonly found in naturally occurring proteins). Any amino acid sequence comprising amino acids that are post-translationally modified (e.g., methylated, phosphorylated, acetylated, amidated, hydroxylated, formylated, or glycosylated) may be described as the initial translated amino acid sequence, i.e., these modifications are not explicitly shown in the amino acid sequence. Any polypeptide that can be represented as a sequence modification of linkages, crosslinks, and end caps, non-peptidyl bonds, etc. is encompassed by this definition. In some cases, a polypeptide may comprise an N-terminal and / or C-terminal protecting group. In some cases, a polypeptide may comprise one or more non-natural amino acids.

[0153] Table 1. List of the 20 proteinogenic amino acids.

[0154] Amino Acids Three letter code Single letter code Alanine Ala A Cysteine Cys C Aspartic acid Asp D Glutamate Glu E Phenylalanine Phe F Glycine Gly G Histidine His H Isoleucine lle I Lysine Lys K Leucine Leu L Methionine Met M Asparagine Asn N Proline Pro P Glutamine Gln Q Arginine Arg R Serine Ser S Threonine Thr T Valine Val V Tryptophan Trp W Tyrosine Tyr Y Unknown or other XA X

[0155] The term "sequence identity" as used herein refers to the amount of amino acids or nucleotides that are exactly matched between two different sequences. For example, the "percentage of sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated as follows: ([the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] / [the total number of amino acid residues in the first amino acid sequence]) × 100%, wherein each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is regarded as a difference at a single amino acid residue (i.e., a single position). Typically, for the purpose of determining the "percentage of sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the maximum number of amino acid residues is taken as the "first" amino acid sequence, and the other amino acid sequence is taken as the "second" amino acid sequence. The amino acid sequence identity percentage can be determined using sequence comparison programs known in the art (e.g., NCBI-BLAST).

[0156] As used herein, the term "amino acid difference" refers to the deletion, insertion or substitution of a single amino acid residue relative to a reference sequence, and in some embodiments, a substitution. For example, such conservative substitutions may be substitutions in which one amino acid in the following groups (a)-(e) is substituted by another amino acid residue in the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0157] Exemplary conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0158] 5.2 Immunoglobulin Single Variable Domains

[0159] The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen binding site is present on and formed by a single immunoglobulin domain. This distinguishes ISVDs from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e., a total of 6 CDRs will participate in the formation of the antigen binding site.

[0160] In view of the above definition, conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD or IgE molecules; known in the art) or antigen binding domains derived from such conventional 4-chain antibodies, Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or diabodies (all known in the art) will generally not be regarded as ISVDs, because in these cases, binding to the corresponding epitope of the antigen generally does not occur through one (single) immunoglobulin domain, but rather through a pair of (associated) immunoglobulin domains (such as light chain and heavy chain variable domains) that bind together to the epitope of the corresponding antigen, i.e., through a VH-VL pair of immunoglobulin domains.

[0161] In contrast, ISVDs are able to specifically bind to antigenic epitopes without pairing with additional immunoglobulin variable domains. The binding site of an ISVD is formed by a single VH, a single VHH, or a single VL domain.

[0162] Therefore, the single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; as long as it is able to form a single antigen binding unit (i.e., a functional antigen binding unit substantially consisting of a single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

[0163] An immunoglobulin single variable domain (ISVD) can be, for example, a heavy chain ISVD, such as VH, VHH, including camelized VH or humanized VHH. In some embodiments, the immunoglobulin single variable domain is a VHH, including a humanized VHH. In some embodiments, the immunoglobulin single variable domain is a VH, including a camelized VH, a human VH, and a camelized human VH. The heavy chain ISVD can be derived from a conventional four-chain antibody or a heavy chain antibody.

[0164] For example, the ISVD can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or a dAb (or an amino acid sequence suitable for use as a dAb), or ISVD (as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment of either thereof.

[0165] In particular, an ISVD can be ISVD (such as VHH, including humanized VHH or camelidized VH) or a suitable fragment thereof. [Note: and is a registered trademark of Ablynx NV]

[0166] The term "VHH domain" (also referred to as VHH, VHH antibody fragment and VHH antibody) has originally been described as the antigen-binding immunoglobulin variable domains of a "heavy chain antibody" (i.e., "antibody without light chain"; Hamers-Casterman et al. 1993, Nature 363:446-448). The term "VHH domain" has been selected to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For further description of VHH, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302).

[0167] Typically, the generation of immunoglobulins involves the immunization of experimental animals, the fusion of immunoglobulin-producing cells to produce hybridomas, and the specificity required for screening. Alternatively, immunoglobulins can be generated by screening natural or synthetic libraries (e.g., by phage display). The generation of immunoglobulin sequences (such as VHH) has been widely described in various publications, including WO 94 / 04678, Hamers-Casterman et al. 1993 (Nature 363: 446-448) and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74: 277-302). In these methods, camelids are immunized with target antigens to induce an immune response against the target antigen. The VHH library obtained from the immunization is further screened for VHHs that bind to the target antigen. In these cases, the generation of antibodies requires purified antigens for immunization and / or screening. Antigens can be purified from natural sources or by recombinant production methods. Immunization and / or screening of immunoglobulin sequences can be carried out using peptide fragments of such antigens.

[0168] Can produce, purify and / or separate immunoglobulin sequences of different origins, comprise mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences.In addition, can produce, purify and / or separate fully human, humanized or chimeric sequence.For example, can produce, purify and / or separate camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences or camelization domain antibodies, for example, as described in the camelization dAb of people such as Ward 1989 (Nature 341:544) (referring to for example WO 94 / 04678 and Davies and Riechmann 1994, Febs Lett., 339:285-290 and 1996, Prot.Eng.9:531-537). In addition, ISVDs are fused to comprise or consist of at least three ISVDs forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. 2001 (J. Biol. Chem., 276: 7346-7350), and, for example, WO 96 / 34103 and WO 99 / 23221). The ISVD sequence may further comprise a tag or other functional part, such as a toxin, a label, a radiochemical, etc.

[0169] The ISVD polypeptide sequence contained in the polypeptide according to the present technology is not limited with respect to the source of the ISVD polypeptide sequence, nor is it limited with respect to the manner in which the ISVD polypeptide sequence is generated or obtained (or has been generated or obtained). Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a specific but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), "camelized" immunoglobulin sequences (in particular camelized VH sequences), and ISVDs that have been obtained by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled artisan; or any suitable combination of any of the foregoing.

[0170] In one aspect, the ISVD polypeptide sequence can be obtained from a mouse, rat, rabbit, cat, dog, goat, sheep, horse, pig, non-human primate such as a cynomolgus monkey (also referred to herein as a "cyno"), or a camelid (such as a llama or alpaca), or a human.

[0171] The term "humanized VHH" comprises an amino acid sequence that corresponds to an amino acid sequence of a naturally occurring VHH domain, but has been "humanized" by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular the framework sequence) with one or more amino acid residues present at one or more corresponding positions in a VH domain of a conventional 4-chain antibody from a human (e.g., as indicated above). This can be done in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the state of the art (e.g., WO2008 / 020079). Furthermore, it should be noted that such humanized VHHs can be obtained in any suitable manner known per se, and are therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising a naturally occurring VHH domain as starting material.

[0172] The term "camelized VH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain but has been "camelized" by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody with one or more amino acid residues that occur at one or more corresponding positions in a VHH domain of a (camelid) heavy chain antibody. This can be done in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the state of the art (e.g., Davies and Riechman 1994 and 1996). As defined herein, such "camelized" substitutions are inserted at amino acid positions that form and / or occur at the VH-VL junction and / or at the so-called camelid hallmark residues, as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann 1994 and 1996). In some embodiments, the VH sequence used as the starting material or starting point for the generation or design of a camelized VH is a VH sequence from a mammal, such as a human VH sequence, such as a VH3 sequence. It should be noted, however, that such camelized VH may be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides that have been obtained using a polypeptide comprising a naturally occurring VH domain as starting material.

[0173] The (general) structure of ISVD comprises four framework regions ("FR"), which are referred to as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3") and "framework region 4" ("FR4"); these framework regions are interrupted by three complementarity determining regions ("CDR"), which are referred to as "complementarity determining region 1" ("CDR1"), "complementarity determining region 2" ("CDR2") and "complementarity determining region 3" ("CDR3"). The framework sequences in ISVD may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example based on standard manuals and the additional disclosures and literature mentioned herein.

[0174] The term "framework sequence" describes an immunoglobulin framework sequence or a framework sequence (a suitable combination) that has been derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence can be a framework sequence derived from a light chain variable domain (e.g., a VL sequence) and / or a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In a specific aspect, the framework sequence is a framework sequence that has been derived from a VHH sequence (wherein the framework sequence may optionally have been partially or fully humanized) or a conventional VH sequence that has been camelized (as defined herein).

[0175] In particular, an ISVD can be defined as an immunoglobulin sequence with the following (general) structure:

[0176] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0177] Herein, “FR1”, “FR2”, “FR3” and “FR4” refer to framework regions 1 to 4; and “CDR1”, “CDR2” and “CDR3” refer to complementarity determining regions 1 to 3.

[0178] The framework sequences present in the ISVD sequence may contain one or more "hallmark residues" such that the ISVD sequence is ISVD, such as VHH, includes humanized VHH or camelized VH.

[0179] As used herein, the term "hallmark residues" refers to residues that appear in Amino acid residues at defined positions in the framework region of ISVD.

[0180] In particular, an ISVD can be defined as an immunoglobulin sequence with the following (general) structure:

[0181] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0182] wherein "FR1", "FR2", "FR3" and "FR4" refer to framework regions 1 to 4; "CDR1", "CDR2" and "CDR3" refer to complementarity determining regions 1 to 3; and wherein the ISVD is further characterized by the presence of one or more "Hallmark residues" (see Table 2 below). In some embodiments, the ISVD comprises a VHH sequence, and the VHH sequence comprises a (partially) humanized VHH sequence and a camelized VH sequence.

[0183] For example, an ISVD may be an immunoglobulin sequence having the following structure:

[0184] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0185] wherein "FR1", "FR2", "FR3" and "FR4" refer to framework regions 1 to 4; "CDR1", "CDR2" and "CDR3" refer to complementarity determining regions 1 to 3; and wherein the ISVD is further characterized by the presence of one or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering selected from the hallmark residues mentioned in Table 2 below.

[0186] Table 2: Signature residues in ISVD (see Table A-11 for a list of SEQ ID NOs).

[0187]

[0188]

[0189] The amino acid residues of the ISVD can be numbered according to the general numbering of VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), such as the VHH domains of camelids from the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see, for example, the publication of this publication). Figure 2 ) is applicable and as further described in paragraph q) on pages 58 and 59 of WO 2008 / 020079. As is well known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues 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. The total number of amino acid residues in VH domains and VHH domains is typically between 110 and 120, often in the range between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.

[0190] In this application, unless otherwise indicated, the FR and CDR sequences of ISVDs (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) are determined according to AbM numbering as described in Kontermann and Dübel (eds. 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

[0191] The determination of the CDR regions can also be performed according to different methods (e.g., Kabat, Chothia or IMGT). For example, in the CDR determination according to Kabat, the FR1 of the ISVD comprises the amino acid residues at positions 1-30, the CDR1 of the ISVD comprises the amino acid residues at positions 31-35, the FR2 of the ISVD comprises the amino acids at positions 36-49, the CDR2 of the ISVD comprises the amino acid residues at positions 50-65, the FR3 of the ISVD comprises the amino acid residues at positions 66-94, the CDR3 of the ISVD comprises the amino acid residues at positions 95-102, and the FR4 of the ISVD comprises the amino acid residues at positions 103-113.

[0192] ISVD has a "three-dimensional structure". For example, in the context of ISVD, the term "three-dimensional structure" refers to a complex structure comprising alpha helices and beta sheets folded into a compact structure that is stabilized by polar and non-polar interactions. The three-dimensional structure forms spontaneously and is maintained due to interactions between amino acid side chains.

[0193] The crystal structures of different VHHs have been reported (Desmyter et al. 1996, Nat. Struct. Biol. 3:803-811; Spinelli et al. 1996, Nature Struct Biol 3:752; Decanniere et al. 1999, Structure 7:361-370). VHHs adopt the standard folding of immunoglobulin variable domains. Immunoglobulin variable domains fold into a native conformation, also considered a beta barrel. The FR regions of immunoglobulin variable domains form nine parallel beta strands folded into two sheets that are stacked relative to each other and stabilized by conserved disulfide bonds that constitute the beta sheet scaffold. The antigen binding site formed by the CDR loops (hypervariable regions) is actually located on top of this core scaffold structure, clustered at one end of the domain.

[0194] The present technology provides improved ISVDs that bind to CX3CR1. The ISVDs of the present technology are based on ISVDs 66B02 (SEQ ID NO: 4) and 54A12 (SEQ ID NO: 1) described in WO 2013 / 130381 (SEQ ID NOs: 1 and 2 of WO 2013 / 130381).

[0195] In one aspect, the sequence-optimized ISVD has the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; and wherein the ISVD specifically binds to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1, and comprises (according to AbM numbering)

[0196] a CDR1 comprising or consisting of the amino acid sequence GSIFSSTAMA (SEQ ID NO: 10);

[0197] A CDR2 comprising the amino acid sequence X1IX3TVGX7TK (SEQ ID NO: 202), wherein the ambiguous residue X can be X1=A or V, X3=N or S, X7=I or V; and

[0198] A CDR3 comprising the amino acid sequence DX1RRGWDTRY (SEQ ID NO: 138), wherein the ambiguous residue X can be X1=P or A; and

[0199] In some embodiments, CDR2 comprises the amino acid sequence X1IX3TVGX7TK (SEQ ID NO:202), wherein X1=A, X3=N or S, X7=I or V; X1=A or V, X3=N, X7=I or V; X1=A or V; X3=N or S; X7=I; X1=A, X3=N, X7=I or V; X1=A, X3=N or S, X7=I or V; or X1=A or V, X3=N, X7=I.

[0200] In some embodiments, CDR2 comprises the amino acid sequence VISTVGITK (SEQ ID NO: 12).

[0201] In some embodiments, CDR3 comprises the amino acid sequence DARRGWDTRY (SEQ ID NO: 14).

[0202] In some embodiments, the ISVDs are sequence optimized to yield 66B02_SO (SEQ ID NO: 5) and 54A12_SO (SEQ ID NO: 2).

[0203] Compared to ISVD 66B02 (SEQ ID NO: 4) and 54A12 (SEQ ID NO: 1), sequence-optimized ISVDs such as 66B02_SO (SEQ ID NO: 5) and 54A12_SO (SEQ ID NO: 2) are improved in at least one of the following features:

[0204] - More human-like (humanized);

[0205] - Reduced binding to pre-existing antibodies;

[0206] - Reduced antigenicity (fewer T cell epitopes);

[0207] - Improved chemical stability

[0208] The sequences of the CDR and FR regions of exemplary sequence-optimized ISVDs are depicted in Table A-2 (based on AbM's CDR determination) and Table A-3 (based on Kabat's CDR determination). The alignment of the ISVD sequences described in WO 2013 / 130381 with the exemplary sequence-optimized ISVDs of the present invention is shown in Table A-2 (based on AbM's CDR determination) and Table A-3 (based on Kabat's CDR determination). Figure 1 Given in.

[0209] 5.3 Peptides

[0210] The polypeptide according to the present technology may comprise or consist of various ISVDs (such as a "first ISVD", "second ISVD" and "third ISVD"), also referred to herein as ISVD building blocks. In some cases, the terms "first ISVD", "second ISVD" and "third ISVD" may indicate the relative position of the specifically enumerated ISVDs to each other, wherein the numbering starts from the N-terminus of the polypeptide. Thus, the "first ISVD" is closer to the N-terminus than the "second ISVD" and the "third ISVD". Thus, the "third ISVD" is closer to the C-terminus than the "first ISVD" and the "second ISVD". Since the numbering is therefore not absolute and only indicates the relative position of two ISVDs, it is not excluded that additional binding units / building blocks may be present in the polypeptide. Furthermore, the possibility that other binding units / building blocks (such as ISVDs) may be placed in between is not excluded.

[0211] The ISVD building blocks in the polypeptides according to the present technology can be directly connected or connected via a peptide linker. In this context, the term "directly" means that the two ISVDs contain immunoglobulin sequences with the following structure:

[0212] [FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD1 -L-[FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD2

[0213] Among them, "FR1", "FR2", "FR3" and "FR4" refer to framework regions 1 to 4; "CDR1", "CDR2" and "CDR3" refer to complementarity determining regions 1 to 3. And "L" between ISVD1 and ISVD2 refers to a single peptide bond connecting ISVDs.

[0214] The connection of two ISVDs via a "peptide linker" means that the two ISVDs comprise immunoglobulin sequences having the following structure:

[0215] [FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD1 -L-[FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD2

[0216] Wherein, "FR1", "FR2", "FR3" and "FR4" refer to framework regions 1 to 4; "CDR1", "CDR2" and "CDR3" refer to complementarity determining regions 1 to 3, and "L" between ISVD1 and ISVD2 refers to the linker connecting the ISVDs (e.g., as described in Table A-7).

[0217] The peptide linker can be a synthetic amino acid sequence consisting of a linear chain of amino acids, and the length can be, for example, 1 to 62 amino acid residues or 1 to 50 amino acid residues, preferably 9 to 40 amino acid residues in length. The linker can ensure that the ISVD connected by the linker can perform the biological activity of the ISVD. In some cases, the linker comprises alanine, glycine and / or serine residues, for example, arranged in repeating units. In the technology of the present invention, the ISVD can be connected via a peptide linker, but can also be directly connected without a peptide linker.

[0218] Some preferred examples of such amino acid linker sequences include, for example, the types described in WO 99 / 42077 (Gly x Ser y ) zA Gly-Ser linker (e.g., (Gly4Ser)3 (SEQ ID NO: 71) or (Gly3Ser2)3 (SEQ ID NO: 136)), a hinge-like region (e.g., a hinge region of a naturally occurring heavy chain antibody or a similar sequence (e.g., as described in WO 94 / 04678)). The Gly-Ser linker comprises one or more repeats of a peptide motif, such as a GGGGS (SEQ ID NO: 66) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 66), wherein n can be 1, 2, 3, 4, 5, 6, 7 or greater). Some commonly used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 69), the 15GS linker (n=3; SEQ ID NO: 71), and the 35GS linker (n=7; SEQ ID NO: 76). For example, see Chen et al. 2013 (Adv. Drug Deliv. Rev. 65: 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. 27: 325-330). In some embodiments, a 9GS linker (SEQ ID NO: 69) is used to connect the ISVDs in the polypeptide to each other.

[0219] For example, a polypeptide according to the present technology may include two peptide linkers between two ISVDs in the polypeptide. For example, a polypeptide may include an immunoglobulin sequence having the following structure:

[0220] [FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD1 -L1-[FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD2 -L2-[FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD3

[0221] Wherein, "FR1", "FR2", "FR3" and "FR4" refer to framework regions 1 to 4; "CDR1", "CDR2" and "CDR3" refer to complementarity determining regions 1 to 3, and "L1" and "L2" between two ISVDs within a polypeptide refer to linkers connecting the ISVDs (e.g., as described in Table A-7). In some embodiments, L1 and L2 are SEQ ID NO: 69.

[0222] The term "monovalent" indicates that only one binding unit / building block, such as one ISVD, is present in the polypeptide. The term "monospecific" refers to binding to one (particular) type of target molecule.

[0223] The terms "bivalent", "trivalent", "tetravalent" or "pentavalent" all fall within the term "multivalent" and indicate the presence of two, three, four or five binding units (such as ISVDs). The terms "bispecific", "trispecific", "tetraspecific" or "pentaspecific" all fall within the term "multispecific" and refer to binding to two, three, four or five different target molecules, respectively.

[0224] In one aspect, the polypeptide comprises at least three ISVDs, wherein each ISVD has the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; and wherein the first ISVD and the second ISVD specifically bind to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1, and comprise (according to AbM numbering) a CDR1 comprising or consisting of the amino acid sequence GSIFSSTAMA (SEQ ID NO: 10);

[0225] A CDR2 comprising the amino acid sequence X1IX3TVGX7TK (SEQ ID NO: 202), wherein the ambiguous residue X can be X1=A, V; X3=N, S; X7=I, V; and

[0226] A CDR3 comprising the amino acid sequence DX1RRGWDTRY (SEQ ID NO: 138), wherein the ambiguous residue X may be X1=P, A; and

[0227] wherein the third ISVD specifically binds to albumin and comprises (according to AbM numbering)

[0228] A CDR1 as shown in GFTFRSFGMS (SEQ ID NO:53) or having two or one amino acid differences from SEQ ID NO:53;

[0229] A CDR2 as shown in SISGSGSDTL (SEQ ID NO: 55) or having two or one amino acid differences from SEQ ID NO: 55; and

[0230] a CDR3 as shown in GGSLSR (SEQ ID NO:57) or having two or one amino acid differences from SEQ ID NO:57; and

[0231] Wherein the at least three ISVDs are directly linked or linked via a peptide linker.

[0232] In some embodiments, the first ISVD and the second ISVD comprise a CDR2 comprising the amino acid sequence X1IX3TVGX7TK (SEQ ID NO: 202), wherein X1=A, X3=N or S, X7=I or V; X1=A or V, X3=N, X7=I or V; X1=A or V; X3=N or S; X7=I; X1=A, X3=N, X7=I or V; X1=A, X3=N or S, X7=I or V; or X1=A or V, X3=N, X7=I.

[0233] In some embodiments, CDR2 comprises the amino acid sequence VISTVGITK (SEQ ID NO: 12).

[0234] In some embodiments, CDR3 comprises the amino acid sequence DARRGWDTRY (SEQ ID NO: 14).

[0235] The combination of CDR1, CDR2 and CDR3 of the first ISVD and / or the second ISVD may be selected from the following embodiments, wherein exemplary combinations of CDR1, CDR2 and CDR3 are represented by a row of Table 3.

[0236] Table 3: CDR1 to 3 of ISVDs that specifically bind to the fractal chemokine receptor CX3CR1 and / or single nucleotide polymorphism (SNP) variants of CX3CR1.

[0237] CDR1 (SEQ ID NO) CDR2 (SEQ ID NO) CDR3 (SEQ ID NO) 54A12_SO(AbM) 10 12 14 66B02_SO(AbM) 10 19 21 54A12_SO(Kabat) 24 26 14 66B02_SO(Kabat) 24 33 21

[0238] In one aspect, the amino acid sequence of an ISVD that binds to a fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 may have 90% or more (e.g., 91%, 92%, 93%, 94% or more), 95% or more (e.g., 96%, 97%, 98% or more), or even 99% or more sequence identity with any of the amino acid sequences of SEQ ID NOs: 1 to 8. Although the CDRs of the ISVD may have three, two, or one amino acid differences compared to the specific CDRs set forth herein, the CDRs of the ISVD retain biological activity in terms of efficacy and potency. For example, an ISVD with a CDR change of 3, 2, or 1 amino acid retains the binding specificity of the ISVD to a fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 compared to the specific CDRs set forth herein.

[0239] In some embodiments, the ISVD that specifically binds to CX3CR1 is located at the N-terminus of the polypeptide. Therefore, in some embodiments, the polypeptide comprises or consists of the following in order from the N-terminus of the polypeptide: an ISVD that specifically binds to CX3CR1, an ISVD that specifically binds to CX3CR1, an ISVD that provides an increased half-life for the polypeptide. The inventors surprisingly found that this configuration increases the expression yield of the polypeptide. In addition, this configuration of the polypeptide provides good chemistry, manufacturing and control (CMC) features, including upstream and downstream manufacturability. CMC activities are important activities when developing new drug products. This involves defining manufacturing practices and product specifications that must be followed and met to ensure product safety and consistency between batches.

[0240] In one aspect, the polypeptide comprises at least three ISVDs, wherein each ISVD has the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; wherein the first ISVD and the second ISVD specifically bind to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 (such as human fractal chemokine receptor CX3CR1) and the third ISVD specifically binds to albumin (such as human serum albumin); wherein the at least three ISVDs are directly connected or connected via a peptide linker (L1, L2); wherein the C-terminal ISVD of the polypeptide carries a C-terminal extension of FR4 of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length; and wherein the order of the ISVDs and the peptide linker as described in Table 4 below indicates the relative position of the ISVDs and the peptide linker to each other within the polypeptide from the N-terminus (first column) to the C-terminus (last column) of the polypeptide.

[0241] Table 4: Exemplary polypeptides of the present technology.

[0242]

[0243]

[0244] In one aspect, the amino acid sequence of the polypeptide comprises: ISVD1 directly followed by L1 directly followed by ISVD2 directly followed by L2 directly followed by ISVD3 (as described in a row of Table 4) and optionally directly followed by a C-terminal extension of FR4 of ISVD3 (e.g., one, two or three alanine (Ala) residues); and the polypeptide may have 90% or more (e.g., 91%, 92%, 93%, 94% or more), 95% or more (e.g., 96%, 97%, 98% or more) or even 99% or more sequence identity with any amino acid sequence of the polypeptide represented in a row of Table 4. For example, a polypeptide having 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid changes in the amino acid sequence retains the good CMC characteristics of the polypeptide (including upstream and downstream manufacturability) and / or the biological activity of the polypeptide in terms of efficacy and potency compared to a specific amino acid sequence shown herein (e.g., SEQ ID NO: 82).

[0245] In one aspect, the polypeptide comprises three ISVDs (1) to (3) and two peptide linkers, wherein each peptide linker is located between two ISVDs within the polypeptide; wherein (1), (2) and (3) indicate their relative positions relative to each other within the polypeptide from the N-terminus to the C-terminus of the polypeptide; and

[0246] wherein ISVD (1) and (2) specifically bind to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1, and ISVD (1) and (2) comprise (according to AbM numbering) a CDR1 as shown in GSIFSSTAMA (SEQ ID NO: 10); a CDR2 as shown in VISTVGITK (SEQ ID NO: 12); and a CDR3 as shown in DARRGWDTRY (SEQ ID NO: 14);

[0247] wherein the third ISVD specifically binds albumin and comprises (according to AbM numbering) a CDR1 as shown in GFTFRSFGMS (SEQ ID NO: 53); a CDR2 as shown in SISGSGSDTL (SEQ ID NO: 55); and a CDR3 as shown in GGSLSR (SEQ ID NO: 57); and wherein the third ISVD has a C-terminal extension of 1 to 3 alanine or 1 to 3 glycine residues.

[0248] The determination of the CDR region can also be performed according to Kabat. Therefore, the present technology also provides a polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), wherein each of the at least three ISVDs has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions and CDR1 to CDR3 refer to complementarity determining regions; and wherein the polypeptide comprises

[0249] (a) a first ISVD that specifically binds to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and comprises (according to Kabat numbering)

[0250] CDR1 as shown in SEQ ID NO:24;

[0251] CDR2 as shown in SEQ ID NO:26; and

[0252] CDR3 as shown in SEQ ID NO: 14; and

[0253] (b) a second ISVD that specifically binds to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and comprises (according to Kabat numbering)

[0254] CDR1 as shown in SEQ ID NO:24;

[0255] CDR2 as shown in SEQ ID NO:26; and

[0256] CDR3 as shown in SEQ ID NO: 14; and

[0257] (c) a third ISVD that specifically binds to albumin and (according to Kabat numbering) comprises a CDR1 as shown in SEQ ID NO: 60 or having two or one amino acid differences with SEQ ID NO: 53;

[0258] A CDR2 as shown in SEQ ID NO:62, or having two or one amino acid difference with SEQ ID NO:55; and a CDR3 as shown in SEQ ID NO:57, or having two or one amino acid difference with SEQ ID NO:57; and wherein (a), (b) and (c) are directly linked or linked via a peptide linker.

[0259] In some embodiments, the C-terminal ISVD of a polypeptide as described herein carries a C-terminal extension of FR4 of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.

[0260] In some embodiments, the order of (a), (b), and (c) indicates the relative positions of the ISVDs to each other within a polypeptide, from the N-terminus to the C-terminus of the polypeptide.

[0261] In some embodiments, the peptide linker comprises 3 to 62 amino acids. The amino acids in the peptide linker can be selected from alanine (A), glycine (G) and serine (S). In a specific aspect, the peptide linker is selected from a 9GS linker, a 20GS linker and a 35GS linker, such as SEQ ID NO:69.

[0262] The term "C-terminal extension" refers to the C-terminal extension (tail) relative to the ISVD sequence. In particular, the C-terminal ISVD of the polypeptide may carry a C-terminal extension of FR4 of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length. The C-terminal extension comprises an amino acid sequence that is not naturally associated with the ISVD sequence (i.e., FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4).

[0263] In some embodiments, the ISVD at the C-terminus of the polypeptide comprises a C-terminal extension (X) n , wherein n is 1 to 10, or 1 to 5, such as 1, 2, 3, 4 or 5 (and including 1 or 2, such as 1); and each X is independently selected and can be independently selected from a (in some embodiments, naturally occurring) amino acid residues of alanine (A), glycine (G), valine (V), leucine (L) and isoleucine (I). In an embodiment, n is 1 and X is alanine (A).

[0264] In some embodiments, the polypeptide comprises an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94% or more), 95% or more (e.g., 96%, 97%, 98% or more), or even 99% or more sequence identity to SEQ ID NO: 82. In some embodiments, the polypeptide comprises SEQ ID NO: 82. In some embodiments, the polypeptide consists of SEQ ID NO: 82.

[0265] As used herein, the equilibrium dissociation constant (K) of an antigen and an antigen binding protein is D The term "affinity" refers to the strength of binding between an antigenic determinant and an antigen binding site on an antigen binding protein: K DThe smaller the value of , the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (alternatively, affinity can also be expressed as an affinity constant (K A ), which is 1 / K D ). Affinity can be determined in a manner known per se depending on the specific antigen of interest. The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, e.g., Ober et al. 2001, Intern. Immunology 13: 1551-1559). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows real-time biospecific interactions to be analyzed by detecting changes in protein concentration in a biosensor matrix, in which one molecule is immobilized on a biosensor chip and another molecule passes over the immobilized molecule under flow conditions, thereby generating a k on , k off The measured value, and thus the K D (or K A ) value. For example, this can be done using the well-known System (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, New Jersey). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. 8: 125-131) and Johnnson et al. (1991, Anal. Biochem. 198: 268-277).

[0266] Another well-known biosensor technology for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377: 209-217). As used herein, the term "biolayer interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on the biosensor tip (signal beam). Changes in the number of molecules bound to the biosensor tip result in a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Because the interactions can be measured in real time, association and dissociation rates as well as affinity can be determined. For example, BLI can be measured using the well-known The system was performed (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0267] Alternatively, you can use The platform (Sapidyne Instruments, Boise, USA) measures affinity in a kinetic exclusion assay (KinExA) (see, e.g., Drake et al. 2004, Anal. Biochem. 328: 35-43). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. The equilibrium solution of the antibody / antigen complex is passed through a column with beads pre-coated with antigen (or antibody), thereby allowing free antibody (or antigen) to bind to the coated molecule. The detection of the antibody (or antigen) thus captured is completed with a fluorescently labeled protein that binds to the antibody (or antigen).

[0268] Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

[0269] Meso Scale Discovery-Solution Equilibrium Titration (MSD-SET) is a high-throughput procedure that allows reliable affinity screening of unpurified antibody fragments (Estep P. et al. 2013 MAbs. 5(2):270-8).

[0270] The terms "specificity", "specifically binds" or "specific binding" refer to the number of different target molecules (such as antigens) from the same organism to which a particular binding unit (such as an ISVD) can bind with sufficiently high affinity (see below). In some cases, "selectivity", "selectively binds" or "selectively binds" are also used in this context. A binding unit (such as an ISVD) specifically binds to its designated target. The specificity / selectivity of a binding unit can be determined based on affinity. Affinity indicates the strength or stability of a molecular interaction. Affinity is usually given by KD or dissociation constant, with units of moles / liter (or M). Affinity can also be expressed as the association constant KA, which is equal to 1 / KD and has units of (mol / L) -1 (or M -1 ).

[0271] As used herein, the term "fractal chemokine receptor CX3CR1" refers to C-X3-C motif chemokine receptor 1 (CX3CR1) (also known as fractal chemokine receptor or G protein-coupled receptor 13 (GPR13)), which is a transmembrane protein and receptor for fractal chemokines (also known as CX3CL1).

[0272] In some embodiments, human CX3CR1 and cynomolgus monkey CX3CR1 comprise amino acid sequences having greater than 90% sequence identity or greater than 95% sequence identity to SEQ ID Nos: 84 and 85 (Table A-9).

[0273] In some embodiments, human CX3CR1 and cynomolgus monkey CX3CR1 are as depicted in SEQ ID NOs: 84 and 85, respectively (Table A-9).

[0274] As used herein, the term "single nucleotide polymorphism (SNP) variant" refers to an allele variant of a gene (e.g., CX3CR1) containing at least one single nucleotide polymorphism (SNP). "SNP" means a single nucleotide variation between the genomes of individuals of the same species. In some cases, SNP can be a single nucleotide deletion or insertion. Typically, SNPs occur relatively frequently in the genome, and therefore contribute to genetic diversity. SNPs are considered to be more stable than other polymorphisms in mutation, making them suitable for association studies, where the linkage disequilibrium between markers and unknown variants is used to draw mutations that cause diseases. SNPs can have two, three or four alleles, or (although there may be three or four different forms of SNPs, corresponding to different nucleotides), thereby facilitating genotyping (by simple plus / minus determination rather than length measurement) and automation. The flanks of SNP positions are usually highly conserved sequences. For the alleles of each SNP site, an individual may be homozygous or heterozygous. Heterozygous SNP alleles can be differentiation polymorphisms. SNPs can appear in protein-coding nucleic acid sequences ("cSNPs"). Such SNPs can lead to amino acid changes in the encoded protein, which may have functional consequences, i.e., lead to "variant" proteins or polypeptides. Alternatively, such SNPs can be "silent", in which it does not lead to amino acid changes. SNPs can also occur in introns and intergenic regions, but can lead to phenotypic changes. For example, SNPs that lead to abnormal splicing can lead to non-functional proteins. Alternatively, SNPs may not have phenotypic effects. Variant proteins or polypeptides contain at least one amino acid residue that is different from the corresponding amino acid sequence of a polypeptide referred to as "wild type" or "normal" in the art. Such variant polypeptides can be caused by codon changes or nonsense mutations, or by any SNP that causes changes in protein structure, function, activity, regulation or expression.

[0275] In order to identify potential SNPs, the genome of a subject (e.g., a genome of a patient suffering from a CX3CR1-related disease or condition) can be sequenced and analyzed (e.g., by a SNP genotyping method). In addition, the SNP can be derived from the dbSNP database (www.ncbi.nlm.nih.gov / snp / ), wherein the SNP obtains a unique dbSNP reference SNP (rs or RefSNP) number assigned to the locus accession for the variant type.

[0276] For example, the single nucleotide polymorphism (SNP) variant of CX3CR1 may comprise at least one SNP selected from the group consisting of SNPrs3732378 (also known as rs60081475, rs52789411, rs17792900) and rs3732379 (also known as rs59717546, rs52808794, rs17792918) (sbSNP database, https: / / www.ncbi.nlm.nih.gov / snp, "CX3CR1" and clinical significance = "pathogenic"), or a SNP selected from the group consisting of or comprising: rs170 38679, rs41535248, rs55975803, rs56181422, rs139019894, rs199811198, rs201442030, rs202143296, rs376411124, rs1575205636 (sbSNP database, https: / / www.ncbi.nlm.nih.gov / snp, "CX3CR1" and clinical significance = "benign" or "likely benign"), rs938203, rs2669849 and rs1050592 (Tremblay et al. 2006, Genes Immun. 7:632-9).

[0277] The term "albumin" or "serum albumin" refers to a family of globular proteins commonly found in the plasma of animals and humans. Serum albumin is encoded by the albumin gene. Preferably, the albumin is human serum albumin. For example, human serum albumin as described in NCBI reference sequence: NP_000468.1.

[0278] In some embodiments, the polypeptides described herein have a lower affinity and / or do not bind to the cell surface glycoprotein MUC18 (alternative name: melanoma cell adhesion molecule (MCAM); https: / / www.uniprot.org / uniprot / P43121). In some embodiments, the polypeptides described herein bind to the cell surface glycoprotein MUC18 with a lower affinity (e.g., 10 -4 M or lower K D , such as 10-3 M, 10 -2 In some embodiments, the polypeptides described herein bind to melanoma cell adhesion molecule (MCAM) with a lower affinity than the binding of A041600087 (SEQ ID NO: 83) to MCAM, for example as measured by antibody specificity profiling (e.g., by protein microarray).

[0279] The term "antigenicity" as used herein in the context of a polypeptide refers to the presence of a B cell or T cell epitope on a polypeptide. "Antigenicity" describes the ability of a polypeptide (antigen) to bind or interact with a final cell-mediated response product (such as a B cell or T cell receptor). An antigenic determinant or epitope is a structural feature on a polypeptide (antigen) that interacts with a B cell receptor (also referred to as an antibody or immunoglobulin). When an epitope is combined with a major histocompatibility complex (MHC) molecule, a T cell receptor recognizes a linear amino acid sequence (also referred to as an epitope) within the polypeptide antigen. For example, antigenicity can be assessed by binding on a human leukocyte antigen DR isotype (HLA-DR) allele.

[0280] In some embodiments, the polypeptides described herein have lower antigenicity than A041600087 (SEQ ID NO: 83), for example as measured by binding on human leukocyte antigen DR isotype (HLA-DR) alleles (e.g., using the NetMHCIIpan-v4.0 software tool (http: / / www.cbs.dtu.dk / services / NetMHCIIpan / )). The term "pre-existing antibodies" refers to antibodies that are not "stimulated" or "induced" by administration of a drug (e.g., a polypeptide containing an ISVD) (e.g., in the case of anti-drug antibodies (ADA)). "Pre-existing antibodies" are already present in the blood or serum of a subject who has never received any drug (e.g., a polypeptide containing an ISVD).

[0281] The polypeptides of the present technology may exhibit limited reactivity to pre-existing antibodies in the subject to be treated (ie, antibodies present in the subject prior to the first treatment with the antibody construct).

[0282] 5.4 Polynucleotides, vectors and hosts

[0283] The term "polynucleotide" or "nucleic acid molecule" as used herein refers to a linear polymer consisting of covalently linked natural or non-natural nucleotide sequences comprising deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or combined DNA-RNA. In particular, the nucleotide sequence may be a naturally occurring nucleotide sequence, a (chemically) modified nucleotide sequence or a synthetic or semi-synthetic nucleotide sequence, and may for example be a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence that has been isolated from a library (and in particular an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0284] Nucleic acid according to the technology of the present invention can be prepared or obtained in a manner known per se, and / or can be separated from a suitable natural source. The nucleotide sequence encoding a naturally occurring (poly)peptide can for example be subjected to site-directed mutagenesis, so that the nucleic acid molecule encoding the polypeptide with sequence variation is provided. In addition, it is clear to those skilled in the art that, in order to prepare nucleic acid, several nucleotide sequences (such as at least one nucleotide sequence encoding the targeting moiety) and for example the nucleic acid encoding one or more joints can also be linked together in a suitable manner.

[0285] Techniques for generating nucleic acids will be clear to the skilled person, and may, for example, include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof), introducing mutations that can result in the expression of a truncated expression product; introducing one or more restriction sites (e.g., to create cassettes and / or regions that are easily digested and / or ligated using appropriate restriction enzymes), and / or introducing mutations by PCR reaction using one or more "mismatched" primers.

[0286] In some embodiments, the nucleic acid sequence of the polynucleotide is sequence optimized for expression of the polypeptide in a suitable host.

[0287] In some embodiments, the polynucleotides of the present technology may be further optimized for in vitro or in vivo administration (eg, for therapeutic applications).

[0288] Also provided are vectors of nucleic acid molecules comprising polypeptides encoding the technology of the present invention. As used herein, vectors are vehicles suitable for carrying genetic material into cells. Vectors include naked nucleic acids (such as plasmids or mRNA) or nucleic acids embedded in larger structures (such as liposomes or viral vectors).

[0289] The vector generally comprises at least one nucleic acid optionally linked to one or more regulatory elements (e.g., one or more suitable promoters, enhancers, terminators, etc.). The vector may be an expression vector, i.e., a vector suitable for expressing the encoded polypeptide or construct under appropriate conditions (e.g., when the vector is introduced into (e.g., human) cells). For DNA-based vectors, this generally includes the presence of elements for transcription (e.g., promoters and poly A signals) and translation (e.g., Kozak sequences).

[0290] In the vector, the at least one nucleic acid and the regulatory element may be "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, if a promoter is able to initiate or otherwise control / regulate the transcription and / or expression of a coding sequence, the promoter is considered to be "operably linked" to a coding sequence (wherein the coding sequence is understood to be "under the control" of the promoter). Typically, when two nucleotide sequences are operably linked, the two nucleotide sequences will be in the same orientation and generally also in the same reading frame. The two nucleotide sequences are also generally substantially contiguous, but this may not be necessary.

[0291] The regulatory elements of the vector may be chosen such that they are able to provide their intended biological function in the intended host cell or host organism.

[0292] For example, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, which means, for example, that the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence (e.g., a coding sequence) to which it is operably linked.

[0293] The polynucleotide or the vector comprising the polynucleotide can be used for transformation / transfection host cells or host organisms, for example, for expression and / or production of polypeptides. Suitable hosts or host cells for production purposes will be clear to the technician, and can be, for example, any suitable fungi, prokaryotic or eukaryotic cells or cell lines or any suitable fungi, prokaryotic or eukaryotic organisms. Hosts or host cells comprising nucleic acids encoding polypeptides of the technology of the present invention are also encompassed herein.

[0294] As used herein, the term "host cell" or "host organism" (collectively referred to as "host") refers to a suitable cell or organism in which a desired polypeptide can be expressed in fully functional form. In the production methods described herein, any host (organism) or host cell can be used, provided that they are suitable for producing polypeptides containing ISVDs. In some embodiments, the host is a non-human host.

[0295] Specific examples of suitable hosts include prokaryotes, such as coryneform bacteria or Enterobacteriaceae. Also included are insect cells, particularly insect cells suitable for baculovirus-mediated recombinant expression, such as cells derived from Trichoplusia ni or Spodoptera frugiperda, including but not limited to BTI-TN-5B1-4 High Five TM Insect cells (Invitrogen), SF9 or Sf21 cells; mammalian cells such as CHO cells, and lower eukaryotic hosts including yeasts such as Pichia (Phaffiform yeast), Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulina, Schizosaccharomyces, Pseudomonas, Pachysolensis, Debaryomyces, Metschnikowia, Rhodosporidium, White Sporangiophora, Botryoascus, Sporebombyx, and Pseudomonas. In some embodiments, yeast is used as a host, such as Pichia pastoris. In some embodiments, the host is Pichia pastoris.

[0296] The host used in the production method will be able to produce polypeptides containing ISVD. The host is usually genetically modified to contain one or more nucleic acid sequences encoding one or more polypeptides containing ISVD. Non-limiting examples of genetic modification include, for example, transformation with a plasmid or vector, or transduction with a viral vector. Some hosts can be genetically modified by fusion technology. Genetic modification includes introducing a separate nucleic acid molecule (e.g., a plasmid or vector) into the host, and directly modifying the genetic material of the host, for example, by integration into the chromosome of the host (e.g., by homologous recombination). A combination of the two usually occurs, for example, the host is transformed with a plasmid, and once homologous recombination occurs, the plasmid will be (at least partially) integrated into the host chromosome. The technician knows suitable methods for genetically modifying the host so that the host can produce polypeptides containing ISVD.

[0297] 5.5 Production and purification

[0298] The term "express", "expression" or "expressing" as used herein in the context of polypeptides refers to the manner in which a polypeptide is synthesized, modified and regulated in a host. Traditional strategies for recombinant protein expression involve transfecting cells with a vector (e.g., DNA) containing a template (e.g., a polynucleotide encoding a polypeptide containing an ISVD), and then culturing these cells to allow them to transcribe and translate the desired polypeptide.

[0299] Specific conditions and gene constructs for expressing nucleic acids and for producing polypeptides are described in the art, for example general culture methods, plasmids, promoters and leader sequences are described in WO 94 / 25591, Gasser et al. 2006 (Biotechnol. Bioeng. 94:535), Gasser et al. 2007 (Appl. Environ. Microbiol. 73:6499) or Damasceno et al. 2007 (Microbiol. Biotechnol. 74:381).

[0300] In some embodiments, the method for producing a polypeptide as described herein (e.g., a sequence having greater than 90% sequence identity with the amino acid sequence of SEQ ID NO: 82) comprises the following steps: expressing the polypeptide in a suitable host cell or host organism (e.g., Pichia pastoris) or in another suitable expression system; and isolating and / or purifying the polypeptide. Thus, the polypeptides of the present technology can be efficiently produced (e.g., with high expression yields in microbial hosts).

[0301] In some embodiments, the titer after expressing a polypeptide as described herein is higher than 6.0 g / L cell-free medium. In some embodiments, the titer after expressing a polypeptide as described herein is higher than 4.0 g / L in cell broth. In some embodiments, the titer after expressing a polypeptide as described herein is higher than the titer after expressing A041600087 (SEQ ID NO: 83). In some embodiments, the titer after expressing a polypeptide as described herein is higher than the titer after expressing A041600085 (SEQ ID NO: 137).

[0302] As used herein, the term "isolate", "isolation" or "isolating" in the context of a polypeptide means that the desired polypeptide product is separated or isolated from a composition comprising cellular components and the desired polypeptide product. For example, host cells can be lysed to extract the expressed desired polypeptide for subsequent purification.

[0303] The terms "purify," "purification," or "purifying," as used herein in the context of polypeptides, refer to a series of steps to separate a desired polypeptide from a complex mixture of components (e.g., components derived from cells, tissues, organisms). The purification process can separate the protein and non-protein portions of the mixture, and ultimately separate the desired protein product (e.g., a polypeptide containing an ISVD) from all other proteins. The separation steps take advantage of differences in protein size, physicochemical properties, binding affinity, and biological activity. For example, (size exclusion) chromatography can be used to separate proteins in solution.

[0304] The term "concentrate," "concentration," or "concentrating" as used herein in the context of proteins refers to increasing the amount of protein in an aqueous sample, e.g., for storage or for biopharmaceutical applications. Methods are available to the skilled artisan that provide information on how to analyze protein concentration using, for example, UV protein spectroscopy, traditional dye-based absorbance measurements, BCA, Lowry and Bradford assays, fluorescent dye-based assays, amine derivatization, and detergent partition assays.

[0305] The first step of the ISVD polypeptide purification process is often referred to as the "capture step". The purpose of the capture step is to first reduce process-related impurities (such as, but not limited to, host cell proteins (HCPs), color, and DNA) and capture the ISVD polypeptide product while maintaining high recovery. In some embodiments, the capture step refers to the first purification step on Protein A chromatography in bind and elute mode.

[0306] The second step of the purification process is often referred to as a "polishing purification step" intended to improve purity. For example, as the second purification step of the ISVD polypeptide purification process, an ion exchange chromatography step in bind and elute mode can be used to remove / reduce product-related variants (such as, but not limited to, high molecular weight (HMW) species, low molecular weight (LMW) species, and other charged variants) as well as some process-related impurities (such as, but not limited to, HCPs, residual protein A, DNA) that are still present after the capture step.

[0307] After the polishing step, an ultrafiltration / diafiltration / ultrafiltration (UF / DF / UF) step can be added to concentrate the polypeptide and exchange the buffer.

[0308] In some embodiments, after production and further purification, the polypeptides of the present technology have improved product quality (e.g., reduced amount of low molecular weight species and / or reduced amount of high molecular weight species and / or aggregation). In some embodiments, the polypeptides of the present technology have improved long-term stability under storage conditions (e.g., as measured by turbidity or opalescence of a polypeptide at a concentration of 100 mg / mL).

[0309] The term "biophysical properties" of a polypeptide refers to physical properties of a biological process, including but not limited to melting temperature (Tm), aggregation temperature (Tagg), the amount of high molecular weight species (HMW), the amount of low molecular weight species (LMW), turbidity and / or opalescence, and particle formation. In one aspect, the measurement of the biophysical properties of a "polypeptide" (e.g., by turbidity / opalescence, reverse phase chromatography, capillary gel electrophoresis, size exclusion high performance liquid chromatography) refers to the measurement of the biophysical properties of a "polypeptide population".

[0310] As used herein in the context of polypeptides, the terms "stability" and "stable" refer to the resistance of a polypeptide to aggregation, to the formation of degradation products and / or to the formation of fragmented products under given transport and / or storage conditions. In addition and / or in addition, a "stable" polypeptide retains biological activity under given transport and / or storage conditions. The stability of the polypeptide can be assessed by the degree of aggregation, degradation and / or fragmentation (as measured by, for example, SE-HPLC, RP-(U)HPLC, IEX-HPLC, sub-visible particle counting, analytical ultracentrifugation, dynamic light scattering, OD320 / OD280 ratio measurement, OD500 measurement, elastic light scattering, etc.) and / or % biological activity (as measured by, for example, ELISA, Biacore, etc.) compared to a reference polypeptide. For example, the reference polypeptide can be a reference standard frozen at -20°C or below -60°C (e.g. -80°C), consisting of the same polypeptide at the same concentration and in the same buffer as the stressed sample, but without applying stress conditions, this reference formulation regularly gives a main peak by SE-HPLC, RP-(U)HPLC and / or IEX-HPLC, and / or retains its biological activity in Biacore and / or ELISA.

[0311] The term "long-term stability under storage conditions" or "stability under storage conditions" as used herein in the context of a polypeptide refers to the stability of a polypeptide during transport and / or storage at -20°C, below -60°C, 5°C for 4 weeks, 3 months, 6 months, 1 year, 2 years, 3 years or longer.

[0312] The term "long-term stability under accelerated or stress conditions" as used herein in the context of a polypeptide refers to the stability of a polypeptide during transport and / or storage at 25°C and 40°C for 2 weeks, 4 weeks, 3 months, 6 months or longer.

[0313] As used herein, the term "turbidity" or "opalescence" refers to the cloudiness or haziness of a fluid caused by intermolecular attraction of a large number of individual particles and / or molecules that are not normally visible to the naked eye. For example, turbidity or opalescence can be measured at a concentration of 100 mg / mL of a polypeptide. Turbidity and opalescence can be measured using OD500 and the opalescence visually compared to an opalescence standard. Turbidity can be measured by a nephelometer (e.g., as provided by BMG, Labtech, Thermo Fisher).

[0314] The term "low molecular weight species" or "LMW" as used herein in the context of a polypeptide refers to a fragment of a polypeptide. For example, LMW species can be measured by peak front on reverse phase chromatography (e.g., RP-UPLC) or by capillary gel electrophoresis (CGE).

[0315] The term "high molecular weight species", "high molecular weight variant" or "HMW" as used herein in the context of a polypeptide refers to a polypeptide aggregate having an apparent molecular weight equal to or higher than the apparent molecular weight observed in a size exclusion high performance liquid chromatography (SE-HPLC) analysis of polypeptide dimers (e.g., 90 kDa as observed for A041600035 SE-HPLC) compared to a molecular weight marker. For example, HMW species can be measured by size exclusion high performance liquid chromatography (SE-HPLC).

[0316] The term "thermostability" as used herein in the context of polypeptides refers to the ability of a polypeptide to resist irreversible changes in its chemical or physical structure at elevated relative temperatures, typically by resisting decomposition or aggregation.

[0317] The term "aggregation" as used herein in the context of a polypeptide refers to the presence of high molecular weight aggregates, i.e., aggregates with an apparent molecular weight equal to or higher than the apparent molecular weight observed in a SE-HPLC analysis of a polypeptide dimer (e.g., 90 kDa as observed for A041600035 SE-HPLC) compared to a molecular weight marker. Aggregation can be assessed by various methods known in the art. Without limitation, examples include high performance size exclusion chromatography (SE-HPLC), subvisible particle counting, analytical ultracentrifugation (AUC), dynamic light scattering (DLS), static light scattering (SLS), elastic light scattering, OD320 / OD280 measurements, OD500 measurements, turbidimetry.

[0318] In some embodiments, after expressing a polypeptide as described herein, the amount of HMW species (as measured by, for example, SE-HPLC) is less than 5%. In some embodiments, the amount of HMW species (as measured by, for example, SE-HPLC) after expressing a polypeptide as described herein is lower than the amount of HMW species after expressing A041600087 (SEQ ID NO: 83). In some embodiments, the amount of HMW species (as measured by, for example, SE-HPLC) after expressing a polypeptide as described herein is lower than the amount of HMW species after expressing A041600085 (SEQ ID NO: 137).

[0319] In some embodiments, after the first purification step on the protein A chromatography, the amount of HMW species of the polypeptide as described herein (as measured by, for example, SE-HPLC) is less than 5%. In some embodiments, after the first purification step on the protein A chromatography, the amount of HMW species of the polypeptide as described herein (as measured by, for example, SE-HPLC) is lower than the amount of HMW species of A041600087 (SEQ ID NO: 83) after the first purification step on the protein A chromatography. In some embodiments, after the first purification step on the protein A chromatography, the amount of HMW species of the polypeptide as described herein (as measured by, for example, SE-HPLC) is lower than the amount of HMW species of A041600085 (SEQ ID NO: 137) after the first purification step on the protein A chromatography. In some embodiments, after the polishing purification step (e.g., ion exchange chromatography step), the amount of HMW species of the polypeptide as described herein (as measured by, for example, SE-HPLC) is less than 1%.

[0320] In some embodiments, for polypeptides as described herein, the amount of variants with missing disulfide bridges at the end of the expression step (as measured by, for example, RP-UHPLC) is less than 5%. In some embodiments, for polypeptides as described herein, the amount of variants with missing disulfide bridges at the end of the expression step (as measured by, for example, RP-UHPLC) is lower than the amount of variants with missing disulfide bridges at the end of the expression step of A041600087 (SEQ ID NO: 83). In some embodiments, for polypeptides as described herein, the amount of variants with missing disulfide bridges at the end of the expression step (as measured by, for example, RP-UHPLC) is lower than the amount of variants with missing disulfide bridges at the end of the expression step of A041600085 (SEQ ID NO: 137). In one aspect, "the amount of variants with missing disulfide bridges" refers to the measurement of a subpopulation of polypeptide variants with missing disulfide bridges compared to the total population of polypeptides.

[0321] In some embodiments, for polypeptides as described herein, the amount of variants with missing disulfide bridges after Cu treatment at the end of the expression step (as measured by, e.g., RP-UHPLC) is less than 5%. In some embodiments, for polypeptides as described herein, the amount of variants with missing disulfide bridges after Cu treatment at the end of the expression step (as measured by, e.g., RP-UHPLC) is lower than the amount of variants with missing disulfide bridges at the end of the expression step of A041600087 (SEQ ID NO: 83). In some embodiments, for polypeptides as described herein, the amount of variants with missing disulfide bridges after Cu treatment at the end of the expression step (as measured by, e.g., RP-UHPLC) is lower than the amount of variants with missing disulfide bridges at the end of the expression step of A041600085 (SEQ ID NO: 137).

[0322] In some embodiments, after the expression step, the sum of the hexose % on the polypeptide as described herein (as determined by MS-ID) is less than 15%. In some embodiments, the sum of the hexose % on the polypeptide as described herein (as determined by MS-ID) after the expression step is lower than the sum of the hexose % on A041600087 (SEQ ID NO: 83) after the expression step. In some embodiments, the sum of the hexose % on the polypeptide as described herein (as determined by MS-ID) after the expression step is lower than the sum of the hexose % on A041600085 (SEQ ID NO: 137) after the expression step.

[0323] In some embodiments, the amount of LMW species (as measured by, e.g., CGE) after expression of a polypeptide as described herein is less than 5%. In some embodiments, the amount of LMW species (as measured by, e.g., CGE) after expression of a polypeptide as described herein is lower than the amount of LMW species after expression of A041600087 (SEQ ID NO: 83). In some embodiments, the amount of LMW species (as measured by, e.g., CGE) after expression of a polypeptide as described herein is lower than the amount of LMW species after expression of A041600085 (SEQ ID NO: 137).

[0324] In some embodiments, after the first purification step on Protein A chromatography, the amount of LMW species of a polypeptide as described herein (as measured by, e.g., CGE) is less than 5%. In some embodiments, the amount of LMW species of a polypeptide as described herein (as measured by, e.g., CGE) after the first purification step on Protein A chromatography is lower than the amount of LMW species of A041600087 (SEQ ID NO: 83) after the first purification step on Protein A chromatography. In some embodiments, the amount of LMW species of a polypeptide as described herein (as measured by, e.g., CGE) after the first purification step on Protein A chromatography is lower than the amount of LMW species of A041600085 (SEQ ID NO: 137) after the first purification step on Protein A chromatography. In some embodiments, after a polishing purification step (e.g., an ion exchange chromatography step), the amount of LMW species of a polypeptide as described herein (as measured by, e.g., CGE) is less than 1%.

[0325] In some embodiments, the downstream (DS) capture recovery of a polypeptide as described herein is greater than 90%. In some embodiments, the DS capture recovery of a polypeptide as described herein is greater than the DS capture recovery of A041600087 (SEQ ID NO: 83). In some embodiments, the DS capture recovery of a polypeptide as described herein is greater than the DS capture recovery of A041600085 (SEQ ID NO: 137).

[0326] In some embodiments, the downstream (DS) capture binding of a polypeptide as described herein is greater than 18 mg / ml. In some embodiments, the DS capture binding of a polypeptide as described herein is greater than the DS capture binding of A041600087 (SEQ ID NO: 83). In some embodiments, the DS capture binding of a polypeptide as described herein is greater than the DS capture binding of A041600085 (SEQ ID NO: 137).

[0327] In some embodiments, the downstream (DS) capture post-pH adjustment recovery rate of the polypeptides as described herein is greater than 90%. In some embodiments, the DS capture post-pH adjustment recovery rate of the polypeptides as described herein is higher than the DS capture pH adjustment recovery rate of A041600087 (SEQ ID NO: 83). In some embodiments, the DS capture post-pH adjustment recovery rate of the polypeptides as described herein is higher than the DS capture pH adjustment recovery rate of A041600085 (SEQ ID NO: 137).

[0328] In some embodiments, the downstream UF / DF / UF recovery rate of the polypeptide as described herein is higher than 90%. In some embodiments, the DSUF / DF / UF recovery rate of the polypeptide as described herein is higher than the DSUF / DF / UF recovery rate of A041600087 (SEQ ID NO: 83). In some embodiments, the DSUF / DF / UF recovery rate of the polypeptide as described herein is higher than the DSUF / DF / UF recovery rate of A041600085 (SEQ ID NO: 137).

[0329] In some embodiments, the turbidity (as measured by OD500) of a polypeptide as described herein after a polishing purification step (e.g., an ion exchange chromatography step) is lower than that of A041600087 (SEQ ID NO: 83). In some embodiments, the turbidity (as measured by OD500) of a polypeptide as described herein is lower than that of A041600085 (SEQ ID NO: 137).

[0330] In some embodiments, for proteins as described herein, after a fine purification step (e.g., an ion exchange chromatography step), the amount of host cell proteins (HCP) (as measured by an immuno-enzymatic assay) is less than 30 ppm. In some embodiments, the amount of host cell proteins (HCP) of polypeptides as described herein (as measured by an immuno-enzymatic assay) is lower than the amount of host cell proteins (HCP) of A041600087 (SEQ ID NO: 83). In some embodiments, the amount of host cell proteins (HCP) of polypeptides as described herein (as measured by an immuno-enzymatic assay) is lower than the amount of host cell proteins (HCP) of A041600085 (SEQ ID NO: 137).

[0331] 5.6 Composition

[0332] The present invention also provides a composition comprising at least one polypeptide of the present invention, at least one polynucleotide encoding the polypeptide of the present invention, or at least one vector containing such a polynucleotide. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprises one or more additional pharmaceutically active polypeptides and / or compounds.

[0333] Therefore, for pharmaceutical use, the polypeptides of the present technology can be formulated as pharmaceutical preparations comprising: (i) at least one polypeptide of the present technology and (ii) at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and / or stabilizer, and (iii) optionally one or more additional pharmaceutically active polypeptides and / or compounds. Therefore, according to another aspect, the present technology relates to a pharmaceutical composition or formulation containing at least one polypeptide of the present technology and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and / or stabilizer, and optionally one or more additional pharmaceutically active substances.

[0334] In all cases, the ultimate 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 an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0335] 5.7 Uses of Peptides

[0336] The polypeptides can be further optimized for in vitro or in vivo administration (e.g., for therapeutic applications), which can be assessed by modulating the activity of CX3CR1 in cells, tissues, organs or organisms. For example, the polypeptides can be used to inhibit the binding of CX3CR1 to fractal chemokines in mammalian cells.

[0337] In one aspect, the present technology provides a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide for use as a medicament. For example, a therapeutically effective amount of the polypeptide can be used to treat a disease or disorder associated with the fractal chemokine receptor CX3CR1 (or a single nucleotide polymorphism (SNP) variant of CX3CR1).

[0338] The terms "therapeutic agent," "pharmaceutical agent," "drug" refer to a compound (eg, an ISVD-containing polypeptide) used to prevent or treat a disease or disorder or to improve the health of a subject.

[0339] The term "disease" or "disorder" refers to a change in a cell, tissue, organ or organism compared to a normal (healthy) cell, tissue, organ or organism. In some cases, physiological functions associated with native organ function, homeostasis, aging or regeneration may be altered, such as abnormal organ development, inflammatory diseases, autoimmune diseases, chronic diseases, infectious diseases or cancer.

[0340] In one aspect, the disease or disorder is associated with a fractal chemokine receptor CX3CR1 (or a single nucleotide polymorphism (SNP) variant of CX3CR1) in a cell, tissue, organ or organism (e.g., a change in the amount or activity of CX3CR1). For example, the disease, disorder or condition is selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, degenerative disorders of the nervous system and demyelinating diseases, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuropathic pain, inflammatory pain, and cancers including ovarian cancer.

[0341] The term "subject" as used herein can be any animal, such as a mammal. In mammals, humans and non-human mammals can be distinguished. Non-human mammals can be, for example, companion animals (e.g., dogs, cats), livestock (e.g., cattle, horses, sheep, goats or pigs) or mammals (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (e.g., cynomolgus monkeys) or camelids (e.g., llamas or alpacas)) that are commonly used for research purposes and / or for producing antibodies. In the context of prevention and / or treatment purposes, the subject can be any animal, and more specifically any mammal, such as a human subject. As will be clear to the technician, the subject to be treated will particularly be suffering from the diseases, disorders and conditions mentioned herein or be in the people at risk of the diseases, disorders and conditions mentioned herein.

[0342] As used herein, the term "administering" refers to administering a substance (e.g., a composition comprising a polypeptide) to a subject to diagnose, treat, or prevent a disease. Methods of administration include parenteral administration (e.g., intravenous (IV), intraperitoneal, subcutaneous, intramuscular, intracavitary, intraarterial, or intrathecal administration) or oral administration. In some cases, the route of administration may be different or the same for the administration of a polypeptide, polynucleotide, or composition. When a polypeptide, polynucleotide, or composition is intended for administration to a subject (e.g., for prevention, treatment, and / or diagnostic purposes), it may comprise an immunoglobulin sequence that is not naturally present in the subject.

[0343] One or more doses may be administered. If more than one dose is administered, the doses may be administered at appropriate intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.

[0344] As used herein, the term "(pharmaceutically or therapeutically or prophylactically) effective amount or dose" of a substance or composition refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect.

[0345] The polypeptides or polynucleotides of the present invention can be used to prevent, treat, alleviate and / or diagnose CX3CR1-related diseases, disorders or conditions, particularly inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allogeneic transplant rejection, systemic sclerosis, degenerative disorders of the nervous system and demyelinating diseases, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain or cancer including ovarian cancer.

[0346] In another aspect, the present technology provides a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide for use in treating or preventing atherosclerosis.

[0347] In another aspect, the present technology provides a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide for treating or preventing atherosclerosis by preventing and / or reducing the formation of new atherosclerotic lesions or plaques and / or by preventing or slowing the progression of existing lesions and plaques.

[0348] In another aspect, the present technology provides a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide for treating or preventing atherosclerosis by changing the composition of the plaque to reduce the risk of plaque rupture and atherothrombotic events.

[0349] In one aspect, the present technology provides a method for treating or reducing the risk of the following diseases or conditions in a human suffering from or at risk of the following diseases or conditions: inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, degenerative disorders of the nervous system and demyelinating diseases, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, or cancer including ovarian cancer, wherein the method comprises administering to the human a therapeutically effective amount of a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide.

[0350] In one aspect, the present technology also provides a method for treating atherosclerosis or reducing the risk of atherosclerosis in a human suffering from or at risk of an atherosclerotic disease or condition, wherein the method comprises administering to the human a therapeutically effective amount of a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide.

[0351] In one aspect, the present technology provides a method for treating atherosclerosis or reducing the risk of atherosclerosis in a human suffering from an atherosclerotic disease or condition or at risk of the disease or condition by preventing and / or reducing the formation of new atherosclerotic lesions or plaques and / or by preventing or slowing the progression of existing lesions and plaques, wherein the method comprises administering to the human a therapeutically effective amount of a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide.

[0352] In one aspect, the present technology also provides a method for treating atherosclerosis or reducing the risk of atherosclerosis in a human suffering from an atherosclerotic disease or condition or at risk of the disease or condition by altering the composition of the plaque to reduce the risk of plaque rupture and atherothrombotic events, wherein the method comprises administering to the human a therapeutically effective amount of a polypeptide as described herein or a pharmaceutical composition comprising the polypeptide.

[0353] In one aspect, the polypeptides as described herein are indicated for use in treating or preventing a disease or condition in which modulation of activity at the CX3CR1 receptor is desired. In one aspect, the present technology also provides a method for treating a disease or condition in which antagonizing the CX3CR1 receptor is beneficial or reducing the risk of the disease or condition, the method comprising administering a polypeptide as described herein to a person suffering from the disease or condition or at risk of the disease or condition.

[0354] Prevention is contemplated to be particularly relevant to the treatment of persons who have suffered a previous episode of the disease or condition in question or who are otherwise considered to be at increased risk for the disease or condition. Persons at risk for developing a particular disease or condition generally include those with a family history of the disease or condition, or those who have been identified through genetic testing or screening as being particularly susceptible to developing the disease or condition.

[0355] It will also be clear to the skilled person that the above methods of treating a disease include the preparation of a medicament for treating said disease.

[0356] In addition, it is clear that the polypeptides of the present technology can be used as active ingredients in drugs or pharmaceutical compositions intended for the treatment of the above-mentioned diseases. Therefore, the present technology also relates to the use of the polypeptides of the present technology in the preparation of pharmaceutical compositions for preventing, treating and / or alleviating any of the diseases, disorders or conditions mentioned above. The present technology further relates to polypeptides of the present technology for therapeutic or preventive purposes and specifically for preventing, treating and / or alleviating any of the diseases, disorders or conditions mentioned above. The present technology further relates to pharmaceutical compositions for preventing, treating and / or alleviating the diseases, disorders or conditions mentioned above, wherein such compositions comprise at least one polypeptide of the present technology.

[0357] The polypeptides of the present technology and / or compositions comprising the polypeptides can be administered to a patient in need thereof in any suitable manner depending on the specific pharmaceutical formulation or composition to be used.

[0358] The polypeptide as described herein and / or the composition comprising the polypeptide is administered according to a treatment regimen suitable for preventing, treating and / or alleviating the disease, disorder or condition to be prevented, treated or alleviated. Clinicians are generally able to determine a suitable treatment regimen based on factors such as the following: the disease, disorder or condition to be prevented, treated or alleviated, the severity of the disease, the severity of the disease symptoms, the specific polypeptide of the present technology to be used, the specific route of administration and the pharmaceutical formulation or composition to be used, the patient's age, sex, weight, diet, general condition, and similar factors well known to clinicians. Typically, the treatment regimen will include administering one or more polypeptides of the present technology or one or more compositions comprising the polypeptide in an effective amount or dosage.

[0359] The efficacy of the polypeptides of the present invention and compositions comprising the polypeptides can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal models known per se, or any combination thereof, depending on the specific disease involved. Suitable assays and animal models will be clear to the skilled person.

[0360] As used herein, the term "half-life" refers to the time required for the serum concentration of a polypeptide to decrease by 50% in vivo, for example, due to polypeptide degradation and / or the polypeptide being cleared or chelated by natural mechanisms. The in vivo half-life of a polypeptide can be determined in any manner known per se (such as by pharmacokinetic analysis). Parameters such as t1 / 2-α, t1 / 2-β, and area under the curve (AUC) can be used to represent the half-life. Reference standard manuals, such as Kenneth, A et al. 1986 (Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists) and Peters et al. 1996 (Pharmacokinetic analysis: A Practical Approach). Reference is also made to M Gibaldi and Perron 1982 (Pharmacokinetics, published by Marcel Dekker, 2nd revised edition). The term "increase in half-life" or "increased half-life" refers to an increase in t1 / 2-β, with or without an increase in t1 / 2-α and / or AUC or both.

[0361] In some embodiments, the polypeptides of the present technology exhibit a sufficiently long half-life (eg, blood half-life) in the subject to be treated that allows for convenient spacing of consecutive treatments.

[0362] In one aspect, when administered to a mammal such as a cynomolgus monkey or a human, the polypeptide as described herein is characterized by a pharmacokinetic (PK) profile that is different from the PK profile observed for a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83 or SEQ ID NO: 137. In particular, the polypeptide may exhibit a more prolonged serum persistence (i.e., a longer half-life) compared to a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83. This effect may be particularly prominent at lower doses ( Fig.18 ). In one aspect, the PK profile of the polypeptides as described herein does not exhibit target mediated drug deposition (TMDD). In one aspect, when administered to a mammal such as a cynomolgus monkey or a human, the polypeptides as described herein are characterized by a PK profile having lower target mediated drug deposition (TMDD) than a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83. The lack of TMDD provides increased selectivity for the polypeptides described herein, and therefore provides a better safety profile.

[0363] In one aspect, when a polypeptide as described herein is compared to a reference polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 83, after administration of a similar effective dose (e.g., 0.1 mg / kg or 1 mg / kg) of the polypeptide, the polypeptide as described herein has higher exposure in a subject for a longer period of time. In one aspect, after administration of a lower dose than the reference polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 83, the polypeptide as described herein has the same exposure as compared to the reference polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 83.

[0364] According to yet other embodiments, there is provided a method of diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction, the method comprising the steps of:

[0365] a) obtaining a sample from a subject; and

[0366] b) contacting the sample in vitro with a polypeptide of the present technology as defined above, and

[0367] c) detecting binding of the polypeptide to the sample, and

[0368] d) comparing the binding detected in step (c) to a standard, wherein a difference in binding relative to said sample is diagnostic of a disease, disorder or condition characterized by CX3CR1 dysfunction.

[0369] According to other embodiments, there is provided a method of diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction, the method comprising the steps of:

[0370] a) obtaining a sample from a subject; and

[0371] b) contacting the sample with a polypeptide of the present technology as defined above;

[0372] c) determining the amount of CX3CR1 in the sample; and

[0373] d) comparing the amount determined in step (c) to a standard, wherein a difference in amount relative to said sample is diagnostic of a disease, disorder or condition characterized by CX3CR1 dysfunction.

[0374] The above-described diagnostic methods can also be used to monitor the effectiveness of therapeutic treatment in a subject.

[0375] According to other embodiments, a kit for diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction and / or for monitoring the effectiveness of a therapeutic treatment is provided, wherein the kit is used in a method as defined above. Such a kit may comprise at least one polypeptide, polynucleotide or composition of the technology of the present invention, and optionally one or more media, detection means and / or in vitro or in vivo imaging agents, and further optionally instructions for use. Suitable in vivo imaging agents include 99mTc, 111indium, 123iodine, and paramagnetic compounds for magnetic resonance imaging.

[0376] The present technology further provides a kit comprising at least one polypeptide, polynucleotide or composition of the present technology, and one or more other components selected from other drugs for treating the diseases and disorders described above, and the devices described above.

[0377] 6. Examples

[0378] Hereinafter, the present technology is described in more detail and specifically with reference to embodiments, however these embodiments are not intended to limit the scope of the present technology.

[0379] Example 1: Sequence optimization of monovalent anti-CX3CR1 ISVD

[0380] The anti-CX3CR1 ISVD building blocks 66B02 (SEQ ID NO: 4) and 54A12 (SEQ ID NO: 1) have been described in WO 2013 / 130381 (SEQ ID NOs: 1 and 2 in WO 2013 / 130381). These ISVD building block sequences were optimized to 66B02_SO (SEQ ID NO: 5) and 54A12_SO (SEQ ID NO: 2) to make them more human (humanized), to reduce their binding to pre-existing antibodies, to reduce antigenicity (remove T cell epitopes) and to remove post-translational modification (PTM) sites. An alignment of the ISVD sequences described in WO 2013 / 130381 with the sequence-optimized exemplary ISVDs of the present technology is shown in Figure 1 Given in.

[0381] Example 2: Characterization of sequence-optimized monovalent ISVDs

[0382] The biophysical properties and potency of the sequence optimized ISVD variants 66B02_SO (SEQ ID NO: 5) and 54A12_SO (SEQ ID NO: 2) were characterized.

[0383] 2.1 Thermal shift determination

[0384] Thermal shift assays (TSA) were performed in 96-well plates on a qPCR machine (LightCycler 480II, Roche). In each row, one ISVD protein was analyzed in the following pH ranges: 4, 5, 6, 7, 8, and 9.

[0385] In each well, 5 μL of ISVD protein sample (0.8 mg / mL in D-PBS) was added to 5 μL of SyproOrange (40× in MilliQ water; Invitrogen, catalog number S6551) and 10 μL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate and 115 mM NaCl, pH range 4 to 9). A temperature gradient (37°C to 99°C at a rate of 0.03°C / s) was applied, which induced the unfolding of the ISVD protein and thus exposed the hydrophobic patches. The increase in fluorescence intensity caused by the binding of Sypro Orange to those hydrophobic patches was measured (Ex / Em=465 / 580 nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 was used as a measure of the melting temperature (Tm).

[0386] The Tm (°C) values ​​obtained for the sequence optimized variants are given in Table B-1. All ISVDs showed acceptable Tm values.

[0387] 2.2 Oligomerization assay

[0388] The oligomerization tendency of monovalent ISVD proteins under stress conditions (1 week at 45° C.) was investigated by analytical size exclusion chromatography (SE-HPLC). For this purpose, ISVD proteins with a 3xFLAG-His6 tag produced in E. coli, purified by IMAC and then by preparative SEC, filtered (0.22 μm), at a concentration of 1 mg / mL (D PBS) were used. The SE-HPLC profiles of two 100 μL aliquots were compared: one sample was incubated at -20° C. (T0) for 1 week and the other sample was incubated at 45° C. for 1 week. The samples were clarified by centrifugation at 20000 RCF for 5 minutes and subsequently analyzed on a Waters Xbridge column (mobile phase 10 mM phosphate + 150 mM arginine + 10% 1-propanol pH 7, flow rate 0.5 mL / min). The relative pre-peak area difference between stressed (+45°C) and unstressed samples (-20°C) was calculated and reported as Δ% HMW (=% HMW 1W 45°C - % HMW TO).

[0389] The obtained Δ% HMW values ​​for the sequence optimized variants are given in Table B-1. The % oligomerization was acceptable for all ISVDs.

[0390] 2.3 Determination of aggregation onset temperature (Tagg determination)

[0391] The temperature at which the ISVD protein starts to aggregate (=temperature of aggregation onset=Tagg) was determined by dynamic light scattering (DLS) using a DynaPro plate reader (Wyatt). For this purpose, an ISVD protein with a 3xFLAG-His6 tag produced in E. coli, purified by IMAC and then by preparative SEC, filtered (0.22 μm) at a concentration of 1 mg / mL (D-PBS) was used. After thawing, the sample was filtered through a 0.1 μm membrane and centrifuged at 14000 rpm for 5 min. 30 μL of the sample (4 replicates) were heated from 40°C to 80°C at a constant rate of 0.25°C / min and the light scattering intensity was continuously recorded. The hydrodynamic radius derived from the measured intensity was plotted against the temperature to determine the temperature at which the radius began to increase (=Tagg, °C).

[0392] The Tagg values ​​of the obtained sequence optimized variants are given in Table B-1. All ISVDs had acceptable Tagg values.

[0393] 2.4 Inhibition of CX3CL1 / Fractal Chemokine Ligand Binding to Human and Cynomolgus Monkey CX3CR1

[0394] The ability of purified ISVD proteins to inhibit the interaction between human CX3CL1 / fractional chemokines and human and cynomolgus monkey (cyno) CX3CR1 expressed on the cell surface of CHO K1 cells was evaluated by flow cytometry. CHO cells overexpressing human or cynomolgus monkey CX3CR1 were generated using techniques known in the art. Cells were harvested and resuspended in assay buffer (PBS, 2% FBS, 0.05% NaN3). Cells were seeded in 384-well Bio-One V bottom plates (Greiner, catalog number 781280), with a total of 2E04 cells seeded per well. After washing, ISVD protein (in assay buffer, starting from 250 nM, 3-fold dilution, 11 points) was premixed with 30 μM HSA (Sigma-Aldrich, catalog number A8763) and human CX3CL1 (R&D Systems, catalog number 365-FR; internal labeling) labeled with AF647 at a final concentration of 2E-10 M (≈EC30) and added to the cells for 4 hours at 4°C. The plate was washed 3 times and the cells were resuspended in PI (Miltenyi Biotec, catalog number 130-093-233, diluted 1000 times in assay buffer). The cell suspension was analyzed with iQue Screener PLUS or iQue 3 (Intellicyt). IC50 was estimated by dose-response modeling. The curves were fitted using 4-parameter logistic regression in GraphPad (GraphPad Software Inc.).

[0395] The IC50 values ​​obtained for the sequence optimized variants are given in Table B-1. The sequence optimized building blocks retained acceptable Tm values ​​for further development.

[0396] Table B-1. Characterization of selected monovalent ISVDs.

[0397]

[0398] NA: Not available

[0399] Example 3: Preparation of multivalent ISVD constructs

[0400] A panel of 24 versions containing sequence-optimized anti-CX3CR1 ISVD building blocks (i.e., 66B02_SO (SEQ ID NO: 5), 66B02_SO (E1D) (SEQ ID NO: 6), 54A12_SO (SEQ ID NO: 2) and / or 54A12_SO (E1D) (SEQ ID NO: 3)) and an albumin binding building block (ALB23002; SEQ ID NO: 37) was generated. The building blocks were fused head-to-tail with a 9GS (SEQ ID NO: 69), 20GS (SEQ ID NO: 73) or 35GS (SEQ ID NO: 76) linker (Table A-7). In this panel, ALB23002 was located at the second or third position. All 24 versions also had an alanine (A) introduced at the C-terminus. Two forms (A041600085 and A041600087) were generated using building blocks 307 (SEQ ID NO: 7) (building block 307 is SEQ ID NO: 223 of WO 2013 / 130381), 307 (D1E) (SEQ ID NO: 8) and ALB23002 (SEQ ID NO: 37) or ALB11 (SEQ ID NO: 42), respectively. A C-terminal alanine (A) was introduced in A041600085 (see Table B-2).

[0401] Table B-2. Summary of exemplary polypeptides against CX3CR1.

[0402]

[0403]

[0404] Example 4: Characterization of multivalent ISVD constructs by expression profiling

[0405] The expression levels of 24 different multivalent ISVD constructs in Pichia pastoris (Komagataella phaffii) were characterized. Briefly, the different ISVD forms were transformed into Pichia pastoris strain NRRL Y-11430 (ATCC 76273) and cultured in the presence of 1000 μg / ml The Pichia clones were grown on YPDS plates (2% peptone (w / v), 1% yeast extract (w / v), 18.2% sorbitol (w / v), 1.5% selective agar (w / v), 2% glucose (w / v)). After selection on agar, twenty clones were picked for each multivalent ISVD construct and used for expression profiling in 96-well deep-well plates. For this, Pichia clones were incubated in 100 μl BGCM medium (10% citric acid 1 M pH 6.0, 0.1% glycerol (v / v), 13.4 g / L yeast nitrogen base (without amino acids) (w / v), 0.00004% (w / v) biotin, 2.6% peptone (w / v), 1.4% yeast extract (w / v)) in master 96-well deep-well plates. After incubation for 24 hours (30°C, 200rpm), 100 μl BGCM culture medium in the second 96-well deep well plate was inoculated with 5 μl of pre-culture. After 24 hours of growth, the culture in the expression plate was induced by adding 75 μl MeOH (0.66% v / v) to each well. During the fermentation period, another 75 μl MeOH (0.66% v / v) was added to each culture to keep induction. After induction for 24h, cells were harvested and supernatant was analyzed. The amount of protein expressed in the culture medium was analyzed on Octet Red384 using ProtA biosensor. After normalization relative to reference, relative normalized expression was drawn for each clone in the corresponding form, and the average normalized expression of each form was determined. The forms were then sorted according to the corresponding ISVD building blocks and joint length.

[0406] exist Figure 2 An overview of the expression profile analysis can be seen in . Within each form, the expression levels of the clones showed considerable variation. Based on the average normalized expression (Table B-3), many trends were found. The form containing the 54A12_SO building block obtained higher expression than the form with the 66B02_SO building block. If the ALB32002 building block is located at the C-terminal position, higher titers can be obtained. As a linker between the first and second building blocks, the 9GS linker is better than 20GS and 35GS. As a linker between the second and third building blocks, the 20GS linker is better than the 9GS linker and the 35GS linker.

[0407] Table B-3. Summary of normalized expression values ​​grouped by multivalent ISVD construct type.

[0408]

[0409]

[0410]

[0411]

[0412]

[0413] Based on the ranking obtained after expression profiling, seven forms were evaluated in fed-batch fermentation. Most forms expressed >2 g / L. Ten ISVD forms were selected (from 24 forms) for further characterization (see Table B-4).

[0414] Forms A041600085 and A041600087 were not included in the expression profiling experiments but were included in further characterization. Example 5: In vitro characterization of multivalent ISVD constructs

[0415] 5.1 Characterization of multivalent ISVD constructs in CX3CR1 binding assay

[0416] Binding of selected ISVD formats and A041600085 on CHO K1 human and cynomolgus monkey CX3CR1 cells in the presence of 30 μM HSA was characterized by flow cytometry and compared to A041600087 (SEQ ID NO: 83).

[0417] CHO cells overexpressing human or cynomolgus CX3CR1 were generated using techniques known in the art. Cells were harvested and resuspended in assay buffer (PBS, 2% FBS, 0.05% NaN3) and seeded individually in 384-well Bio-One V bottom plates (Greiner, catalog number 781280) with a total of 2E04 cells seeded per well. After washing, serial dilutions of multivalent ISVD constructs (starting from 250nM, 3-fold dilutions, 11 points, diluted in assay buffer) were added and incubated at 4°C for 4 hours. Binding was performed in the presence of 30 μM HSA (Sigma-Aldrich, catalog number A8763). The plates were then washed 3 times and the cells were incubated with anti-VHH mouse antibodies (diluted 3300-fold in assay buffer) at 4°C for 30 minutes. Subsequently, the plate was washed 3 times, and the cells were incubated at 4 ° C for 30 minutes in goat anti-mouse Fc APC (Jackson ImmunoResearch, catalog number 115-135-164 / 115-116-071) (diluted 100 times in assay buffer). Finally, the plate was washed 3 times, and the cells were resuspended in PI (Miltenyi Biotec, catalog number 130-093-233, diluted 1000 times in assay buffer). The cell suspension was analyzed with iQueScreener PLUS (Intellicyt). EC50 was estimated by dose-response modeling. The 4-parameter logistic regression curve was fitted using GraphPad (GraphPad Software Inc.). Purified anti-human CX3CR1 antibody [K0124E1] (BioLegend, catalog number 355701 / 355702) was used as a positive control to detect CX3CR1 expression on the cell surface.

[0418] The values ​​obtained are reported in Table B-4. All formats showed binding to human CX3CR1 with EC50 between 0.1 and 0.6 nM, and cross-reactivity with cynomolgus monkey CX3CR1 (maximum 3.3-fold difference in EC50). The binding curves for selected formats are shown in Figure 3.

[0419] Table B-4. In vitro characterization of selected anti-CX3CR1 multivalent ISVD constructs in a CX3CR1 binding assay.

[0420]

[0421] 5.2 Characterization of multivalent ISVD constructs in CX3CL1 / fractal chemokine ligand competition assay

[0422] Ten selected multivalent ISVD constructs, formats A041600085 and A041600087 and "KAND-567" were further characterized in a competition assay with AF-647 labeled soluble CX3CL1 / fractal chemokine ligand (at EC30 concentration) as described in Example 2.4 in the presence of 30 μM HSA.

[0423] The values ​​obtained are reported in Table B-5. All formats showed very similar IC50s ranging from 0.7 to 7.7 nM with a maximum 8-fold difference on cynomolgus monkey CX3CR1. The competition curves of the 5 selected multivalent ISVD constructs are shown in Figure 4.

[0424] Table B-5 In vitro characterization of selected anti-CX3CR1 multivalent ISVD constructs in the CX3CL1 / fractive chemokine ligand competition assay.

[0425]

[0426] NA: Not available

[0427] 5.3 Characterization of multivalent ISVD constructs for inhibition of CX3CL1 / fraclinical chemokine-induced chemotaxis of BA / F3 cells expressing human CX3CR1

[0428] Ten selected multivalent ISVD constructs, forms A041600085 and A041600087 and "KAND-567" were also tested in a chemotaxis assay in which Ba / F3-huCX3CR1 cells were stimulated with soluble CX3CL1 / fractal chemokines in the presence of 30 μM HSA. Ba / F3 cells overexpressing human CX3CR1 were generated using techniques known in the art. The ability of purified ISVD constructs to inhibit CX3CL1-induced migration of BA / F3 cells expressing human CX3CR1 was assessed using a Boyden chamber-based method using NeuroProbe Chemotax plates (Cat. No. 106-5). In brief, the bottom wells of the chamber were filled with 0.5, 1 or 5 nM human CX3CL1 (R&D Systems, Cat. No. 365-FR) in 30 μL assay buffer (RPMI medium containing 30 μM HSA (Sigma, Cat. No. A8763)). BA / F3 cells were pre-incubated with ISVD constructs or inhibitors at 37°C in RPMI culture medium containing 30 μM HSA (Sigma, catalog number SLCD9951) for 30 min. Next, 50 μL of ISVD constructs or inhibitors / cell suspensions (containing 1E05 cells) mixtures were added to the membrane, and the plate was incubated for 3 h at 37°C with 5% CO2. Cells that migrated were quantified using CellTiter Glo, which measures the ATP content of cells. To this end, 30 μL of the remaining cell suspensions were transferred to white Costar 96-well plates (#3917), and the wells were washed with 20 μL of assay culture medium. Next, 50 μL of CellTiter Glo reagents (Promega, catalog number G7571) were added to each well, and the contents were mixed on an orbital shaker at 1000 RPM for 5 min to induce cell lysis. After incubation at room temperature for 10 min, the signal was measured with Envision 2 (RLU-1 second).

[0429] All tested multivalent ISVD constructs effectively blocked huCX3CL1-induced chemotaxis of Ba / F3-huCX3CR1 cells with IC50s ranging from 1.6 to 3.6 nM (when 1 nM CX3CL1 was used). Multiple assays were performed and the geometric mean of the different IC50s are presented in Table B-6. The inhibition of chemotaxis by the five multivalent ISVD constructs selected for further characterization is shown in Figure 1. Figure 5 and compared with A041600087.

[0430] Table B-6. In vitro characterization of selected anti-CX3CR1 multivalent ISVD constructs in a chemotaxis assay.

[0431]

[0432]

[0433] NA: Not available

[0434] Example 6: In vitro characterization of selected multivalent ISVD constructs

[0435] Based on potency, high titers, biophysical properties, and building block diversity, multivalent ISVD constructs A041600025, A041600034, A041600041, A041600035, and A041600085 were selected for further characterization.

[0436] 6.1 Characterization of multivalent ISVD constructs with respect to binding to different huCX3CR1 variants

[0437] Two important SNPs in huCX3CR1, namely V249I (Rs3732379) and T280M (Rs3732378), were described that may affect receptor activity and binding to ISVDs. Both SNPs are common in global populations. Different combinations of these SNPs were introduced into human CX3CR1 and binding to five selected multivalent ISVD constructs was assessed by flow cytometry after transient transfection of huCX3CR1 variants in HEK293T cells.

[0438] In brief, CX3CR1 construct is cloned into pcDNA3.1 (ThermoFisher Scientific, catalog number V79020), and plasmid DNA is prepared from Escherichia coli TOP10 cells. HEK293T cells are inoculated with a concentration of 1.5E06 cells per T75 flask, and incubated overnight at 37°C in the DMEM culture medium (Gibco, catalog number 31966) supplemented with 10% FBS (Sigma, catalog number F7524). The culture medium is then replaced with Opti-MEM culture medium (Gibco, catalog number 31985). The mixture of 9 μg plasmid DNA, 27 μL Fugene 6 (Promega, catalog number E2691) in the Opti-MEM of 1mL in final volume is incubated at room temperature for 15 minutes, and then added to cells. After incubation for 3 hours at 37°C, 10mL DMEM supplemented with 20% FBS is added, and incubation is continued. After 48 hours, the cells were washed with PBS and suspended with 4 mL of trypsin EDTA (Gibco, catalog number 25200-056), and then 6 mL of DMEM medium supplemented with 10% FBS was added. Binding assays were performed as described in Example 5.1. In addition to the binding of ISVDs, binding of AF-647-labeled soluble CX3CL1 / fractal chemokine ligands to different receptor variants was included as a positive control for CX3CR1 expression. The tested multivalent ISVD constructs showed similar binding to different huCX3CR1 variants and no binding to parental HEK293T cells (Table B-7 and Figure 6).

[0439] Table B-7. In vitro characterization of selected multivalent ISVD constructs in CX3CR1 binding assays with different CX3CR1 variants.

[0440]

[0441]

[0442] 6.2 Binding to pre-existing antibodies

[0443] The five selected multivalent ISVD constructs were tested for binding to pre-existing antibodies present in 96 serum samples from healthy volunteers using ProteOn XPR36 (Bio-Rad Laboratories, Inc.) and compared to A041600087.

[0444] The results showed that the five multivalent ISVD constructs bound very little to pre-existing antibodies.

[0445] 6.3 Specificity of multivalent ISVD constructs

[0446] 6.3.1 Specificity for CCR2 and CCR5

[0447] The specificity of the five selected multivalent ISVD constructs for the huCX3CR1 receptor was evaluated by FACS binding experiments on CHO-K1 cells expressing human CCR2 or human CCR5. CHO cells overexpressing huCCR2 or huCCR5 were generated using techniques known in the art. The binding of the multivalent ISVD constructs to huCCR2 or huCCR5 expressed on the cell surface was examined by flow cytometry as described in Example 5.1.

[0448] No binding to huCCR2 or huCCR5 was detected. Binding curves are shown in Figure 7 and Figure 8 Purified anti-human CCR2 [K036C2] (BioLegend, catalog number 357201 / 357202) or anti-human CCR5 [3A9] (BD Biosciences, catalog number 556041) was used as a positive control to detect CCR2 or CCR5 expression on the cell surface.

[0449] 6.3.2 Specificity in human membrane protein arrays

[0450] A041600035 and A041600087 were evaluated for binding in the human membrane protein array (MPA) to analyze specificity and target selectivity.

[0451] MPA is a protein library consisting of 6,000 different human membrane protein clones, each of which is overexpressed from an expression plasmid in living cells. Each clone is transfected individually in a separate well of a 384-well plate and then incubated for 36 hours (Tucker et al. 2018, Proc. Natl. Acad. Sci. USA. 29: 115 (22): E4990-9). Cells expressing each individual MPA protein clone are arranged in duplicate in a matrix format for high-throughput screening.

[0452] Prior to screening on MPA, optimal A041600035 and A041600087 concentrations of 20 μg / mL for screening were determined on HEK-293T cells expressing positive (CX3CR1 or membrane-tethered protein A) and negative (mock transfected) binding controls and then detected by flow cytometry using fluorescently labeled secondary antibodies that bind to ISVD.

[0453] A041600035 and A041600087 ligands were added to MPA at a predetermined concentration of 20 ug / mL and binding across the protein library was measured on the Intellicyt iQue using fluorescently labeled secondary antibodies on unfixed cells. Each array plate contained positive (Fc binding) and negative (empty vector) controls to ensure reproducibility of each plate. Nonspecific fluorescence was determined as any value below 3 standard deviations of the mean background value.

[0454] In a second flow cytometry experiment, serial dilutions of the test antibody in buffer containing 10% human serum albumin were used to confirm the interaction of the test ligand with any target identified by MPA screening, and the target identity was revalidated by sequencing. For A041600035, validated targets that showed a dose response and MFI ≥ 2 times background at the two highest concentrations tested are shown in Fig.17A and for A041600087, it is shown in Fig. 17B middle.

[0455] For A041600035, specific binding to CX3CR1 but not to other human membrane proteins was observed.

[0456] For A041600087, specific binding to CX3CR1 and MCAM (alternative name: MUC18; https: / / www.uniprot.org / uniprot / P43121) was observed.

[0457] Example 7: Manufacturability Assessment of Selected Multivalent ISVD Constructs

[0458] Based on potency, high titers, biophysical properties, and building block diversity, multivalent ISVD constructs A041600085, A041600025, A041600034, A041600041, and A041600035 were selected for further manufacturability evaluation.

[0459] Manufacturability experiments focused on product titer and purity during upstream processing (USP), and purification recovery, purity, and DSP platform matching after downstream processing (DSP).

[0460] 7.1. Product titer and purity during upstream processing (USP)

[0461] The titers were measured in the supernatant and in the cell broth at the end of the fermentation.

[0462] Other criteria measured are related to product purity and are assessed using different methods. HMW species associated with ISVD at the end of fermentation are quantified as the sum of the relative areas before the peak in the SE-HPLC chromatogram after Protein A cleanup of the fermentation sample. At the beginning of process development, less than 5% HMW species is preferred.

[0463] The post-peak area of ​​the RP-UHPLC chromatogram mainly provides information about the amount of variants with missing disulfide bridges. These variants cannot be removed by the purification process (although some spontaneous oxidation has been observed) and should therefore be kept to a minimum. Other variants such as carbamylated variants or low molecular weight variants in special cases are also observed as post-peak, but identification requires additional characterization (e.g., LC-MS). Therefore, the post-peak area is considered to be an indication only of variants with missing disulfide bridges. Preferably, the amount of variants with missing disulfide bridges after copper treatment (WO 2010 / 125187) is less than 5%.

[0464] There is no numerical score or threshold for the banding patterns on SDS-PAGE. The major band should be clearly visible at the expected molecular weight, and no or limited degradation should be observed on the SDS-PAGE gel. The extent of O-glycosylation of these forms is determined by MS-ID. Ideally, the sum of the hexose % is less than 15%.

[0465] The USP manufacturability of exemplary multivalent ISVD constructs is shown in Table B-8. A041600025 showed very good titer and product quality at the end of fermentation. Most USP manufacturability criteria received high quality scores except for LMW species and post-peak percentage on RP-UHPLC before copper treatment (just above the threshold).

[0466] A041600034 showed low titer and poor product quality at the end of fermentation. A high percentage of LMW species was observed, and the post-peak percentage on RP-UHPLC was just above the threshold before and after copper treatment (aCu). Lower titers can negatively impact development timelines, fermentation volumes, and commodity costs.

[0467] A041600035 had the second highest titer at the end of the fermentation and the best quality. In addition to the high percentage of LMW, the product quality was very good.

[0468] A041600041 showed good titer. Only one USP manufacturability criterion had a high quality score (post-peak percentage on RP-UHPLC after copper treatment).

[0469] A041600085 showed good titer. Only one USP manufacturability criterion had a high quality score (post-peak percentage on RP-UHPLC after copper treatment).

[0470] Table B-8. Manufacturability assessment of 5 multivalent ISVD constructs from a USP perspective.

[0471]

[0472] Color: White, high quality; light gray, medium quality; dark gray, low quality.

[0473] a Unless otherwise mentioned, aCu analysis of samples except PA-HPLC was performed after ProtA cleanup.

[0474] b The wet cell weight at the end of the fermentation (usually 350-400 g / L) was used to calculate as follows: Titer Cell-free [g / L] x ((1000 - wet cell weight [g / L]) / 1000) x 1.12.

[0475] c PA-HPLC titers determined using a universal standard curve.

[0476] 7.2 Downstream Processing (DSP) Manufacturability Assessment

[0477] The DSP manufacturability assessment was designed to evaluate the suitability of the multivalent ISVD constructs for purification. Qualitative and quantitative assessments were performed during larger scale purification, including criteria for capture resin binding capacity (>18 mg / mL), capture resin recovery (>90%), eluate pH adjustment recovery (>95%), UF / DF / UF recovery (>85%), and retention of molecular integrity during UF / DF / UF.

[0478] The results of the DSP manufacturability assessment are summarized in Table B-9. Considering the trivalent format of the multivalent ISVD constructs, the target capture resin binding capacity was set at ">18 mg / mL". Of the five multivalent ISVD constructs tested, two candidates had a binding capacity to the capture resin below this predetermined limit, namely A041600085 and A041600041. The lower capture resin binding capacity of these two multivalent ISVD constructs indicated lower recoveries at a load of 20 mg ISVD / mL resin during capture. For A041600035, A041600025, A041600041, limited protein loss was measured during pH adjustment, but for A041600085, A041600034, more significant drops were observed, resulting in recoveries below 95%. Finally, during the final UF / DF / UF step, all five multivalent ISVD constructs were recovered with high yields without compromising their molecular integrity.

[0479] Table B-9. Manufacturability assessment of 5 selected multivalent ISVD constructs in terms of downstream processing (DSP).

[0480]

[0481] Color: White, high quality; light gray, medium quality; dark gray, low quality.

[0482] DSP manufacturability assessment of the five selected multivalent ISVD constructs confirmed that A041600035 and A041600025 met all criteria: capture binding capacity (>18 mg / mL), capture recovery at 20 mg / mL load (>90%), pH adjustment recovery (>95%), UF / DF / UF recovery (>85%), and retention of molecular integrity during UF / DF / UF (Table B-9).

[0483] 7.3 Molecular profiling of exemplary multivalent ISVD constructs

[0484] The molecular profiles of the five selected multivalent ISVD constructs were evaluated to identify and characterize the biochemical properties and structurally related tendencies of the five selected multivalent ISVD constructs.

[0485] 7.3.1 Determination of high molecular weight products

[0486] The level of high molecular weight products (HMW) was determined by size exclusion high performance liquid chromatography (SE-HPLC). The results are shown in Fig. 9 middle.

[0487] A041600034 had a lower sum of HMW after capture (1.5% HMW) compared to the four other candidates. However, for all five selected multivalent ISVD constructs, HMW was reduced to less than 1% after polishing chromatography. In addition, this level of HMW remained stable at less than 1% during UF / DF / UF and until the final step of formulation.

[0488] 7.3.2 Determination of low molecular weight products

[0489] The levels of low molecular weight products (LMW) were determined by capillary gel electrophoresis (CGE). Fig.10 As shown, the polishing step by cation exchange chromatography (CEX) significantly reduced low molecular weight products (LMW), but to varying levels. A041600041 did not decrease below 1% after the polishing step. In all 5 multivalent ISVD constructs, the level of LMW remained stable during UF / DF / UF until the final step of formulation.

[0490] 7.3.3 Visual inspection - turbidity

[0491] Visual inspection by direct observation of the bulk final product ( Fig.11 ). The results were classified as "clear", "slightly turbid", or "turbid". At a mid-range concentration of 50 mg / mL, all multivalent ISVD constructs tested were "clear". At a target concentration of 100 mg / mL, four of the five multivalent ISVD constructs were found to be "slightly turbid", and A041600025 was observed to have significant turbidity and / or opalescence and was therefore classified as "turbid".

[0492] Use OD500( Fig.12 ) and further analysis of this turbidity and / or opalescence was performed by comparison to a turbidity standard curve. Both confirmed direct observation of the samples. A041600025 was confirmed as "turbid", while the other multivalent ISVD constructs were seen as "slightly turbid" at 100 mg / mL. In addition, the final products of the five multivalent ISVD constructs were found to have a slight color (yellow) and no particles by direct visual inspection.

[0493] 7.3.4 Measurement of host cell proteins

[0494] Host cell proteins (HCP) were measured by immuno-enzymatic assay for 5 multivalent ISVD constructs after different DSP steps. The HCP content in A04160035 was below 30 ppm. The HCP range for the other 4 multivalent ISVD constructs was between 30 and 60 ppm.

[0495] 7.3.5 Conclusions of Molecular Profiling Analysis

[0496] Table B-10 summarizes the results of the molecular profiling data.

[0497] Table B-10. Molecular profiling assessment of multivalent ISVD constructs in terms of DSP.

[0498]

[0499] Color: White, high quality; light gray, medium quality; dark gray, low quality.

[0500] Example 8: Further characterization of selected multivalent ISVD constructs

[0501] 8.1 Characterization of multivalent ISVD constructs for blocking huCX3CL1-induced ERK phosphorylation in BA / F3 cells expressing human CX3CR1

[0502] The ability of multivalent ISVD constructs A041600034, A041600035, A041600085 and A041600087 and "KAND-567" to inhibit CX3CL1-induced ERK phosphorylation in BA / F3 cells expressing human CX3CR1 was assessed in an HTRF-based assay using the Advanced phospho-ERK (Thr202 / Tyr204) Cell Kit from Cisbio Bioassays (Cat. No. 64AERPEG | 64AERPEH | 64AERPET). Briefly, ISVD constructs or inhibitors were premixed with 1.3 nM CX3CL1 (approximately EC30) in assay buffer (RPMI + 30 μM HSA) and 50 μL was added to each well of a Costar 96-well plate (#3596). Next, 50 μL of cell suspension (6E04 BA / F3 cells in assay buffer) was added to each well and incubated at room temperature for 10 min. The plate was centrifuged at 300 g for 2 min, and the assay medium was removed by flipping the plate. Next, 50 μL of lysis / ERK blocking solution (prepared according to the manufacturer's instructions) was added, and the plate was incubated at room temperature for 5 min while shaking at 900 RPM, and then incubated on the bench for another 40 min at room temperature. Before reading, the plate was shaken again at 900 RPM for 30 seconds, and 16 μL of lysate (without pipetting up and down) was transferred to a HTRF-compatible 384-well plate (PerkinElmer; ProxiPlate-384 Plus, white 384 shallow well microplate; #6008280). Donor and receptor antibodies were mixed in a 1:1 ratio in the assay buffer, and 4 μL of this mixture was added to each well and incubated at room temperature for 3 h in the dark. The signal was measured with a TECAN F200 and the ratio was determined by dividing the TRF signal at 665 nm by the TRF signal at 620 nm and multiplying by 1E04.

[0503] The values ​​obtained are reported in Table B-11. The multivalent ISVD constructs blocked huCX3CL1-induced phosphorylation of ERK in cells expressing human CX3CR1 with IC50s ranging from 2.7 to 5.4 nM (see Fig.13 ). Multiple determinations were performed and the geometric means of the different IC50s are shown in Table B-11.

[0504] Table B-11. In vitro characterization of selected anti-CX3CR1 multivalent ISVD constructs in the pERK assay.

[0505]

[0506] 8.2 Characterization of the multivalent ISVD A041600035 with respect to blocking human or mouse CX3CL1-induced phosphorylation of ERK in CHO K1 cells expressing human CX3CR1

[0507] The pERK assay for A041600035 was repeated by stimulating CHO K1 cells expressing human CX3CR1 with human or mouse CX3CL1. An alternative antibody recognizing mouse CX3CR1 (AB5715, Biolegend, Cat. No. 149002) was used as a positive control to block CX3CL1-induced phosphorylation of ERK in CHO K1 mouse CX3CR1 cells.

[0508] A041600035 can block the activity of two ligands on human CX3CR1 to the same extent with an IC50 of 6nM (see Table B-12 and Figure 14). These experiments also show that the effect of human CX3CL1 inducing ERK phosphorylation is about 10 to 40 times that of mouse CX3CL1 (for both human or mouse CX3CR1). The alternative antibody (AB5715, Biolegend, catalog number 149002) identifying mouse CX3CR1 blocks the ERK phosphorylation (see Table B-12 and Figure 14) induced by CX3CL1 in CHO K1 mouse CX3CR1 cells with an IC50 of 0.8 to 1.6nM.

[0509] Table B-12. Inhibition of ERK phosphorylation induced by human (hu) or mouse (m) fractal chemokine (CX3CL1).

[0510]

[0511] Italics and Underlining : Indicative value

[0512] 8.3 Affinity determination

[0513] The in-solution affinity of A041600035 for cells expressing human and cynomolgus monkey CX3CR1 was determined by the solution equilibrium kinetic exclusion KD measurement method MSD-SET (Meso Scale Discovery - Solution Equilibrium Titration) in the presence of 30 μM HSA. The affinity was determined on 3 independent assay occasions.

[0514] The geometric mean of 3 independent experiments is shown in Table B- 13. There was no difference in affinity for human and cynomolgus monkey CX3CR1.

[0515] Table B-13. In-solution affinity (KD) of A041600035 for human and cynomolgus monkey CX3CR1.

[0516] Human CX3CR1 (N=3) Cynomolgus monkey CX3CR1 (N=3) 1.3E-10M 1.1E-10M

[0517] 8.4 Characterization of A041600035 in blocking fractal chemokine-mediated chemotaxis of monocytes from healthy subjects and patients with lupus nephritis.

[0518] Pan-monocytes from healthy donors (HemaCare) were thawed at 37°C, washed with RPMI containing 10% FBS, resuspended in RPMI1640 containing 10% FBS and 25ug / mL DNAse I (Sigma-Aldrich), and left at 37°C for 1h under 5% CO2. The monocytes were spun down and stained with anti-human CD14-APC (Biolegend) and anti-human CD16-PE (Biolegend) at 1:100 dilution for 10min at room temperature in RPMI1640 containing 10% FBS. The monocytes were then washed and resuspended in RPMI1640 containing 1% FBS at a concentration of 5×10E6 / mL. The monocyte suspension was incubated with A041600035, A041600087 or negative control (IRR00163; VHH reference) or culture medium at different concentrations at room temperature in a 1:1 volume ratio for 10 min. Full-length recombinant fractal chemokine (R&D systems) was diluted at 50 ng / mL in RPMI1640 containing 1% FBS, and 500 uL of the diluted fractal chemokine was added to the lower chamber of a transwell (24-well, Corning). Then 100 uL of the monocyte suspension was slowly added to the upper chamber of the transwell. The transwell plate was placed in an incubator and cultured at 37°C for 3 h under 5% CO2. The upper chamber was then removed and the lower chamber was slowly mixed by pipetting. Flow cytometry analysis was performed to count monocyte subsets (classical: CD14+CD16-, intermediate: CD14-CD16+, and nonclassical: CD14-CD16+) in 100uL of suspension from the lower chamber. Arbitrary chemotaxis index is shown as fold change relative to medium control wells: (number of cells) / (number of cells in medium control) (Table B-14; Figure 15).

[0519] Table B-14. Descriptive table of arbitrary migration index of different monocyte subsets from healthy volunteers (n=3 for all cases) for A041600035

[0520]

[0521] Adjusted p-values ​​were obtained by Dunnett's test at fixed concentration following two-way ANOVA for the factor compound and concentration and their interactive effects on log-transformed arbitrary migration index parameters of the correlation index.

[0522] *IRR00163 vs. SAR445611, adjusted P value < 0.05; **IRR00163 vs. SAR445611, adjusted P value < 0.001GM = geometric mean

[0523] Blood samples from patients with lupus nephritis (LN disease stage 2-5) were provided by Dr. Ian R. Rifkin from Boston Medical Center, Massachusetts. PBMCs were isolated from fresh blood using SepMate (Stemcell) and pan-monocytes were enriched using a pan-monocyte enrichment kit (Miltenyi Biotech). The isolated monocytes were used in triplicate for transwell assays, and arbitrary chemotaxis indices were calculated as described above (Tables B-15 and Fig.16 ).

[0524] Table B-15. Arbitrary migration index of different monocyte subsets from healthy volunteers and LN patients (n=3 for all cases) for A041600035.

[0525]

[0526] P values ​​were obtained by mixed effects modeling with Bonferroni-Holm adjustment of log-transformed arbitrary migration index values ​​for each population (corrected for multiple monocyte type testing). Abbreviations: CD = cluster of differentiation, CI = confidence interval, LN = lupus nephritis

[0527] Example 9: NTS-induced glomerular remodeling in hCX3CR1 knock-in transgenic mice for selected multivalent ISVD constructs Evaluation in a non-accelerated model of nephritis

[0528] 9.1 Nephrotoxic nephritis (NTN) model

[0529] The non-accelerated NTN model in mice is described in the literature as a model of acute glomerulonephritis, in which nephritis is induced by administering anti-glomerular serum (i.e., nephrotoxic antiserum [NTS]). NTS binds to and deposits in the glomeruli of the kidneys and damages the glomerular filtration barrier, leading to proteinuria and inflammation (Ougaard et al. 2018, Int. J. Nephrol. 2018: 8424502: 1-12). In view of the lack of mouse cross-reactivity in the multivalent ISVD construct, NTN studies were performed on hCX3CR1 KI transgenic mice. The hCX3CR1 KI (C57BL / 6) strain was developed by Boehringer Ingelheim. Female hCX3CR1 KI (C57BL / 6) mice received a single injection of sheep anti-rat glomerular serum, with a 7-day observation period (Example 9.4) or a 21-day (Example 9.5) observation period after NTS administration.

[0530] 9.2 Immunohistochemical procedures

[0531] Glomerular infiltrating cell-mediated tissue renal injury is a key pathogenic early event that is expected to be detectable in this model (Ougaard et al. 2018, Int. J. Nephrol. ID 8424502: 1-12). The different biomarkers used in the literature (Hochheiser et al. 2013, J. Clin. Invest. 123: 4242-4254; Guo et al. 2019, Faseb J. 33: 2359-2371; Sung et al. 2017, J. Immunol. 198: 2589-2601) were used to analyze the therapeutic efficacy of multivalent ISVD constructs for chamber-specific monocytes / macrophages and T cell type infiltration in the renal cortex. Treatment with dexamethasone (administered daily at a unique dose) and an unrelated VHH (IRR00163) that does not bind to any mouse protein was used as a control to evaluate the therapeutic effect.

[0532] CD11b and Mac-2 antibodies were used to differentially detect infiltrating blood-derived monocytes and macrophages that may be distinct from F4 / 80-positive intrinsic and more mature macrophages found in the interstitial and juxtaglomerular regions (Geissmann et al. 2003, Immunity 19:71-82; Steinmetz et al. 2009, J. Immunol. 183:4693-704; Bideak et al. 2018, Kidney Int. 93:826-41). Interestingly, elevated expression of galectin-3 protein (recognized by Mac-2 antibody) and serum galectin-3 levels were reported in renal tissue of LN patients compared with healthy controls and identified as a possible biomarker of LN disease activity (Kang et al. 2009, Lupus 18:22-28). Additional CD4 and CD8 immunostaining was used to detect subsets of lymphocytic T cells that are also thought to be important in the pathogenesis of LN (Couzi et al. 2007, Arthritis Rheum. 56:2362-2370).

[0533] 9.2.1 FFPE kidney tissue preparation

[0534] On the 7th and / or 21st day of animal autopsy, the left kidney dedicated for immunohistochemistry study was divided into two pieces along the longitudinal axis (in the horizontal part in the middle of the kidney) in its middle part. The two halves were placed in a pH 6.9RS solution (Sigma Aldrich) buffered with 4% w / v formaldehyde at room temperature for 2 days. The fixation step of the kidney blocks stored in the appropriate box was stopped by rinsing 2x 5min in PBS buffer (1x PBS Gibco pH7.4, catalog number 10010-031), followed by dehydration and embedding in paraffin.

[0535] Paraffin-embedded tissue blocks containing hCX3CR1 KI mouse kidneys were sectioned at 3 μm thickness on an ultramicrotome and the sections were transferred to slides suitable for IHC. Immunostaining was performed on dewaxed slides using the Ventana Discovery XT or Ventana ULTRA automated system (Ventana Medical Systems, Inc, USA) according to the manufacturer's instructions.

[0536] 9.2.2 Immunostaining

[0537] Different primary antibodies (i.e., anti-F4 / 80, anti-CD11b, and anti-Mac-2 / Galectin-3 antibodies) were used to detect myeloid cell types on murine FFPE kidney tissue in different IHC assays performed on the Ventana Discovery XT or Ventana ULTRA automated systems. Lymphocyte T cells were detected using anti-CD4 and / or anti-CD8 antibodies. Detection systems with different amplification levels (manufactured by Ventana Medical System Inc) were used depending on the primary antibody used.

[0538] Usually use F4 / 80 (D2S9R) Antibodies (rabbit monoclonal immunoglobulin G (IgG) (Cell Signaling Technology, reference number 70076) were used to detect macrophage cell types. The detection system was biotin-free Discovery anti-rabbit UltraMap TM Horseradish peroxidase (HRP) conjugate (760-4315, Ventana Medical Systems, Inc, USA).

[0539] The monocyte / macrophage cell types are usually detected using Mac-2 / galectin-3 antibody (rat monoclonal IgG2a antibody) (clone M3 / 38, Cedarlane, reference CL8942AP). The detection system is a secondary antibody (linker) corresponding to Rabbit@ratIgG (clone R18-2, Abcam, reference ab125900) and a biotin-free Discovery anti-rabbit OmniMap horseradish peroxidase (HRP) conjugate (760-4311, Ventana Medical Systems, Inc, USA).

[0540] Mononuclear cell types are usually detected using CD11b [EPR1344] rabbit antibody (rabbit monoclonal IgG antibody) (Abcam, reference ab133357). The detection system is Discovery anti-rabbit HQ (760-4815, Ventana Medical Systems, Inc, USA) and anti-HQ HRP multimer (760-4820, Ventana Medical Systems, Inc, USA).

[0541] CD4 rat monoclonal IgG1 antibody (clone 4SM95, Invitrogen, reference 14-9766-82) is used to detect the cell surface receptors on the subpopulation of lymphocyte T cells. The detection system is a secondary antibody corresponding to Rabbit@rat IgG (clone R18-2, Abcam, reference ab125900), Discovery anti-rabbit HQ (760-4815, Ventana Medical Systems, Inc, the U.S.) and Discovery anti-HQ HRP polymer (760-4820, Ventana Medical Systems, Inc, the U.S.).

[0542] CD8a rat monoclonal IgG2a antibody (clone 4SM15, Invitrogen, reference 14-0808-82) is used to detect the cell surface receptors on the subpopulation of lymphocyte T cells. The detection system is a secondary antibody corresponding to Rabbit@rat IgG (clone R18-2, Abcam, reference ab125900), Discovery anti-rabbit HQ (760-4815, Ventana Medical Systems, Inc, the U.S.) and Discovery anti-HQ HRP polymer (760-4820, Ventana Medical Systems, Inc, the U.S.).

[0543] Immunostaining was performed for CD11b, F4 / 80 and Mac-2 / galectin-3 markers using a universal 3,3' diaminobenzidine (DAB) colorimetric detection kit (760-159, Ventana Medical Systems, Inc, USA). For CD4 and CD8 markers, a Discovery purple kit (760-229) was used. A counterstaining step was also performed with hematoxylin II (790-2208, Ventana Medical Systems, Inc, USA), and a blue reversion reagent (760-2037, Ventana Medical Systems, Inc, USA) was applied. The stained slides were dehydrated and covered with coverslips using cytoseal XYL (8312-4, Richard-Allanscientific, USA).

[0544] 9.3 Data Analysis

[0545] 9.3.1 Image Analysis

[0546] Quantitative image analysis is performed on image analysis platform (HALO, Indica Labs) using multiple IHC modules based on automatic cell counting. In the whole cortex regarded as 2 different target areas (in this case, the average density value of positive cells in each treatment group is analyzed) or only in glomerulus (in this case, the value of the average number of positive cells in glomerulus is analyzed). For CD11b / CD4 double staining, HALO AIDense Net neural network tool is used to automatically segment glomerulus (130 to 214 glomeruli were detected in each animal at the 7th day, and 143 to 284 glomeruli were detected in each animal at the 21st day, for analysis). For single Mac-2 / galectin-3 (the 7th day) or dual CD8 and Mac-2 / galectin-3 (the 21st day) staining, 80 glomeruli (7 days research) or 100 glomeruli (21 days research) of each animal are analyzed (manually depicted in the whole cortex).

[0547] 9.3.2 Statistical analysis

[0548] One-way ANOVA matching was performed for each IHC parameter based on log-transformed responses to mitigate heterogeneity of variance. With log-transformation, effect interpretation was in terms of ratios (fold changes) of GM. For comparison of 2-day and 4-day treatment effects, Dunnett correction (for p-values ​​and CI limits) was applied to each IHC parameter to control family-wise error rate. Results of the analysis for each IHC parameter including fold reduction and significance score are presented.

[0549] Used for Windows 10Enterprise 64-bit Analyses were performed in SAS Enterprise Guide v8.2.0 using software version 9.4. Fold reduction and significance scores were written in RStudio v1.1.453 with R v4.1.3 using packages tidyverse, rstatix, ggplot2, ggpubr, plotly, and EnvStats.

[0550] 9.4 Evaluation of renal immunohistochemical immune cell markers in a 7-day non-accelerated model of NTS-induced glomerulonephritis in knock-in hCX3CR1 transgenic mice in response to A041600035 administration

[0551] 9.4.1 Study Design

[0552] NTS (sheep anti-rat glomerular serum; PTX-001S-Ms, Probetex) was injected intraperitoneally on day 1 to induce non-accelerated NTN. The dose level and route of administration of NTS were based on the literature on this model (Ougaard et al. 2018, Int. J. Nephrol. ID 8424502: 1-12; Hochheiser et al. 2013, J. Clin. Invest. 123: 4242-4254; Guo et al. 2019, Faseb J. 33: 2359-2371) and were further effectively determined in optimization studies. The study included five treatment groups, and each treatment group initially included 10 female hCX3CR1KI mice injected with 240 μl NTS on day 1. Fig.19 A protocol for in vivo experimental study design is outlined in .

[0553] Treatments were administered as detailed in Table B-16. A041600035 was administered intraperitoneally (IP) on days 0, 2, 4, and 6. Dexamethasone (0.3 mg / kg) was administered daily by oral (PO) gavage.

[0554] Table B-16. Treatment Groups and Dosing Details for the 7-Day Non-Accelerated NTN Study.

[0555] Group treat Planned dose level Number of mice 1 NTS / IRR00163 30 mg / kg; IP on days 0, 2, 4, and 6 10 2 NTS / A041600035 Dose 1; IP on days 0, 2, 4, and 6 10 3 NTS / A041600035 Dose 2; IP on days 0, 2, 4, and 6 10 4 NTS / A041600035 Dose 3; IP on days 0, 2, 4, and 6 10 5 NTS / Dexamethasone 0.3 mg / kg once a day orally 10*

[0556] *One animal in the dexamethasone group was euthanized before day 7.

[0557] 9.4.2 Microscope observation

[0558] When compared with 2 untreated hCX3CR1 KI mice in the first trial, for all hCX3CR1 KI mice in the NTS / IRR00163 group, the distribution pattern and density of F4 / 80, CD11b and Mac-2 / galectin-3 positive cells on renal tissue were observed under a microscope (Figure 20). As expected, NTS injection induced significant overall accumulation of F4 / 80 positive cells in the entire cortical area and almost only around the glomerulus and tubulointerstitial area at the 7th day. In contrast, compared with F4 / 80, CD11b and Mac-2 / galectin-3 positive cells were detected mainly in the more local subregions of glomerulus and tubulointerstitial at the 7th day. It is worth noting that Mac-2 / galectin-3 protein expression is also significantly increased in the renal tubular epithelium in the renal interstitium, as reported in renal pathology cases (Henderson et al. 2008, Am. J. Pathol. 172: 288-298).

[0559] 9.4.3 Quantitative analysis

[0560] Five IHC marker / location variables (hereafter referred to as IHC parameters) were analyzed: 1) "Average density of F4 / 80+ cells in the entire cortex (per mm 2 )”; 2) “Average density of CD11b+ cells in the entire cortex (per mm 2 )”; 3) “Average density of CD4+ cells in the entire cortex (per mm 2 )”; 4) “Mean number of CD11b+ cells per glomerulus in the cortex”; 5) “Mean number of Mac-2 / galectin-3+ cells per glomerulus in the cortex”.

[0561] IHC analysis of tissues in the study revealed a significant dose-dependent reduction in the number of monocytes / macrophages (CD11b+; Mac-2 / galectin-3+) per glomerulus in response to A041600035 treatment compared to mice receiving negative controls 7 days after NTS administration, with a fold reduction (FD) of more than 2.0 (Table B-17). In contrast, daily administration of dexamethasone at 0.3 mg / kg IP for 7 consecutive days induced a more limited protective effect on the average number of positive cells per cortical glomerulus for both CD11b (estimated mean 1.35 FD, p=0.0105) and Mac-2 / galectin-3 (estimated mean 1.50 FD, p=0.0058) markers.

[0562] No changes were detected in the mean density of CD4+ cells in the cortex with any treatment.

[0563] Table B-17. Treatment effect on fold reduction of mean number of CD11b+ and Mac-2 / Galectin3+ cells per glomerulus in the cortex at day 7 compared to the NTS IRR00163 group.

[0564]

[0565] a FD compared to NTS / IRR00163 (reduction factor)

[0566] Based on IHC evaluation of CD11b, Mac-2 / galectin-3, and F4 / 80 immune cell markers, this study demonstrated that A041600035 could induce a dose-dependent reduction in the number of monocyte / macrophage cell types in kidney tissue from the non-accelerated NTN model in hCX3CR1 KI mice at day 7 after NTS injection. The fold reductions observed for CD11b and Mac-2 / galectin-3 IHC markers were more pronounced for the 2 highest concentrations of A041600035 compared to the dexamethasone tested doses.

[0567] 9.5 Evaluation of renal immunohistochemical immune cell markers in a 21-day non-accelerated model of NTS-induced glomerulonephritis in knock-in hCX3CR1 transgenic mice in response to A041600035 administration

[0568] 9.4.1 Study Design

[0569] NTS (sheep anti-rat glomerular serum; PTX-001S-Ms, Probetex) was injected by intraperitoneal route on day 1 to induce non-accelerated NTN. The dose level and route of administration of NTS are based on the literature on this model (Ougaard et al. 2018, Int. J. Nephrol. ID 8424502: 1-12; Hochheiser et al. 2013, J. Clin. Invest. 123: 4242-4254; Guo et al. 2019, Faseb J. 33: 2359-2371), and further effectively determined in optimization studies. The study included eleven treatment groups, and each treatment group except the initial group containing 2 animals (without any NTS injection) included 7 female hCX3CR1 KI mice injected with 240 μl NTS on day 1. For all treatment groups, dosing began on day 0, and a subset of animals were necropsied on day 7 (corresponding to groups 1 to 6) and then on day 21 (corresponding to groups 7 to 11) after the unique NTS injection. Fig.21 The protocol for the in vivo experimental study design is outlined in . Treatments were administered as detailed in Table B-18.

[0570] Table B-18. Treatment groups and dosing details for the 7-day and 21-day non-accelerated NTN mouse model.

[0571]

[0572] *One animal was missed from IHC studies due to issues during tissue processing.

[0573] 9.4.2 Microscope observation

[0574] As already described in Example 9.3, at an early time point of 7 days after the unique NTS injection, when compared to 2 naive hCX3CR1 KI mice not treated with NTS, for all hCX3CR1 KI mice treated with NTS and IRR00163 Nb, significant changes in the distribution pattern and density of F4 / 80, CD11b and Mac-2 / galectin-3 and CD4 positive cells on the renal tissue were observed microscopically. At 21 days after injection, NTS qualitatively induced similar microscopic changes, with a significant increase in the density of CD4 positive cells in the entire renal cortex of hCX3CR1 KI mice treated with NTS and IRR00163 (Figure 22). At day 21, a significant overall accumulation of F4 / 80 positive cells could still be clearly detected in the entire cortical area and almost exclusively around the glomerulus and tubulointerstitial areas. The density of CD11b and Mac-2 / galectin-3 positive cells increased in the more local (compared to F4 / 80) subregions of the glomerulus and tubulointerstitium. It is worth noting that Mac-2 / galectin-3 protein expression was still increased in the tubular epithelium within the renal interstitium at day 21, as reported in cases of renal pathology (Henderson et al. 2008, Am. J. Pathol. 172: 288-298). Compared with other markers at day 21, the overall change of CD8 markers induced at day 21 after NTS injection was limited.

[0575] 9.4.3 Quantitative analysis

[0576] Considering the 5 treatment groups, various IHC markers / positional variables (hereafter referred to as IHC parameters) have been selected for analysis at day 7 and day 21 after the first IP injection or oral gavage. The following 6 have been selected for analysis at day 7: "Average density of F4 / 80+ cells in the entire cortex (mm 2 )”, “Average density of CD4+ cells in the whole cortex (mm 2 )”, “Average density of CD11b+ cells in the entire cortex (mm 2)”, “mean number of CD4+ cells per glomerulus in the cortex”, “mean number of CD11b+ cells per glomerulus in the cortex”, and “mean number of Mac-2 / galectin-3+ cells per glomerulus in the cortex”. The analysis at day 21 additionally included “mean density of CD8+ cells in the entire cortex (mm 2 )” and “mean number of CD8+ cells per glomerulus in the cortex” IHC markers. Each of these parameters has been evaluated by comparing the “dexamethasone (0.5 mg / kg)”, “cyclophosphamide (30 mg / kg)”, “mycophenolate mofetil (100 mg / kg)” and “NTS / A041600035” treated groups with the “NTS / IRR00163” (30 mg / kg) control group.

[0577] IHC analysis of tissues (including days 7 and 21 after NTS administration) revealed a unique pharmacological effect of A041600035 in significantly and significantly reducing CD11b+ and Mac-2 / galectin-3 monocytes / macrophages per glomerulus. In contrast, dexamethasone and, to a lesser extent, mycophenolate mofetil reduced the CD4+ lymphocyte T cell population. (Table B-19).

[0578] Table B-19. Summary of treatment effects on the fold reduction of the mean density of F4 / 80+, CD11b+, Mac-2 / Galectin-3, CD4+, and CD8+ cells per glomerulus at days 7 and 21.

[0579]

[0580] NA 1 : Not evaluated due to intense staining on endothelial cells

[0581] NA 2 : Not evaluated due to operational issues

[0582] *Significance code of adjusted p value: *: p < 0.05; 2 *: p < 0.01; 3 *: p < 0.001, and 4 *: p<0.0001

[0583] a FD compared to NTS / IRR00163 (reduction factor)

[0584] Collectively, these results show that A041600035 has a profound effect on CD11b+ and Mac-2 / galectin-3+ infiltrating blood-derived monocytes and macrophages in the renal cortex and is more pronounced in the glomerular region of NTN animals at 7 and 21 days after NTS injection. In the 7-day study, only a less pronounced but significant effect was observed on F4 / 80+ macrophages, which may reflect activity on a blood-derived subset of this mixed population of infiltrating and tissue-resident macrophages. No effects of A041600035 on CD4 and CD8 T cell populations were detected.

[0585] The effects of A041600035 were distinct from those of dexamethasone, especially at 21 days after NTS injection. In contrast, dexamethasone treatment induced a significant reduction in both CD4- and CD8-positive T-cell populations at 21 days after NTS injection, but had no effect on monocyte and macrophage markers. In the same model, mycophenolate mofetil treatment had a significant effect only on the CD4-positive T-cell population at 21 days after NTS injection and when measuring the entire renal cortex.

[0586] In this model, no significant effects on any of the selected IHC markers were detected following cyclophosphamide treatment.

[0587] Example 10: Determination of pharmacokinetic (PK) curve

[0588] A041600035 or A041600087 was administered to cynomolgus monkeys as a single dose by intravenous infusion. Blood samples were collected at different time points after administration. The concentration of A041600035 or A041600087 was measured by ligand binding assay. Concentration-time curves were plotted ( Fig.18 ).

[0589] The different pK curves observed for A041600087 at higher doses compared to lower doses may indicate target mediated drug deposition (TMDD). This TMDD was not observed for A041600035. In addition, at similar doses, the exposure of A041600035 was higher than that of A041600087.

[0590] 7. Industrial Applicability

[0591] The technology of the present invention can be developed in industry. Application and practical development in industry can be realized by the general knowledge of technicians based on this specification.

[0592] 8. Sequence Listing

[0593] The term "ID" in the table below (last column) refers to "SEQ ID NO" as used herein.

[0594] Table A-1. Monovalent anti-CX3CR1 ISVD sequences having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions and CDR1 to CDR3 refer to complementarity determining regions.

[0595]

[0596] Table A-2. Anti-CX3CR1 ISVD sequence part: according to AbM Numbered CDRs and frameworks.

[0597]

[0598]

[0599]

[0600] Table A-3. Anti-CX3CR1 ISVD sequence part: Kabat Numbered CDRs and frameworks.

[0601]

[0602]

[0603] Table A-4. Serum albumin binding ISVD sequences.

[0604]

[0605]

[0606] Table A-5. Anti-albumin ISVD sequence part: according to AbM Numbered CDRs and frameworks.

[0607]

[0608] Table A-6. Anti-albumin ISVD sequence part: according to Kabat Numbered CDRs and frameworks.

[0609] Name [Additional information] sequence ID ALB23002_FR1[Albumin, FR1, Kabat] EVQLVESGGGVVQPGGSLRLSCAASGFTFR 59 ALB23002_CDR1[Albumin, CDR1, Kabat] SFGMS 60 ALB23002_FR2[Albumin, FR2, Kabat] WVRQAPGKGPEWVS 54 ALB23002_CDR2[Albumin, CDR2, Kabat] SISGSGSDTLYADSVKG 62 ALB23002_FR3[Albumin, FR3, Kabat] RFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 63 ALB23002_CDR3[Albumin, CDR3, Kabat] GGSLSR 57 ALB23002_FR4[Albumin, FR4, Kabat] SSQGTLVTVSS 58

[0610] Table A-7. Peptide linker sequences.

[0611]

[0612]

[0613] Table A-8. Exemplary multivalent ISVD polypeptides.

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622] Table A-9. CX3CR1 sequences.

[0623]

[0624] Table A-10. Additional sequences.

[0625] Name [Additional information] sequence ID C-terminus of FR4 VTVSS 135

[0626] Table A-11. Combinations of Hallmark residues in ISVD polypeptides (see Table 2).

[0627]

[0628]

[0629] 9. References

[0630] ·Imai et al.,Cell 1997Nov 14;91(4):521-30,PMID:9390561

[0631] ·Umehara et al., Arterioscler Thromb Vasc Biol.2004Jan; 24(1):34-40; PMID:12969992

[0632] ·Faure et al.,Science 2000Mar 24;287(5461):2274-7;PMID:10731151

[0633] ·McDermott et al.Circ Res.2001Aug 31;89(5):401-7,PMID:11532900

[0634] @Moatti et al.Blood 2001Apr 1;97(7):1925-8,PMID:11264153

[0635] ·McDermott,2003;J.Clin.Invest.111:1241,PMID:12697743

[0636] ·Ghilardi,2004,Stroke 35:1276,PMID:15118174

[0637] ·Combadière,2003;Circulation,107:1009,PMID:12600915

[0638] ·Lesnik,2003;J.Clin.Invest.111:333,PMID:12569158

[0639] ·Wakita et al.2017,Mol.Pharmacol.92:502,PMID:28842393

[0640] ·Tanaka et al.2021,Modern Rheumatol 31 783,PMID:33427546

[0641] ·WO 2013 / 130381

[0642] ·Low et al.2020,Mabs,12(1):1709322,PMID:31924119

[0643] ·Hamers-Casterman et al.Nature 363:446-448,1993

[0644] ·review article by Muyldermans(Reviews in Molecular Biotechnology74:277-302,2001)

[0645] ·WO 94 / 04678

[0646] ·Ward et al(see for example WO 94 / 04678 and Riechmann,Febs Lett.,339:285-290,1994and Prot.Eng.,9:531-537,1996)

[0647] @Conrath et al.,J.Biol.Chem.,Vol.276,10.7346-7350,2001,as well as tofor example WO 96 / 34103 and WO 99 / 23221

[0648] ·WO 2008 / 020079

[0649] ·Davies and Riechman(1994 and 1996)

[0650] ·Kabat et al.(“Sequence of proteins of immunological interest”,USPublic Health Services,NIH Bethesda,MD,Publication No.91)

[0651] @Riechmann and Muyldermans,2000(J.Immunol.Methods 240(1-2):185-195

[0652] ·Kontermann and Dübel(Eds.2010,Antibody Engineering,vol 2,SpringerVerlag Heidelberg Berlin,Martin,Chapter 3,pp.33-51)

[0653] ·WO 94 / 25591,Gasser et al.Biotechnol.Bioeng.94:535,2006;Gasser etal.Appl.Environ.Microbiol.73:6499,2007;or Damasceno etal.Microbiol.Biotechnol.74:381,2007

[0654] ·Estep P.et al.2013 MAbs.5(2):270-8.PMID:23575269

Claims

1. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), wherein each of the at least three ISVDs has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions and CDR1 to CDR3 refer to complementarity determining regions; and wherein the polypeptide comprises (a) a first ISVD that specifically binds to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and comprises (according to AbM numbering) CDR1 as the amino acid sequence of SEQ ID NO: 10; CDR2 being the amino acid sequence of SEQ ID NO: 12; and CDR3 having the amino acid sequence of SEQ ID NO: 14; and (b) a second ISVD that specifically binds to the fractal chemokine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and comprises (according to AbM numbering) CDR1 as the amino acid sequence of SEQ ID NO: 10; CDR2 being the amino acid sequence of SEQ ID NO: 12; and CDR3 having the amino acid sequence of SEQ ID NO: 14; and (c) a third ISVD that specifically binds to albumin and comprises (according to AbM numbering) A CDR1 that is the amino acid sequence of SEQ ID NO:53 or an amino acid sequence that has 2 or 1 amino acid differences from SEQ ID NO:53; A CDR2 that is the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence that has 2 or 1 amino acid differences from SEQ ID NO: 55; and A CDR3 that is the amino acid sequence of SEQ ID NO:57 or an amino acid sequence that has 2 or 1 amino acid differences from SEQ ID NO:57; and Wherein (a), (b) and (c) are directly linked or linked via a peptide linker.

2. The polypeptide according to the preceding claim, wherein the C-terminal ISVD of the polypeptide carries a C-terminal extension of FR4 of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length; optionally, wherein the C-terminal extension of FR4 is a single alanine (A) residue.

3. A polypeptide according to any one of the preceding claims, wherein the order of (a), (b) and (c) indicates their relative positions to each other within the polypeptide, viewed from the N-terminus to the C-terminus of the polypeptide.

4. The polypeptide according to any one of the preceding claims, wherein the ISVD in (a), (b) and (c) is selected from a VH domain and a VHH domain.

5. The polypeptide according to the preceding claim, wherein the ISVD in (a), (b) and (c) is selected from the group consisting of a camelized VH domain, a humanized VHH domain, a domain antibody and a dAb.

6. The polypeptide of any of the preceding claims, wherein in (a) and (b), the ISVD comprises one or more selected from the group consisting of: aspartic acid (D) at amino acid position 1 according to Kabat numbering, proline (P) at amino acid position 14 according to Kabat numbering, glycine (G) at amino acid position 16 according to Kabat numbering, arginine (R) at amino acid position 83 according to Kabat numbering, leucine (L) at amino acid position 89 according to Kabat numbering, and leucine (L) at amino acid position 108 according to Kabat numbering.

7. The polypeptide according to any one of the preceding claims, wherein in (a) and (b), the ISVD comprises one or more selected from the group consisting of: a serine (S) at amino acid position 11 according to Kabat numbering and a glutamine (Q) at position 44 according to Kabat numbering.

8. The polypeptide of any one of the preceding claims, wherein in (a) and (b), the ISVD comprises one or more selected from the group consisting of a threonine (T) at amino acid position 32 according to Kabat numbering, a serine (S) at amino acid position 52 according to Kabat numbering, and a threonine (T) at amino acid position 53 according to Kabat numbering.

9. The polypeptide of any one of the preceding claims, wherein in (a) and (b), the ISVD comprises one or more selected from the group consisting of: valine (V) at amino acid position 50 according to Kabat numbering, isoleucine (I) at amino acid position 56 according to Kabat numbering, and alanine (A) at amino acid position 96 according to Kabat numbering.

10. The polypeptide according to any one of the preceding claims, wherein in (a), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:3, or amino acids having at least 90% sequence identity to SEQ ID NO:3, and in (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:2, or amino acids having at least 90% sequence identity to SEQ ID NO:2, wherein for the purpose of determining the % sequence identity, the amino acid sequence of the CDR regions is disregarded.

11. The polypeptide according to any one of the preceding claims, wherein in (a), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence that has two or one amino acid differences with SEQ ID NO: 2, and in (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence that has two or one amino acid differences with SEQ ID NO:

2.

12. The polypeptide according to any one of the preceding claims, wherein in (a), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:3, and in (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:

2.

13. The polypeptide according to any one of the preceding claims, wherein in (a), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:3, and in (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:2, and wherein the order of (a), (b) and (c) indicates their relative positions to each other within the polypeptide from the N-terminus to the C-terminus of the polypeptide.

14. The polypeptide of any one of the preceding claims, wherein in (c), the ISVD specifically binds human serum albumin.

15. A polypeptide according to any one of the preceding claims, wherein in (c), the ISVD specifically binds human serum albumin and comprises (according to AbM numbering) CDR1 being the amino acid sequence of SEQ ID NO:53; CDR2 having the amino acid sequence of SEQ ID NO: 55; and CDR3 having the amino acid sequence of SEQ ID NO:

57.

16. The polypeptide of any one of the preceding claims, wherein in (c), the ISVD comprises or consists of: An amino acid sequence as shown in SEQ ID NO:37, or an amino acid sequence having a sequence identity of greater than 90% identity or greater than 95% identity to SEQ ID NO:

37.

17. The polypeptide according to any one of the preceding claims, wherein in (c), the ISVD comprises or consists of the amino acid sequence as shown in SEQ ID NO:

37.

18. The polypeptide according to any one of the preceding claims, wherein in (a) and (b), the ISVD consists of the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 2, and in (c), the ISVD consists of the amino acid sequence as shown in SEQ ID NO:

37.

19. The polypeptide according to the preceding claim, wherein the C-terminal extension of FR4 is a single Ala residue.

20. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises two peptide linkers, wherein each peptide linker is located between two ISVDs within the polypeptide; Optionally, wherein the two peptide linkers comprise or consist of the amino acid sequence as shown in SEQ ID NO:

69.

21. The polypeptide of any one of the preceding claims, wherein the polypeptide comprises an amino acid sequence having greater than 90% identity or greater than 95% identity to SEQ ID NO:

82.

22. The polypeptide according to the preceding claim, wherein the polypeptide comprises or consists of the amino acid sequence as shown in SEQ ID NO:

82.

23. The polypeptide according to any one of the preceding claims, wherein the single nucleotide polymorphism (SNP) variant of CX3CR1 comprises at least one SNP selected from SNP rs938203, rs2669849, rs1050592, rs3732379 and rs3732378.

24. The polypeptide according to any one of the preceding claims, wherein the polypeptide is characterized by increased production after expression in the yeast Pichia pastoris compared to a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO:

83.

25. The polypeptide according to any one of the preceding claims, wherein the polypeptide is characterized by an improvement in at least one biophysical property compared to a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83; wherein the biophysical property is selected from (a) improved stability after purification of the polypeptide, for example as measured by turbidity / opalescence of the polypeptide at a concentration of 100 mg / mL; (b) a reduced amount of low molecular weight species after expressing the polypeptide in a suitable host, such as measured by the peak front on reverse phase chromatography (e.g., RP-UPLC); (c) the amount of low molecular weight species reduced after purification of the polypeptide, for example as measured by capillary gel electrophoresis (CGE); (d) a reduction in the amount of high molecular weight species after expressing the polypeptide in a suitable host, for example as measured by size exclusion high performance liquid chromatography (SE-HPLC); (e) the amount of high molecular weight species reduced after purification of the polypeptide, e.g., as measured by size exclusion high performance liquid chromatography (SE-HPLC); and (f) A combination of two or more of the biophysical properties described.

26. The polypeptide according to any one of the preceding claims, wherein the polypeptide is characterized in that the recovery rate after a purification step is higher compared to the recovery rate of a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83, wherein the purification step is selected from: i. Capture step on resin; ii. a pH adjustment step after the capture step on the resin; iii. Fine purification step; iv. ultrafiltration / diafiltration / ultrafiltration step; and v. A combination of two or more of the purification steps described.

27. The polypeptide according to any of the preceding claims, wherein the polypeptide has a reduced amount of missing disulfide bridges after expression of the polypeptide in a suitable host compared to a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO: 83, wherein the amount of the polypeptide with a reduced amount of missing disulfide bridges is measured by reverse phase high performance liquid chromatography (RP-HPLC).

28. The polypeptide of any of the preceding claims, wherein the polypeptide binds to the cell surface glycoprotein MUC18 with lower affinity than the binding of a reference polypeptide consisting of the amino acid sequence as shown in SEQ ID NO:

83.

29. A polynucleotide encoding a polypeptide according to any one of the preceding claims.

30. A host or host cell comprising a polynucleotide according to the preceding claim.

31. A composition comprising a polypeptide or polynucleotide according to any one of the preceding claims; optionally, wherein the composition is a pharmaceutical composition; and further optionally, wherein the composition comprises at least one pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant.

32. A method for producing a polypeptide or composition according to any one of the preceding claims, comprising the steps of: - expressing the polypeptide in a suitable host cell or host organism or in another suitable expression system; and further optionally - isolating and / or purifying said polypeptide.

33. The polypeptide, polynucleotide or composition of any preceding claim for use in inhibiting the binding of CX3CR1 to a fractal chemokine in a mammalian cell.

34. A polypeptide, polynucleotide or composition according to any preceding claim for use as a medicament.

35. A polypeptide, polynucleotide or composition according to any one of the preceding claims, for use in preventing, treating and diagnosing a disease or disorder; optionally, wherein the disease or disorder is a disease or disorder associated with fractal chemokine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1.

36. A polypeptide, polynucleotide or composition for use according to the preceding claims, wherein the disease or disorder is selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, degenerative disorders of the nervous system and demyelinating diseases, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain and cancer including ovarian cancer.

37. A method for inhibiting the binding of CX3CR1 to a fractal chemokine in a mammalian cell, the method comprising administering to the cell a polypeptide according to any one of the preceding claims, thereby inhibiting signalling mediated by the fractal chemokine.

38. A method of treating a disease or disorder, wherein the disease or disorder is selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, degenerative and demyelinating disorders of the nervous system, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, and cancer including ovarian cancer, and wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of a polypeptide, polynucleotide or composition according to any one of the preceding claims.

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