CX3CR1-binding compounds, their uses and related methods

Abstract

The present technology relates to polypeptides comprising at least three immunoglobulin single variable domains (ISVDs). In particular, the present technology relates to polypeptides comprising optimized ISVD binding to the fractalkine receptor CX3CR1 and optimized ISVD binding to albumin. The present technology also relates to compositions of the polypeptides; polynucleotides encoding the polypeptides; hosts or host cells comprising the polynucleotides; methods for producing the polypeptides or compositions; and polypeptides, polynucleotides, or compositions for use in the prevention, treatment, and diagnosis of diseases or disorders.

Description

[Technical Field]

[0001] The present technology relates to polypeptides comprising an immunoglobulin single variable domain (ISVD) that binds to CX3CR1. In particular, the present technology relates to polypeptides comprising an ISVD optimized for binding to the fractalkine receptor CX3CR1 and an ISVD optimized for binding to albumin. The present technology also relates to compositions of the polypeptides, polynucleotides encoding the polypeptides, hosts or host cells comprising the polynucleotides, methods for producing the polypeptides or compositions, and polypeptides, polynucleotides, or compositions for use in the prevention, treatment, and diagnosis of diseases or disorders. [Background technology]

[0002] The C-X3-C motif chemokine receptor 1 (CX3CR1), also known as the fractalkine receptor or G protein-coupled receptor 13 (GPR13), is a receptor for the transmembrane protein and chemokine fractalkine (also known as CX3CL1). CX3CR1-CX3CL1 signaling exerts distinct 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 isotope transplant rejection, and rheumatoid arthritis (see, e.g., Umehara et al. 2004, Arterioscler. Thromb. Vasc. Biol. 24:34-40).

[0003] In addition, polymorphisms in CX3CR1 have been shown to be clinically important. For example, functional CX3CR1 analysis showed reduced fractalkine binding in patients homozygous for the CX3CR1 variant (V249I / T280M), a variant haplotype affecting two amino acids (isoleucine-249 and methionine-280). The CX3CR1 variant (V249I / T280M) has been associated with interindividual differences in susceptibility to HIV infection (e.g., Faure et al. 2000, Science 287:2274-7), atherosclerosis, and stroke (e.g., McDermott et al. 2001, Circ. Res. 89:401-7; Moatti et al. 2001, Blood 97:1925-8). HIV-infected patients homozygous for the CX3CR1 variant (V249I / T280M) progressed to AIDS more rapidly than patients with other haplotypes (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 (e.g., McDermott et al. 2001). 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 therapeutic approach. For example, several independent mouse genetic studies have demonstrated the beneficial effects of CX3CR1 deficiency on atherosclerosis (Combadiere et al. 2003, Circulation 107:1009; Lesnik et al. 2003, J. Clin. Invest. 111:333).

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

[0006] Ketomolimab (also known as E6011), a humanized anti-fractalkine (CX3CL1) monoclonal antibody, was developed by Eisai Co., Ltd. (Tanaka et al. 2021, Modern Rheumatol. 31:783). Both biological and clinical development has been discontinued.

[0007] Anti-CX3CR1 VHHs are described in WO 2013 / 130381. Based on this, BI 655088 (herein designated A041600087) was developed as a therapeutic compound for the treatment of atherosclerosis (https: / / adisinsight.springer.com / drugs / 800044864; see Low et al. 2020, Mabs, 12:1709322). However, as of today, no therapeutic biologics that bind to CX3CR1 are available and / or being developed to treat diseases associated with the fractalkine receptor CX3CR1 or single nucleotide polymorphism (SNP) variants of CX3CR1.

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

[0009] Thus, there is a need in the art for improved CX3CR1 binding compounds for modulating CX3CR1 activity to prevent, treat and / or diagnose disease. Summary of the Invention [Means for solving the problem]

[0010] The present inventors have developed polypeptides containing half-life extending moieties that target CX3CR1 and exhibit increased potency and / or efficacy in modulating CX3CR1 activity compared to available anti-CX3CR1 therapeutic compounds. The polypeptides can be efficiently produced (e.g., high expression yields in microbial hosts; good recovery after purification) and have demonstrated improved safety (long-term) under storage conditions (e.g., as measured by turbidity or opalescence at a polypeptide concentration of 100 mg / mL). Furthermore, the polypeptides have been shown to have limited reactivity against pre-existing antibodies in the treated subject (i.e., antibodies present in the subject before the first treatment with the polypeptide). In certain embodiments, such polypeptides exhibit a half-life sufficiently long enough to allow conveniently spaced sequential treatments in a subject in need thereof (e.g., to prevent, treat, or diagnose a disease or disorder associated with the fractalkine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1). Furthermore, such polypeptides may be shown to have improved product quality (e.g., reduced amounts of low molecular weight species and / or reduced amounts of low molecular weight species and / or reduced aggregation) after production and / or further purification.

[0011] In some embodiments, the present technology provides polypeptides 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 extending moiety that specifically binds to a serum protein such as albumin. In further embodiments, the polypeptide comprises or consists of two ISVDs that specifically bind to CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 and one ISVD that specifically binds to albumin, wherein the three ISVDs are optionally linked directly or via a peptide linker.

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

[0013] Also provided are hosts or host cells comprising a polynucleotide encoding a polypeptide according to the subject technology or comprising a vector encoding a polypeptide according to the subject technology. Methods for producing the polypeptides and their use as pharmaceuticals in the prevention, treatment, and diagnosis of diseases or disorders (e.g., inflammatory disease, atherosclerosis, or stroke) are also provided.

[0014] The present technology satisfies or addresses at least some of the above needs and aims to solve the above problems in the art by providing improved products and methods as defined by the independent claims. Particular embodiments are set out in the respective dependent claims and in the specific embodiments set out below. The present technology provides:

[0015] Item 1. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of the at least three ISVDs having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions and CDR1 to CDR3 refer to complementarity determining regions; the polypeptide (a) specifically binds to the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 (according to AbM numbering); CDR1 as shown in SEQ ID NO: 10; CDR2 as set forth in SEQ ID NO: 12; and CDR3 shown in SEQ ID NO: 14 The first ISVD, including: (b) specifically binds to the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 (according to AbM numbering); CDR1 as shown in SEQ ID NO: 10; CDR2 as set forth in SEQ ID NO: 12; and CDR3 shown in SEQ ID NO: 14 a second ISVD containing; and (c) specifically binds to albumin (according to AbM numbering) CDR1 as set forth in SEQ ID NO: 53 or having 2 or 1 amino acid differences from SEQ ID NO: 53; CDR2 as set forth in SEQ ID NO: 55 or having two or one amino acid difference from SEQ ID NO: 55; and a third ISVD comprising a CDR3 as set forth in SEQ ID NO: 57 or having two or one amino acid difference from SEQ ID NO: 57; Including, A polypeptide in which (a), (b) and (c) are linked directly or via a peptide linker.

[0016] Item 2. The polypeptide of the previous item, wherein the C-terminal ISVD of the polypeptide has a C-terminal extension of FR4 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.

[0017] Item 3. The polypeptide of any one of the preceding items, wherein the order of (a), (b), and (c) indicates their relative positions with respect to each other within the polypeptide, considering the polypeptide from N-terminus to C-terminus.

[0018] Item 4. The polypeptide of any one of the preceding items, wherein the ISVDs in (a), (b) and (c) are selected from a VH domain and a VHH domain.

[0019] Item 5. The polypeptide according to the previous item, wherein the ISVD in (a), (b) and (c) is selected from a camelized VH domain, a humanized VHH domain, a domain antibody and a dAb.

[0020] Item 6. The polypeptide according to the previous item, wherein the ISVDs in (a), (b) and (c) are humanized VHH domains.

[0021] Item 7. The polypeptide of any one of the preceding items, wherein in (a) and / or (b), the ISVD comprises one or more of an aspartic acid (D) at amino acid position 1 according to Kabat numbering, a proline (P) at amino acid position 14 according to Kabat numbering, a glycine (G) at amino acid position 16 according to Kabat numbering, an arginine (R) at amino acid position 83 according to Kabat numbering, a leucine (L) at amino acid position 89 according to Kabat numbering, and a leucine (L) at amino acid position 108 according to Kabat numbering.

[0022] Item 8. The polypeptide of any one of the preceding items, wherein in (a) and / or (b), the ISVD comprises one or more of a serine (S) at amino acid position 11 according to the Kabat numbering and a glutamine (Q) at position 44 according to the Kabat numbering.

[0023] Item 9. The polypeptide of any one of the preceding items, wherein in (a) and / or (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.

[0024] Item 10. The polypeptide of any one of the preceding items, wherein in (a) and / or (b), the ISVD comprises one or more of a valine (V) at amino acid position 50 according to the Kabat numbering, an isoleucine (I) at amino acid position 56 according to the Kabat numbering, and an alanine (A) at amino acid position 96 according to the Kabat numbering.

[0025] In item 11.(a) and / or (b), the framework regions FR1 to FR4 are FR1 as shown in SEQ ID NO:9; FR2 as shown in SEQ ID NO: 11; FR3 as set forth in SEQ ID NO: 13; and FR4 shown in SEQ ID NO: 15 Item 10. A polypeptide according to any one of the preceding items, comprising or consisting of:

[0026] Item 12. The polypeptide of any one of the preceding items, wherein in (a) and / or (b), the ISVD comprises an amino acid sequence having a sequence identity of more than 90% identity or more than 95% identity to SEQ ID NO: 2, and wherein for determining the % sequence identity, the amino acid sequence of the CDR regions is disregarded.

[0027] Item 13. The polypeptide of any one of the preceding items, wherein in (a) and / or (b), the ISVD comprises an amino acid sequence having a sequence identity of greater than 90% identity or greater than 95% identity to SEQ ID NO:2.

[0028] Item 14. The polypeptide of any one of the preceding items, wherein in (a) the ISVD consists of the amino acid sequence set forth 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 the amino acid sequence set forth in SEQ ID NO: 2 or amino acids having at least 90% sequence identity with SEQ ID NO: 2, and wherein the amino acid sequence of the CDR regions is disregarded for purposes of determining the % sequence identity.

[0029] Item 15. The polypeptide of any one of the preceding items, wherein in (a) the ISVD consists of the amino acid sequence set forth 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 the amino acid sequence set forth in SEQ ID NO: 2 or amino acids having at least 90% sequence identity with SEQ ID NO: 2.

[0030] Item 16. The polypeptide according to any one of the preceding items, wherein in (a) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 2, and in (b) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having two or one amino acid differences from SEQ ID NO: 2.

[0031] Item 17. The polypeptide according to any one of the preceding items, wherein in (a) and / or (b), the ISVD comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0032] Item 18. The polypeptide of any one of the preceding items, wherein in (a) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 3, and in (b) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0033] Item 19. The polypeptide of any one of the preceding items, wherein in (a) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 3, and in (b) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 2, and the order of (a), (b) and (c) indicates their relative positions to each other within the polypeptide, considering from the N-terminus to the C-terminus of the polypeptide.

[0034] Item 20. The polypeptide of any one of the preceding items, wherein in (c), the ISVD specifically binds to human serum albumin.

[0035] In item 21.(c), the framework regions FR1 to FR4 are FR1 as shown in SEQ ID NO: 52; FR2 as shown in SEQ ID NO: 54; FR3 as set forth in SEQ ID NO: 56; and FR4 shown in SEQ ID NO: 58 2. The polypeptide of any one of the preceding items, comprising:

[0036] 22. The polypeptide of any one of the preceding items, wherein in item 22.(c), the ISVD comprises or consists of the amino acid sequence set forth 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, and wherein for purposes of determining the % sequence identity, the amino acid sequence of the CDR regions is disregarded.

[0037] 23. The polypeptide of any one of the preceding items, wherein in item 23(c), the ISVD comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37, or an amino acid sequence having greater than 90% identity or greater than 95% identity to SEQ ID NO: 37.

[0038] Item 24. The polypeptide of any one of the preceding items, wherein in (c) the ISVD comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37.

[0039] Item 25. The polypeptide according to any one of the preceding items, wherein in (a) and (b), the ISVD consists of the amino acid sequence set forth 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 set forth in SEQ ID NO: 37.

[0040] Item 26. The polypeptide of any one of the preceding items, wherein in (a) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 3, in (b) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 2, and in (c) the ISVD consists of the amino acid sequence set forth in SEQ ID NO: 37.

[0041] Item 27. The polypeptide of any one of the preceding items, wherein the ISVD located at the C-terminus of the polypeptide has a C-terminal extension on the C-terminus of FR4, wherein the C-terminus of FR4 is SEQ ID NO: 135, and wherein the C-terminal extension of FR4 comprises 1, 2, 3, 4, or 5 amino acids.

[0042] Item 28. The polypeptide according to the previous item, wherein the C-terminal extension of FR4 comprises or consists of one, two or three alanine (Ala) residues.

[0043] Item 29. The polypeptide according to the previous item, wherein the C-terminal extension of FR4 is a single Ala residue.

[0044] Item 30. The polypeptide according to any one of the preceding items, wherein the peptide linker comprises 3 to 62 amino acids.

[0045] Item 31. The polypeptide according to the previous item, wherein the amino acids in the peptide linker are selected from alanine (A), glycine (G) and serine (S).

[0046] Item 32. The polypeptide according to the previous item, wherein the peptide linker is selected from a 9GS linker, a 20GS linker, and a 35GS linker.

[0047] Item 33. The polypeptide according to the previous item, wherein the peptide linker comprises or consists of a 9GS linker.

[0048] Item 34. Two peptide linkers, each peptide linker located between two ISVDs in the polypeptide; Item 10. The polypeptide of any one of the preceding items, wherein the two peptide linkers comprise or consist of the amino acid sequence set forth in SEQ ID NO: 69.

[0049] Item 35. The polypeptide of any one of the previous items, comprising an amino acid sequence having a sequence identity of greater than 90% identity or greater than 95% identity to SEQ ID NO: 82.

[0050] Item 36. The polypeptide of any one of the previous items, comprising an amino acid sequence having a sequence identity of greater than 90% identity or greater than 95% identity to SEQ ID NO: 82, wherein for purposes of determining sequence identity, the CDR regions are ignored.

[0051] Item 37. The polypeptide according to the previous item, comprising the amino acid sequence set forth in SEQ ID NO: 82.

[0052] Item 38. The polypeptide according to the previous item, consisting of the amino acid sequence set forth in SEQ ID NO: 82.

[0053] Item 39. A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 82.

[0054] Item 40. The polypeptide of any one of the preceding items, wherein the single nucleotide polymorphism (SNP) variant of CX3CR1 comprises at least one SNP selected from the group consisting of or including SNPs rs938203, rs2669849, rs1050592, rs3732379, and rs3732378.

[0055] Item 41. In (a) the first ISVD and / or in (b) the second ISVD are characterized by reduced antigenicity compared to an ISVD consisting of the amino acid sequence shown in SEQ ID NO: 1; Optionally, the polypeptide of any one of the preceding items, wherein antigenicity is assessed by binding to human leukocyte antigen DR isotype (HLA-DR) alleles.

[0056] Item 42. The polypeptide according to any one of the preceding items, characterized by an increased yield after expression in the yeast Pichia pastoris compared to a reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83.

[0057] Item 43. Characterized by at least one improved biophysical property compared to a reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83; Biophysical properties i. improved post-purification stability of the polypeptide as measured, for example, by turbidity / opalescence of the polypeptide at a concentration of 100 mg / mL; ii. a reduction in the amount of low molecular weight species following expression of the polypeptide in a suitable host, e.g., as measured by a pre-peak in reverse phase chromatography (e.g., RP-UPLC), e.g., following expression of the polypeptide in Pichia pastoris; iii. A reduction in the amount of low molecular weight species following purification of the polypeptide, as measured, for example, by capillary gel electrophoresis (CGE); iv. A reduction in the amount of high molecular weight species following expression of the polypeptide in a suitable host, as measured, for example, by size exclusion high performance liquid chromatography (SE-HPLC), for example, following expression of the polypeptide in Pichia pastoris; v. A reduction in the amount of high molecular weight species after purification of the polypeptide, as measured, for example, by size-exclusion high performance liquid chromatography (SE-HPLC); and vi. A combination of two or more of said biophysical properties The polypeptide of any one of the preceding items, selected from:

[0058] Item 44. Characterized by at least one improved biophysical property compared to a reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83; Biophysical properties i. Improved post-purification stability of the polypeptide as measured by turbidity / opalescence of the polypeptide at a concentration of 100 mg / mL; ii. A reduction in the amount of low molecular weight species following expression of the polypeptide in Pichia pastoris, as measured by pre-peak in reverse phase chromatography (e.g., RP-UPLC); iii. A reduction in the amount of low molecular weight species after purification of the polypeptide as measured by capillary gel electrophoresis (CGE); v. A reduction in the amount of high molecular weight species following expression of the polypeptide in Pichia pastoris, as measured by size-exclusion high performance liquid chromatography (SE-HPLC); v. A reduction in the amount of high molecular weight species after purification of the polypeptide, as measured by size-exclusion high performance liquid chromatography (SE-HPLC); and vi. A combination of two or more of said biophysical properties The polypeptide of any one of the preceding items, selected from:

[0059] Item 45. The method is characterized by a higher recovery rate after a purification step compared to the recovery rate of a reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83, wherein the purification step comprises: i. A capture step on the resin; ii. A pH adjustment step after the capture step on the resin; iii. Polishing process; iv.Ultrafiltration / diafiltration / ultrafiltration process; v. A combination of two or more of the above purification steps The polypeptide of any one of the preceding items, selected from:

[0060] Item 46. The polypeptide according to the previous item, wherein the capture step (i) is a purification step on Protein A chromatography.

[0061] Item 47. The polypeptide of any one of the preceding items, wherein the polishing step (iii) is an ion exchange chromatography step.

[0062] Item 48. The polypeptide of any one of the preceding items, wherein the amount of missing disulfide bridges of the polypeptide is reduced after expression of the polypeptide in a suitable host compared to a reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83, and the amount of the polypeptide with reduced amount of missing disulfide bridges is measured by reverse-phase high-performance liquid chromatography (RP-HPLC).

[0063] Item 49. The polypeptide according to the previous item, wherein the host is Pichia.

[0064] Item 50. The polypeptide of any one of the previous items, which binds to the cell surface glycoprotein MUC18 with a lower affinity compared to binding by a reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83.

[0065] Item 51. A polynucleotide encoding a polypeptide according to any one of the preceding items.

[0066] Item 52. A (non-human) host or host cell containing a polynucleotide according to the previous item.

[0067] Item 53. A composition comprising a polypeptide or polynucleotide according to any one of the preceding items, optionally a pharmaceutical composition; and further optionally comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant.

[0068] Item 54. - expressing the polypeptide in a suitable host cell or host organism or another suitable expression system; Further optionally, - isolating and / or purifying the polypeptide; A method for producing a polypeptide or composition according to any one of the preceding items, comprising:

[0069] Item 55. A polypeptide, polynucleotide or composition according to any one of the preceding items for use in inhibiting the binding of CX3CR1 to fractalkine in mammalian cells.

[0070] Item 56. A polypeptide, polynucleotide or composition according to any one of the preceding items for use as a medicament.

[0071] Item 57. A polypeptide, polynucleotide or composition according to any one of the preceding items for use in the prevention, treatment and / or diagnosis of a disease or disorder.

[0072] Item 58. A polypeptide, polynucleotide, or composition for use according to the previous item, wherein the disease or disorder is a disease or disorder associated with the fractalkine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1.

[0073] Item 59. The polypeptide, polynucleotide or composition for use according to the previous item, wherein the disease or disorder is selected from inflammatory diseases, cardiac 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-Schönlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative and demyelinating disorders, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, and cancer including ovarian cancer.

[0074] Item 60. A method for inhibiting the binding of CX3CR1 to fractalkine in a mammalian cell, comprising administering to the cell a polypeptide according to any one of the preceding items, thereby inhibiting signal transduction mediated by fractalkine.

[0075] Item 61. A method for treating a disease or disorder, wherein the disease or disorder is selected from inflammatory diseases, cardiac 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-Schönlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative and demyelinating disorders, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, and cancer, including ovarian cancer, the method comprising administering to a subject in need thereof a pharmaceutically active amount of the polypeptide, polynucleotide, or composition according to any one of the preceding items.

[0076] Item 62. Use of a 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 fractalkine in mammalian cells.

[0077] 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.

[0078] 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 fractalkine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1.

[0079] 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, cardiac 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-Schönlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative and demyelinating disorders, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, and cancer, including ovarian cancer. [Brief explanation of the drawings]

[0080] [Figure 1] Alignment of the sequences of ISVD components described in WO 2013 / 130381 (66B02, 54A12 and 307) and exemplary ISVD components of the present technology (66B02_SO and 54A12_SO). [Figure 2] Expression profiling of exemplary multivalent ISVD constructs described in Example 4. The amount of expressed protein was quantified using a ProtA biosensor on an Octet RED384. [Figure 3A] Binding of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) to CHOK1 cells expressing (A) human CX3CR1 and (B) cyno CX3CR1. [Figure 3B] Same as above. [Figure 4A]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) cyno CX3CR1. [Figure 4B] Same as above. [Figure 5] Inhibition of CX3CL1 induced exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) as measured by luminescence (RLU). [Figure 6A] Binding of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) (see top panel) and human CX3CL1 / fractalkine (full length)-Alexa647 (see bottom panel) 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. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 7] Binding to CHO K1 human CCR2 by exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) (see top panel) and purified anti-human CCR2 (see bottom panel). [Figure 8]Binding to CHO K1 human CCR5 by exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085, A041600087) (see top panel) and purified mouse anti-human CCR5 (see bottom panel). [Figure 9] Levels of high molecular weight products (HMW) of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) during large-scale purification, post-capture (Load Polish), post-polish (Load UF / DF / UF), and in the formulation product (Formulation) as determined by size-exclusion high performance liquid chromatography (SE-HPLC). [Figure 10] Levels of low molecular weight products (LMW) of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) during large-scale purification, post-capture (Load Polish), post-polish (Load UF / DF / UF), and in the formulation product (Formulation) as determined by capillary gel electrophoresis (GCE). [Figure 11] Visual inspection of exemplary multivalent ISVD constructs (top panel: A041600085, A041600034, A041600035; bottom panel: A041600025, A041600041) is by direct observation of the final formulation 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 cloudy," with significant turbidity and / or opalescence observed for A041600025, and therefore classified as "turbid." [Figure 12]Turbidity measurements of exemplary multivalent ISVD constructs (A041600025, A041600034, A041600035, A041600041, A041600085) by OD500 measurement of the final formulation product. [Figure 13] Characterization of exemplary multivalent ISVD constructs (A041600034, A041600035, A041600085) for blocking human CX3CL1-induced ERK phosphorylation in BA / F3 cells expressing human CX3CR1, as measured by the ratio of pERK signals, as described in Example 8.1. [Figure 14A] 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: Phosphorylation of ERK induced by human CX3CL1 in CHO K1 cells expressing human CX3CR1. B: Phosphorylation of ERK induced by mouse CX3CL1 in CHO K1 cells expressing human CX3CR1. C: Phosphorylation of ERK induced by human CX3CL1 in CHO K1 cells expressing mouse CX3CR1. D: Phosphorylation of ERK induced by mouse CX3CL1 in CHO K1 cells expressing mouse CX3CR1. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 15A] Monocyte chemotaxis assay of exemplary multivalent ISVD constructs (A041600035, A014600087) and negative controls (neg.ctrl 1: IRR00164, an ISVD that does not bind to CX3CR1; neg.ctrl 2: no ISVD / antibody) in transwell cultures from healthy donors (n=3) as measured by arbitrary migration index (fold change relative to media control wells) of (A) classical monocytes, (B) intermediate monocytes, and (C) non-classical monocytes. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 16] Monocyte chemotaxis assay in transwell cultures from healthy donors (n=5; top panel) and lupus nephritis (LN) donors (n=6; bottom panel) as measured by arbitrary migration index of total monocytes (left), CD16+ monocytes (center), and CD16- monocytes (right). *=p-value<0.05; ***=p-value<0.001; ns=p-value>0.05. [Figure 17A] To profile specificity and target selectivity, binding of exemplary multivalent ISVD constructs (A) A041600035 and (B) A041600087 was assessed on a human membrane protein array (MPA). Binding across protein libraries (vector, DCC, DSC1, MCAM, FCGR2B, CX3CR1) and a positive control (Protein A) was measured on an Intellicyt iQue using fluorescently labeled secondary antibodies on unfixed cells. [Figure 17B] Same as above. [Figure 18] Pharmacokinetic (PK) profiles of exemplary multivalent ISVD constructs A041600035 and A041600087 in cynomolgus monkeys for normalized IV single doses of 0.1 mg / kg and 1 mg / kg. [Figure 19] Design of the 7-day non-accelerated NTN mouse model. [Figures 20A-20B] Representative images of changes in F4 / 80 (A), CD11b / CD4 (B), and Mac-2 / Galectin-3 (C) distribution patterns on the renal cortex of one hCX3CR1 KI mouse treated with IRR00163 compared to one naive mouse 7 days after NTS-specific injection. [Figure 20C] Same as above. [Figure 21] Design of the 21-day non-accelerated NTN mouse model. [Figures 22A-22B] Representative illustration of changes in F4 / 80 (A), CD11b-CD4 (B), and Mac-2 / Galectin-3-CD8 (C) distribution patterns on the renal cortex of hCX3CR1 KI mice treated with NTS and IRR00163 compared to naive mice 21 days after NTS-specific injection. [Figure 22C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0081] 5.1 Definition All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0082] In accordance with the present technology, each occurrence of the word "comprising" may be optionally replaced with the word "consisting of." The articles "a / an" and "the" are used herein to refer to one or to more than one (e.g., at least one) of the grammatical object of the article, unless clearly dictated otherwise to the contrary. By way of example, "an element" means one element or more than one element. The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0083] The term "about," as used herein, refers to a ±10% deviation from the stated value. When the term "about" is used herein in reference to a number, it is to be understood that another embodiment still includes the number unmodified 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.

[0084] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, as would be apparent to one of ordinary skill in the art. For example, Sambrook et al. 1989 (Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 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 Ed., University of California Press, Berkeley, CA), Roitt et al. 2001 (Immunology, 6th Ed., Mosby / Elsevier, Edinburgh), Roitt et al. 2001 (Roitt's Essential Immunology, 10th Ed., Blackwell Publishing, UK), and Janeway et al. 2005 (Immunobiology, 6th Ed. Ed.,Garland Science See standard handbooks such as those published by University Press, ...

[0085] Unless otherwise indicated, all methods, steps, techniques and operations not described in detail may be carried out or performed in a manner known per se, as would be clear to one skilled in the art. Reference is again made, for example, to the standard handbooks and general background art mentioned herein, as well as to the further references cited herein, and to the following reviews, for example: 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 (Tumor 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.

[0086] The terms "polypeptide," "protein," or "peptide" are used to refer 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 containing post-translationally modified amino acids (e.g., methylation, phosphorylation, actylation, amidation, hydroxylation, formylation, or glycosylation) may be described as the amino acid sequence as originally translated, i.e., these modifications are not explicitly indicated in the amino acid sequence. Any polypeptide that can be expressed as a sequence modified linkages, cross-links and end caps, non-peptide bonds, etc., is encompassed by this definition. In some cases, a polypeptide may include N-terminal and / or C-terminal protecting groups. In some cases, a polypeptide may include one or more non-natural amino acids.

[0087] [Table 1]

[0088] The term "sequence identity," as used herein, refers to the amount of exact amino acid or nucleotide matches between two different sequences. The "percentage of sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated by dividing 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 by the total number of amino acid residues in the first amino acid sequence and multiplying by 100%, where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence is considered a difference of a single amino acid residue (i.e., at a single position) compared to the first amino acid sequence. Typically, to determine the "percentage of sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated the "first" amino acid sequence, and the other amino acid sequence is designated the "second" amino acid sequence. The percent amino acid sequence identity may be determined using sequence comparison programs known in the art, such as NCBI-BLAST.

[0089] The term "amino acid difference," as used herein, refers to the deletion, insertion, or substitution of a single amino acid residue relative to a reference sequence, and in some embodiments, is a substitution. Such conservative substitutions can be, for example, substitutions in which one amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small aliphatic, non-polar, or slightly 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, non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0090] Exemplary conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0091] 5.2 Immunoglobulin Single Variable Domains The term "immunoglobulin single variable domain" (ISVD), used interchangeably with "single variable domain," 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, dis-scFv), in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0092] In view of the above definition, fragments of conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD or IgE molecules known in the art), or of Fv fragments such as Fab fragments, F(ab')2 fragments, disulfide-linked Fv fragments, or scFv fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art) are typically not considered ISVDs, since in these cases binding to the respective epitope of an antigen typically occurs not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains, such as light and heavy chain variable domains, i.e., a VH-VL pair of immunoglobulin domains that jointly bind to the respective epitope of the antigen.

[0093] In contrast, ISVDs are capable of specifically binding to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD is formed by a single VH, a single VHH, or a single VL domain.

[0094] Thus, a single variable domain may 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 VHH-sequence) or a suitable fragment thereof, as long as it is capable of forming a single antigen-binding unit (i.e. a functional antigen-binding unit which consists essentially of a single variable domain and which single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0095] The immunoglobulin single variable domain (ISVD) can be, for example, a heavy chain ISVD, such as a VH, VHH, including a camelized VH or a humanized VHH. In some embodiments, it is a VHH, including a humanized VHH. In some embodiments, it is a VH, including a camelized VH, a human VH, and a camelized human VH. The heavy chain ISVD can be derived from a traditional four-chain antibody or from a heavy chain antibody.

[0096] For example, the ISVD may be a single domain antibody (or a suitable amino acid sequence for use as a single domain antibody), a "dAb" or dAb (or a suitable amino acid sequence for use as a dAb), a NANOBODY® ISVD (as defined herein, including but not limited to, a VHH); other single variable domain, or any suitable fragment of any one of these.

[0097] In particular, the ISVD may be a NANOBODY® ISVD (such as a VHH comprising a humanized or camelized VH) or a suitable fragment thereof. [Note: NANOBODY® and NANOBODIES® are registered trademarks of Ablynx NV]

[0098] The term "VHH domain," also known as VVHH, VHH antibody fragment, and VHH antibody, was originally described as the antigen-binding immunoglobulin variable domain of a "heavy chain antibody" (i.e., an antibody without a light chain; Hamers-Casterman et al. 1993, Nature 363:446-448). The term "VHH domain" was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (referred to herein as "VH domains") and from the light chain variable domains present in conventional four-chain antibodies (referred to herein as "VL domains"). For a further description of VHHs, see the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302).

[0099] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to generate hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be generated by screening naive or synthetic libraries, for example, by phage display. The generation of immunoglobulin sequences, such as VHHs, has been extensively 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 a target antigen to elicit an immune response against the target antigen. The resulting repertoire of VHHs is then further screened for VHHs that bind to the target antigen. In these examples, antibody generation requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or by recombinant production approaches. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such sources.

[0100] Immunoglobulin sequences of different origins may be produced, purified and / or isolated, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. Also, fully human, humanized or chimeric sequences may be produced, purified and / or isolated. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs described in Ward et al. 1989 (Nature 341:544) (e.g., WO 94 / 04678 and Davies and Riechmann 1994, Feb. Lett., 339:285-290 and 1996, Prot. Eng. 9:531-537), may be produced, purified and / or isolated. Furthermore, ISVDs may be fused to comprise or consist of at least three ISVDs forming multivalent and / or multispecific constructs (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation see Conrath et al. 2001 (J. Biol. Chem., 276:7346-7350) and also, for example, WO 96 / 34103 and WO 99 / 23221). The ISVD sequence may further comprise a tag or other functional moiety, such as a toxin, label, radiochemical, etc.

[0101] The ISVD polypeptide sequence included in a polypeptide according to the present technology is not limited with respect to the origin of the ISVD polypeptide sequence or the method by which the ISVD polypeptide sequence is (or was) 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 specific, but non-limiting, embodiments, 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 (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, and particularly partially or fully humanized VHH sequences), "camelized" immunoglobulin sequences (and particularly camelized VH sequences), and ISVDs 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 manipulating immunoglobulin sequences well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0102] In one aspect, the ISVD polypeptide sequence can be obtained from a mouse, rat, rabbit, cat, dog, goat, sheep, horse, pig, a 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.

[0103] The term "humanized VHH" includes amino acid sequences that correspond to the amino acid sequence of a naturally occurring VHH domain, but that have been "humanized" by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) with one or more amino acid residues (e.g., as shown above) that are present at the corresponding positions in a VH domain from a conventional human four-chain antibody. This can be performed by methods known per se, and will be clear to those skilled in the art, for example, based on the further explanations herein and the prior art (e.g., WO 2008 / 020079). Again, 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 obtained using a polypeptide comprising a naturally occurring VHH domain as starting material.

[0104] The term "camelized VH" includes an amino acid sequence which corresponds to the amino acid sequence of a naturally occurring VH domain, but which 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 four-chain antibody with one or more amino acid residues which are present at the corresponding positions in a VHH domain of a (camelid) heavy-chain antibody. This can be performed in a manner known per se, and will be clear to those skilled in the art, for example, on the basis of the further explanations herein and the prior art (e.g., Davies and Riechmann 1994 and 1996). Such "camelizing" substitutions are inserted at amino acid positions which form and / or are present at the VH-VL interface and / or 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 starting material or starting point for generating or designing a camelized VH is a VH sequence of mammalian origin, such as a human VH sequence, e.g., a VH3 sequence. However, it should be noted that such a camelized VH can be obtained in any suitable manner known per se and is therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VH domain as starting material.

[0105] The (general) structure of an ISVD comprises four framework regions ("FR"), termed "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"), termed "complementarity-determining region 1" ("CDR1"); "complementarity-determining region 2" ("CDR2"); and "complementarity-determining region 3" ("CDR3"). The framework sequences in an 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 handbooks and the further disclosures and literature referred to herein.

[0106] The term "framework sequence" describes (a suitable combination of) immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization). For example, the framework sequences may be framework sequences 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 particular aspect, the framework sequences are either framework sequences derived from a VHH-sequence (wherein said framework sequences are optionally partially or fully humanized) or are camelized conventional VH sequences (as defined herein).

[0107] In particular, ISVD is a (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (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).

[0108] The framework sequences present in the ISVD sequence may contain one or more "hallmark residues" such that the ISVD sequence is a NANOBODY® ISVD, e.g., a VHH, including a humanized VHH or a camelized VH.

[0109] The term "hallmark residue," as used herein, refers to an amino acid residue that occurs at a given position in the framework region of a NANOBODY® ISVD.

[0110] In particular, ISVD is a (general) structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (where "FR1," "FR2," "FR3," and "FR4" refer to framework regions 1-4; "CDR1," "CDR2," and "CDR3" refer to complementarity determining regions 1-3; where an ISVD may be defined as an immunoglobulin sequence having, further characterized by the presence of one or more "hallmark residues" (see Table 2 below). In some embodiments, the ISVD comprises a VHH sequence, including a (partially) humanized VHH sequence and a camelized VH sequence.

[0111] For example, ISVD is structured FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (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; 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 Kabat numbering selected from the hallmark residues set forth in Table 2 below).

[0112] [Table 2]

[0113] The amino acid residues of the ISVD may be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from camelids in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see, e.g., Figure 2 therein), and as further described in WO 2008 / 020079, pages 58 and 59, paragraph q). As is well known in the art for VH and VHH domains, the total number of amino acid residues in each of the CDRs can vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than permitted by Kabat numbering). This generally means that the Kabat numbering 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 and VHH domains will usually be in the range of 110-120, often 112-115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein.

[0114] In the present application, unless otherwise specified, the FR and CDR sequences of an ISVD (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) are determined according to the AbM numbering system described in Kontermann and Duebel (Eds. 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this system, FR1 contains amino acid residues 1 to 25, CDR1 contains amino acid residues 26 to 35, FR2 contains amino acid residues 36 to 49, CDR2 contains amino acid residues 50 to 58, FR3 contains amino acid residues 59 to 94, CDR3 contains amino acid residues 95 to 102, and FR4 contains amino acid residues 103 to 113.

[0115] The CDR regions may be determined according to different methods (e.g., Kabat, Chothia, or IMGT). For example, in the CDR determination according to Kabat, FR1 of the ISVD comprises amino acid residues 1 to 30, CDR1 of the ISVD comprises amino acid residues 31 to 35, FR2 of the ISVD comprises amino acid residues 36 to 49, CDR2 of the ISVD comprises amino acid residues 50 to 65, FR3 of the ISVD comprises amino acid residues 66 to 94, CDR3 of the ISVD comprises amino acid residues 95 to 102, and FR4 of the ISVD comprises amino acid residues 103 to 113.

[0116] ISVDs have a "three-dimensional structure." In the context of ISVDs, the term "three-dimensional structure" refers to a complex structure containing, for example, alpha helices and beta sheets folded into a compact structure stabilized by both polar and nonpolar interactions. The three-dimensional structure is formed naturally and maintained as a result of interactions between amino acid side chains.

[0117] 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; Decanier et al. 1999, Structure 7:361-370). VHHs adopt the standard fold of immunoglobulin variable domains. Immunoglobulin variable domains fold into a native conformation, also known as a beta-barrel. The FR regions of immunoglobulin variable domains pack together to form nine parallel beta strands folded into two sheets stabilized by conserved disulfide bonds that form a beta-sheet scaffold. The antigen-binding site formed by the CDR loops (hypervariable regions) actually rests on this core scaffold and is clustered at one end of the domain.

[0118] The present technology provides improved ISVDs that bind to CX3CR1, which 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 in WO 2013 / 130381).

[0119] In one aspect, the sequence-optimized ISVD has the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the ISVD specifically binds to the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1, (according to AbM numbering): CDR1 comprising or consisting of the amino acid sequence GSIFSSTAMA (SEQ ID NO: 10); a CDR2 comprising the amino acid sequence X1IX3TVGX7TK (SEQ ID NO: 202), wherein the ambiguous residues X can be: X1=A or V, X3=N or S, and X7=I or V; and CDR3 comprising the amino acid sequence DX1RRGWDTRY (SEQ ID NO: 138), wherein the ambiguous residue X can be X1=P or A. Includes.

[0120] In some embodiments, CDR2 comprises the amino acid sequence X1IX3TVGX7TK (SEQ ID NO: 202), where 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.

[0121] In some embodiments, CDR2 comprises the amino acid sequence VISTVGITK (SEQ ID NO: 12). In some embodiments, the CDR3 comprises the amino acid sequence DARRGWDTRY (SEQ ID NO: 14). In some embodiments, the ISVDs were sequence optimized to yield 66B02_SO (SEQ ID NO: 5) and 54A12_SO (SEQ ID NO: 2).

[0122] 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 characteristics compared to ISVDs 66B02 (SEQ ID NO: 4) and 54A12 (SEQ ID NO: 1): - More human-like (humanized); - Reduced binding by existing antibodies; - Reduced antigenicity (fewer T-cell epitopes); - Improved chemical stability

[0123] The sequences of the CDR and FR regions of exemplary sequence-optimized ISVDs are shown in Table A-2 (CDR determination based on AbM) and Table A-3 (CDR determination based on Kabat). An alignment of the ISVD sequences described in WO 2013 / 130381 and exemplary ISVDs sequence-optimized in the present technology is shown in Figure 1.

[0124] 5.3 Polypeptides Polypeptides according to the present technology may comprise or consist of various ISVDs, also referred to herein as ISVD components, such as a "first ISVD," a "second ISVD," and a "third ISVD." In some cases, the terms "first ISVD," "second ISVD," and "third ISVD" may indicate the relative positions of the specifically listed ISVDs relative to one another, with numbering starting 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." Accordingly, the "third ISVD" is closer to the C-terminus than the "first ISVD" and the "second ISVD." Thus, the numbering is not absolute and merely indicates the relative positions of two ISVDs, and does not exclude the possibility that additional binding units / components may be present in the polypeptide. Furthermore, it does not exclude the possibility that other binding units / components, such as ISVDs, may be positioned therebetween.

[0125] The ISVD components in the polypeptides according to the present technology can be linked directly or via a peptide linker. The term "directly" in this context refers to two ISVDs comprising immunoglobulin sequences having the following structure: [FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD1 -L-[FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD2 (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 the "L" between ISVD1 and ISVD2 refers to the single peptide bond connecting the ISVDs).

[0126] Linking two ISVDs via a "peptide linker" refers to two ISVDs containing immunoglobulin sequences having the following structure: [FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD1 -L-[FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4] ISVD2 (In the formula, "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)).

[0127] The peptide linker may be a synthetic amino acid sequence consisting of a linear chain of amino acids, for example, having a length of 1 to 62 amino acid residues, or 1 to 50 amino acid residues, preferably 9 to 40 amino acid residues. The linker may ensure that the ISVDs connected by the linker can perform their biological activity. In some cases, the linker contains, for example, alanine, glycine, and / or serine residues arranged in the repeat unit. In the present technology, the ISVDs may be linked via a peptide linker or directly without a peptide linker.

[0128] Some preferred examples of such amino acid linker sequences include a Gly-Ser linker, e.g., (Gly x Ser y ) zThese include those of the type described in WO 99 / 42077 (e.g., (Gly4Ser)3 (SEQ ID NO: 71) or (Gly3Ser2)3 (SEQ ID NO: 136), hinge-like regions such as the hinge regions of naturally occurring heavy chain antibodies, or similar sequences (e.g., those described in WO 94 / 04678). Gly-Ser linkers include 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), where n can be 1, 2, 3, 4, 5, 6, 7, or more). 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). See, for example, 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 link ISVDs together in a polypeptide.

[0129] For example, a polypeptide according to the present technology may include two peptide linkers located between two ISVDs within the polypeptide. For example, the polypeptide may include an immunoglobulin sequence having the following structure: [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 (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 in a polypeptide refer to linkers (e.g., as described in Table A-7) connecting the ISVDs.) In some embodiments, L1 and L2 are SEQ ID NO: 69.

[0130] The term "monovalent" indicates that there is only one binding unit / component, such as one ISVD, in a polypeptide. The term "monospecific" refers to binding to one (specific) type of target molecule.

[0131] The terms "bivalent," "trivalent," "tetravalent," or "pentavalent" all refer to the term "multivalent" and indicate the presence of two, three, four, or five binding units (e.g., ISVDs). The terms "bispecific," "trispecific," "tetraspecific," or "pentaspecific" all refer to the term "multispecific" and refer to binding to two, three, four, or five different target molecules, respectively.

[0132] In one aspect, the polypeptide comprises at least three ISVDs, each ISVD having the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; a first ISVD and a second ISVD specifically bind to the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1, (according to AbM numbering) CDR1 comprising or consisting of the amino acid sequence GSIFSSTAMA (SEQ ID NO: 10); a CDR2 comprising the amino acid sequence X1IX3TVGX7TK (SEQ ID NO: 202), wherein the ambiguous residues X can be X1=A, V; X3=N, S; and X7=I, V; and CDR3 comprising the amino acid sequence DX1RRGWDTRY (SEQ ID NO: 138), wherein the ambiguous residue X can be X1=P, A; Including, The third ISVD specifically binds albumin (according to AbM numbering). CDR1 as set forth in GFTFRSFGMS (SEQ ID NO: 53) or having two or one amino acid difference from SEQ ID NO: 53; CDR2 as set forth in SISGSGSDTL (SEQ ID NO: 55) or having two or one amino acid difference from SEQ ID NO: 55; and CDR3 as set forth in GGSLSR (SEQ ID NO: 57) or having two or one amino acid difference from SEQ ID NO: 57 Including, The at least three ISVDs are linked either directly or via peptide linkers.

[0133] In some embodiments, the first ISVD and the second ISVD comprise a CDR2 comprising the amino acid sequence X1IX3TVGX7TK (SEQ ID NO: 202), where 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.

[0134] In some embodiments, CDR2 comprises the amino acid sequence VISTVGITK (SEQ ID NO: 12). In some embodiments, the CDR3 comprises the amino acid sequence DARRGWDTRY (SEQ ID NO: 14).

[0135] The combination of CDR1, CDR2 and CDR3 of the first ISVD and / or second ISVD may be selected from the following embodiments, where an exemplary combination of CDR1, CDR2 and CDR3 is represented by one row of Table 3.

[0136] [Table 3]

[0137] In one embodiment, the amino acid sequence of an ISVD that binds to the fractalkine 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. The CDRs of the ISVD may have three, two, or one amino acid difference compared to the specific CDRs shown herein, yet retain biological activity in terms of efficacy and potency. For example, ISVDs with variance in the CDRs of three, two, or one amino acid compared to the specific CDRs shown herein retain their binding specificity for the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1.

[0138] In some embodiments, the ISVD that specifically binds to CX3CR1 is located at the N-terminus of the polypeptide. Thus, in these embodiments, the polypeptide comprises or consists of, in order, starting from the N-terminus of the polypeptide: an ISVD that specifically binds to CX3CR1, an ISVD that specifically binds to CX3CR1, and an ISVD that provides the polypeptide with increased half-life. The inventors surprisingly found that such a configuration increases the expression yield of the polypeptide. Furthermore, such a configuration of the polypeptide results in favorable chemistry, manufacturing, and control (CMC) properties, including upstream and downstream manufacturability. CMC activity is an important activity in developing novel pharmaceutical products. This includes defining manufacturing practices and product specifications that must be followed and met to ensure batch-to-batch product safety and consistency.

[0139] In one aspect, the polypeptide comprises at least three ISVDs, each ISVD having the general structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the first and second ISVDs specifically bind to the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 (e.g., human fractalkine receptor CX3CR1), and the third ISVD specifically binds to albumin (e.g., human serum albumin); the at least three ISVDs are linked directly or via peptide linkers (L1, L2), and the C-terminal ISVD of the polypeptide has a C-terminal extension of FR4 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length; and the order of the ISVDs and peptide linkers listed in Table 4 below indicates their relative position relative to each other within the polypeptide, considering from the N-terminus (first column) to the C-terminus (last column) of the polypeptide.

[0140] [Table 4]

[0141] [Table 5]

[0142] [Table 6]

[0143] In one aspect, the amino acid sequence of the polypeptide comprises an ISVD1 directly followed by an L1, an ISVD2 directly followed by an L2, and an ISVD3 directly followed by an ISVD3 (listed in row 1 of Table 4), optionally followed by a C-terminal extension of FR4 of ISVD3 (e.g., one, two, or three alanine (Ala) residues); 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 to any of the amino acid sequences of the polypeptides represented in row 1 of Table 4. For example, polypeptides having 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations in the amino acid sequence compared to a particular amino acid sequence shown herein (e.g., SEQ ID NO: 82) retain their favorable CMC properties, including upstream and downstream manufacturability and / or biological activity in terms of efficacy and potency.

[0144] In one embodiment, the polypeptide comprises three ISVDs (1) to (3) and two peptide linkers, each peptide linker being located between two ISVDs within the polypeptide, and the order of (1), (2), and (3) indicating their relative positions with respect to each other within the polypeptide, considering the N-terminus to the C-terminus of the polypeptide; ISVDs (1) and (2) specifically bind to the fractalkine receptor CX3CR1, and / or single nucleotide polymorphism (SNP) variants of CX3CR1 and ISVDs (1) and (2) comprise (according to AbM numbering): CDR1 set forth in GSIFSSTAMA (SEQ ID NO: 10); CDR2 set forth in VISTVGITK (SEQ ID NO: 12); and CDR3 set forth in DARRGWDTRY (SEQ ID NO: 14); The third ISVD specifically binds albumin and comprises (according to AbM numbering) a CDR1 set forth as GFTFRSFGMS (SEQ ID NO: 53), a CDR2 set forth as SISGSGSDTL (SEQ ID NO: 55); and a CDR3 set forth as GGSLSR (SEQ ID NO: 57), wherein the third ISVD has a C-terminal extension of 1 to 3 alanine residues or 1 to 3 glycine residues.

[0145] The determination of the CDR regions may be performed according to Kabat. Accordingly, the present technology also provides a polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of the at least three ISVDs having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions and CDR1 to CDR3 refer to complementarity determining regions, and the polypeptide is (a) specifically binds to the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 (according to Kabat numbering); CDR1 as shown in SEQ ID NO: 24; CDR2 as set forth in SEQ ID NO: 26; and CDR3 shown in SEQ ID NO: 14 The first ISVD, including: (b) specifically binds to the fractalkine receptor CX3CR1 and / or a single nucleotide polymorphism (SNP) variant of CX3CR1 (according to Kabat numbering); CDR1 as shown in SEQ ID NO: 24; CDR2 as set forth in SEQ ID NO: 26; and CDR3 shown in SEQ ID NO: 14 a second ISVD containing; and (c) specifically binds to albumin (according to Kabat numbering) CDR1 as set forth in SEQ ID NO: 60 or having two or one amino acid difference from SEQ ID NO: 53; CDR2 as set forth in SEQ ID NO: 62 or having two or one amino acid difference from SEQ ID NO: 55; and CDR3 as set forth in SEQ ID NO: 57 or having 2 or 1 amino acid difference from SEQ ID NO: 57 3. Includes the third ISVD, including (a), (b) and (c) are linked directly or via a peptide linker.

[0146] In some embodiments, the C-terminal ISVD of a polypeptide described herein has a C-terminal extension of FR4 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.

[0147] In some embodiments, the order of (a), (b), and (c) indicates the relative positions of the ISVDs to each other within a polypeptide, considering the polypeptide from the N-terminus to the C-terminus.

[0148] In some embodiments, the peptide linker comprises 3 to 62 amino acids. The amino acids in the peptide linker may be selected from alanine (A), glycine (G), and serine (S). In certain aspects, the peptide linker is selected from a 9GS linker, a 20GS linker, and a 35GS linker, e.g., SEQ ID NO: 69.

[0149] The term "C-terminal extension" refers to a C-terminal extension (tail) to the ISVD sequence. In particular, the C-terminal ISVD of a polypeptide can have a C-terminal extension of FR4 comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. The C-terminal extension comprises an amino acid sequence not naturally associated with the ISVD sequence (i.e., FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4).

[0150] In some embodiments, the ISVD at the C-terminal end of the polypeptide comprises a C-terminal extension (X n wherein n is 1 to 10, or 1 to 5, e.g., 1, 2, 3, 4, or 5 (and including 1 or 2, e.g., 1); each X is independently selected and can be independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), and isoleucine (I), which in some embodiments are naturally occurring amino acid residues. In one embodiment, n is 1 and X is alanine (A).

[0151] 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.

[0152] Antigen-binding protein (K D The term "affinity," as used herein, refers to the binding strength between an antigenic determinant and an antigen-binding site on an antigen-binding protein, as expressed by the equilibrium constant for the dissociation of an antigen with an antigen: K D The smaller the value of K, the stronger the binding strength between the antigenic determinant and the antigen-binding molecule (or affinity is expressed as 1 / K D The affinity constant (K A )). The affinity can be determined in a manner known per se depending on the particular antigen of interest. The affinity of a molecular interaction between two molecules may be measured by different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al. 2001, Intern. Immunology 13:1551-1559). The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by the detection of changes in protein concentration in a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions to measure k on , k off measurements, hence K D (or K A) values. This can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnson et al. (1991, Anal. Biochem. 198:268-277).

[0153] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). The term "biolayer interferometry" or "BLI," as used herein, 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 a biosensor chip (signal beam). Changes in the number of molecules bound to the biosensor tip cause 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. Interactions can be measured in real time, allowing the association and dissociation rates and affinity to be determined. BLI can be performed, for example, using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0154] Alternatively, affinity can be measured in a kinetic exclusion assay (KinExA) (e.g., Drake et al. 2004, Anal. Biochem. 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). The term "KinExA" as used herein refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of antibody / antigen complexes is passed through a column containing beads precoated with the antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus-captured antibody (or antigen) is achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0155] The GYROLAB® Immunoassay System provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).

[0156] Meso Scale Discovery-Solution Equilibrium Titration (MSD-SET) is a high-throughput procedure that enables reliable affinity screening of unpurified antibody fragments (Estep P. et al. 2013 MAbs. 5(2):270-8).

[0157] The terms "specificity," "specifically binds," or "specific binding" refer to the number of different target molecules, such as antigens from the same organism, that a particular binding unit, such as an ISVD, can bind with sufficiently high affinity (see below). In some cases, "selectivity," "selectively binds," or "selective binding" are also used in this context. Binding units, such as ISVDs, specifically bind to their designated targets. 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 generally given by the KD or dissociation constant, which has units of mol / liter (or M). Affinity can also be expressed as the association constant, KA, where KA is equal to 1 / KD, (mol / l) -1 (or M -1 ) units.

[0158] The term "fractalkine receptor CX3CR1," as used herein, refers to C-X3-C motif chemokine receptor 1 (CX3CR1), also known as fractalkine receptor or G protein-coupled receptor 13 (GPR13), which is a receptor for the transmembrane protein and chemokine fractalkine (also known as CX3CL1).

[0159] In some embodiments, human CX3CR1 and cynomolgus 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).

[0160] In some embodiments, human CX3CR1 and cynomolgus CX3CR1 are as set forth in SEQ ID NOs: 84 and 85, respectively (Table A-9).

[0161] The term "single nucleotide polymorphism (SNP) variant" as used herein refers to an allelic variant of a gene (e.g., CX3CR1) containing at least one single nucleotide polymorphism (SNP). "SNP" refers to a single nucleotide variation between the genomes of individuals of the same species. In some cases, a SNP can be a deletion or insertion of a single nucleotide. SNPs generally occur at relatively high frequencies in genomes, thereby contributing to genetic diversity. SNPs are considered to be more mutationally stable than other polymorphisms and are used in association studies, where linkage imbalance between a marker and an unknown variant is used to map disease-causing mutations. SNPs may have two, three, or four alleles (although it may be possible to have three or four different forms of the SNP corresponding to different nucleotides), thereby facilitating genotyping and automation (by a simple plus / minus assay rather than length measurement). SNP locations are generally flanked by highly conserved sequences. Individuals can be homozygous or heterozygous for alleles at each SNP site. Heterozygous SNP alleles may be differentially differentiated polymorphisms. SNPs may occur in protein-coding nucleic acid sequences ("cSNPs"). Such SNPs may result in amino acid changes in the encoded protein, which may have functional effects, i.e., result in "variant" proteins or polypeptides. Alternatively, such SNPs may be "silent" in that they do not result in an amino acid change. SNPs may also occur in introns and intergenic regions, but may result in phenotypic changes. For example, SNPs that result in aberrant splicing may result in a non-functional protein. Alternatively, SNPs may have no phenotypic effect. A variant protein or polypeptide contains at least one amino acid residue that differs from the corresponding amino acid sequence of a polypeptide referred to in the art as "wild-type" or "normal." Such variant polypeptides may result from codon changes, nonsense mutations, or any SNP that results in an altered protein structure, function, activity, regulation, or expression.

[0162] To identify potential SNPs, the genome of a subject (e.g., a patient suffering from a CX3CR1-related disease or condition) can be sequenced and analyzed (e.g., by SNP genotyping). In addition, SNPs can be derived from the dbSNP database (www.ncbi.nlm.nih.gov / snp / ), and they obtain a unique dbSNP reference SNP (rs or RefSNP) number assigned to the locus reference for the variant type.

[0163] For example, single nucleotide polymorphism (SNP) variants of CX3CR1 are selected from the group including or consisting of SNPs rs3732378 (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 rs17038679, rs41535248, rs55975803, rs The gene may comprise at least one SNP selected from the group including or consisting of: 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).

[0164] The term "albumin" or "serum albumin" refers to a family of globular proteins commonly found in animal and human plasma. Serum albumin is encoded by the albumin gene. Preferably, the albumin is human serum albumin. For example, human serum albumin as set forth in NCBI Reference Sequence: NP_000468.1.

[0165] In some embodiments, the polypeptides described herein bind with low 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 with low affinity (e.g., 10 ~4M For example, 10~3M, 10~ 2 or even lower KD) to melanoma cell adhesion molecule (MCAM). In some embodiments, the polypeptides described herein bind to melanoma cell adhesion molecule (MCAM) with a lower affinity compared to the binding to MCAM by A041600087 (SEQ ID NO: 83), for example, as measured by antibody specificity profiling (e.g., by protein microarray).

[0166] In the context of polypeptides, the term "antigenicity," as used herein, refers to the presence of B-cell or T-cell epitopes on a polypeptide. "Antigenicity" describes the ability of a polypeptide (antigen) to bind to or interact with the products of a final cell-mediated response, 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 known as an antibody or immunoglobulin. T-cell receptors, when combined with major histocompatibility complex (MHC) molecules, recognize linear amino acid sequences within polypeptide antigens, also called epitopes. For example, antigenicity can be assessed by binding to human leukocyte antigen DR isotype (HLA-DR) alleles.

[0167] In some embodiments, the polypeptides described herein have lower antigenicity compared to A041600087 (SEQ ID NO: 83), e.g., as measured by binding to human leukocyte antigen DR isotype (HLA-DR) alleles (e.g., using the NetMHCIIpan-v4.0 software tool (http: / / www.cbs.dtu.dk / services / NetMHCIIpan / )).

[0168] The term "pre-existing antibodies" refers to antibodies that are not "elicited" or "induced" by administration of a drug, for example, by a polypeptide containing an ISVD (as in the case of anti-drug antibodies (ADAS)). "Pre-existing antibodies" are already present in the blood or serum of a subject who has never received a drug, such as a polypeptide containing an ISVD.

[0169] The polypeptides of the present technology may exhibit limited reactivity to pre-existing antibodies in the treated subject (i.e., antibodies present in the subject prior to the first treatment with the antibody construct).

[0170] 5.4 Polynucleotides, Vectors and Hosts The term "polynucleotide" or "nucleic acid molecule," as used herein, refers to a linear polymer composed of covalently linked natural or non-natural nucleotide sequences, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in a DNA-RNA combination. Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence, a (chemically) modified nucleotide sequence, or a synthetic or semi-synthetic nucleotide sequence, such as a sequence isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using techniques for DNA synthesis known per se.

[0171] The nucleic acids according to the present technology can be prepared or obtained by methods known per se and / or isolated from suitable natural sources. A nucleotide sequence encoding a naturally occurring (poly)peptide can, for example, be subjected to site-directed mutagenesis to provide a nucleic acid molecule encoding a polypeptide with sequence variations. As will be clear to those skilled in the art, several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and, for example, nucleic acids encoding one or more linkers, can also be linked together in a suitable manner to prepare a nucleic acid.

[0172] Techniques for generating nucleic acids will be apparent to those of skill in the art and may include, for example, 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 result in expression of truncated expression products; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using suitable restriction enzymes), and / or introducing mutations by PCR reactions using one or more "mismatched" primers.

[0173] In some embodiments, the nucleic acid sequence of the polynucleotide is a sequence optimized for expression of the polypeptide in a suitable host.

[0174] In some embodiments, the polynucleotides of the present technology may be further optimized for in vitro or in vivo administration (e.g., therapeutic uses).

[0175] Also provided is a vector comprising the nucleic acid molecule encoding the polypeptide of the present technology.As used herein, a vector is a vehicle suitable for carrying genetic material into cells.Vector includes naked nucleic acid such as plasmid or mRNA, or nucleic acid embedded in larger structure such as liposome or viral vector.

[0176] A vector generally comprises at least one nucleic acid optionally linked to one or more regulatory elements, such as one or more suitable promoters, enhancers, terminators, etc. The vector is 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 a (e.g., human) cell. In the case of a DNA-based vector, this usually includes the presence of elements for transcription (e.g., a promoter and polyA signal) and translation (e.g., a Kozak sequence).

[0177] In a 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, a promoter is considered to be "operably linked" to a coding sequence if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence (wherein the coding sequence should be understood to be "under the control of" the promoter). Generally, when two nucleotide sequences are operably linked, they are in the same orientation and usually also in the same reading frame. They are usually essentially contiguous, although this may not be required.

[0178] Regulatory elements of the vector may be selected so that they are capable of providing their intended biological function in the intended host cell or host organism. For example, a promoter, enhancer or terminator must be "operable" in the intended host cell or host organism, meaning, for example, that the promoter must be capable of initiating or otherwise controlling / regulating the transcription and / or expression of a nucleotide sequence, e.g., a coding sequence, to which it is operably linked.

[0179] The polynucleotide or a vector comprising the polynucleotide can be used to transform / transfect a host cell or host organism, for example, for expression and / or production of the polypeptide. Suitable hosts or host cells for production purposes will be apparent to those skilled in the art and may be, for example, any suitable fungal, prokaryotic, or eukaryotic cell or cell line, or any suitable fungal, prokaryotic, or eukaryotic organism. Hosts or host cells comprising a nucleic acid encoding a polypeptide of the present technology are also encompassed herein.

[0180] The term "host cell" or "host organism" (collectively "host"), as used herein, refers to a suitable cell or organism capable of expressing a fully functional form of a desired polypeptide. Any host (organism) or host cell suitable for producing a polypeptide containing an ISVD can be used in the production methods described herein. In some embodiments, the host is a non-human host.

[0181] Examples of suitable hosts include prokaryotes such as Corynebacterium or Enterobacterium; insect cells, particularly insect cells suitable for baculovirus-mediated recombinant expression, such as cells derived from Trioplusiani or Spodoptera frugiperda, including but not limited to BTI-TN-5B1-4 High Five™ insect cells (Invitrogen), SF9 or Sf21 cells; mammalian cells such as CHO cells, as well as cells from Pichia (Komagataella), Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, and the like. Also included are lower eukaryotic hosts, including yeasts such as Bacillus subtilis, ...

[0182] The host used in the production method is capable of producing a polypeptide containing an ISVD. It is typically genetically modified to contain one or more nucleic acid sequences encoding a polypeptide containing one or more ISVDs. 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 techniques. Genetic modification includes the introduction of separate nucleic acid molecules into the host, for example, a plasmid or vector, and direct modification of the host's genetic material, for example, by integration into the host's chromosome, e.g., by homologous recombination. Often, a combination of both occurs; for example, the host is transformed with a plasmid, which is integrated (at least partially) into the host chromosome upon homologous recombination. Those skilled in the art are aware of suitable methods for genetic modification of a host to enable the host to produce a polypeptide containing an ISVD.

[0183] 5.5 Production and Purification In the context of a polypeptide, the terms "express," "expression," or "expressing," as used herein, refer to the process by which a polypeptide is synthesized, modified, and regulated in a host. Conventional strategies for recombinant protein expression involve transfecting cells with a vector containing a template (e.g., a polynucleotide encoding a polypeptide containing an ISVD) and then culturing the cells to transcribe and translate the desired polypeptide.

[0184] Specific conditions and genetic constructs for the expression of nucleic acids and production of polypeptides are described in the art, e.g., general culture methods, plasmids, promoters and leader sequences as 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).

[0185] In some embodiments, methods for producing a polypeptide described herein (e.g., a sequence having greater than 90% sequence identity to the amino acid sequence of SEQ ID NO: 82) include expressing the polypeptide in a suitable host cell or host organism (e.g., the yeast Pichia pastoris) or another suitable expression system, and isolating and / or purifying the polypeptide. The polypeptides of the present technology can thereby be efficiently produced (e.g., with high expression yields in microbial hosts).

[0186] In some embodiments, the titer after expression of a polypeptide described herein is greater than 6.0 g / L of cell-free medium. In some embodiments, the titer after expression of a polypeptide described herein is greater than 4.0 g / L of cell broth. In some embodiments, the titer after expression of a polypeptide described herein is greater than the titer after expression of A041600087 (SEQ ID NO: 83). In some embodiments, the titer after expression of a polypeptide described herein is greater than the titer after expression of A041600085 (SEQ ID NO: 137).

[0187] In the context of polypeptides, the terms "isolate," "isolation," or "isolating," as used herein, mean that a desired polypeptide product is separated or separated from cellular components and compositions comprising the desired polypeptide product. For example, host cells can be lysed to extract the expressed desired polypeptide for subsequent purification.

[0188] In the context of polypeptides, the terms "purify," "purification," or "purifying," as used herein, refer to a series of steps for liberating a desired polypeptide from a complex mixture of components (e.g., components derived from a cell, tissue, or organism). The purification process may separate the protein and non-protein portions of the mixture, ultimately separating the desired protein product (e.g., a polypeptide containing an ISVD) from all other proteins. Separation steps exploit differences in protein size, physicochemical properties, binding affinity, and biological activity. For example, (size exclusion) chromatography may be used to separate proteins in solution.

[0189] In the context of proteins, the terms "concentrate," "concentration," or "concentrating," as used herein, refer to increasing the amount of protein in an aqueous sample, e.g., for storage or for biopharmaceutical applications. Methods are available to those skilled in the art to 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 partitioning assays.

[0190] The first step in an ISVD polypeptide purification process is often referred to as the "capture step." The purpose of the capture step is to have a first reduction of process-related impurities (e.g., but not limited to, host cell proteins (HCPs), color, and DNA) and to capture the ISVD polypeptide product while maintaining high recovery. In some embodiments, the capture step refers to the first purification step of Protein A chromatography in bind-and-elute mode.

[0191] The second step of the purification process is often called a "polishing step" for the purpose of improving purity. For example, as the second purification step of the ISVD polypeptide purification process, an ion exchange chromatography step in a bind-and-elute mode can be used to remove / reduce product-related variants (e.g., but not limited to, high molecular weight (HMW) species, low molecular weight (LMW) species, and other charge variants) and some process-related impurities (e.g., but not limited to, HCP, residual protein A, DNA) still present after the capture step.

[0192] After the polishing step, an ultrafiltration / diafiltration / ultrafiltration (UF / DF / UF) step may be added to concentrate the polypeptide and exchange the buffer.

[0193] In some embodiments, the polypeptides of the present technology improve product quality after production and further purification (e.g., reduced amount of low molecular weight species and / or reduced amount of high molecular weight species and / or reduced amount of aggregation). In some embodiments, the polypeptides of the present technology have improved long-term stability under storage conditions (e.g., as measured by polypeptide turbidity or opalescence at a concentration of 100 mg / mL).

[0194] The term "biophysical property" of a polypeptide refers to a physical property of a biological process, including, but not limited to, melting point (Tm), aggregation temperature (Tagg), amount of high molecular weight species (HMW), amount of low molecular weight species (LMW), turbidity and / or opalescence, and particle formation. In one aspect, measuring a biophysical property of a "polypeptide" (e.g., by turbidity / opalescence, reversed-phase chromatography, capillary gel electrophoresis, size-exclusion high performance liquid chromatography) refers to measuring a biophysical property of a "population of polypeptides."

[0195] In the context of polypeptides, the terms "stability" and "stable," as used herein, refer to the resistance of a polypeptide to aggregation, the formation of degradation products, and / or the formation of fragmentation products under given transport and / or storage conditions. Alternatively and / or additionally, a "stable" polypeptide retains biological activity under given transport and / or storage conditions. The stability of the polypeptide can be assessed by the extent of aggregation, degradation, and / or fragmentation (e.g., SE-HPLC, RP-(U)HPLC, IEX-HPLC, subvisible particle counting, analytical ultracentrifugation, dynamic light scattering, measuring the OD320 / OD280 ratio, measuring OD500, elastic light scattering, etc.), and / or the % biological activity compared to a reference polypeptide (e.g., as measured by ELISA, Biacore, etc.). For example, the reference polypeptide may be a reference standard frozen at -20°C or below -60°C (such as -80°C) consisting of the same polypeptide at the same concentration and in the same buffer as the stressed sample, but without the application of stress conditions, wherein the reference preparation regularly gives a major peak by SE-HPLC, RP-(U)HPLC and / or IEX-HPLC and / or retains its biological activity in Biacore and / or ELISA.

[0196] In the context of a polypeptide, the term "long-term stability under storage conditions" or "stability under storage conditions," as used herein, refers to the stability of the polypeptide during transport and / or storage at -20°C, below -60°C, or 5°C for periods of 4 weeks, 3 months, 6 months, 1 year, 2 years, 3 years or more.

[0197] In the context of a polypeptide, the term "long-term stability under accelerated or stressed conditions," as used herein, refers to the stability of the polypeptide during transportation and / or storage at 25°C and 40°C for 2 weeks, 4 weeks, 3 months, 6 months or more.

[0198] The term "turbidity" or "opalescence," as used herein, refers to the cloudiness or turbidity of a fluid caused by the intermolecular attraction of many individual particles and / or molecules, which are generally not visible to the naked eye. For example, turbidity or opalescence can be measured at a polypeptide concentration of 100 mg / mL. Turbidity and opalescence can be measured using OD500, and opalescence is visually compared to an opalescence standard. Turbidity can be measured by a nephelometer (e.g., provided by BMG, Labtech, Thermofischer).

[0199] In the context of a polypeptide, the term "low molecular weight species" or "LMW" as used herein refers to a fragment of a polypeptide. For example, LMW species can be measured by prepeak on reversed-phase chromatography (e.g., RP-UPLC) or capillary gel electrophoresis (CGE).

[0200] In the context of a polypeptide, the term "high molecular weight species," "high molecular weight variant," or "HMW," as used herein, refers to an aggregate of a polypeptide that has an apparent molecular weight, relative to molecular weight markers, equal to or greater than the apparent molecular weight observed in size-exclusion high-performance liquid chromatography (SE-HPLC) analysis of a dimer of the polypeptide (e.g., 90 kDa observed for A041600035 SE-HPLC). For example, HMW species can be measured by size-exclusion high-performance liquid chromatography (SE-HPLC).

[0201] In the context of polypeptides, the term "thermostable," as used herein, refers to the ability of a polypeptide to resist irreversible changes in its chemical or physical structure, often by resisting decomposition or polymerization, at high relative temperatures.

[0202] In the context of polypeptides, the term "aggregation," as used herein, refers to the occurrence of high molecular weight aggregates, i.e., aggregates having an apparent molecular weight equal to or higher than that observed in SE-HPLC analysis for a dimer of the polypeptide (e.g., 90 kDa observed for A041600035 SE-HPLC, compared to molecular weight markers). Aggregation can be assessed by various methods known in the art. Non-limiting examples include high performance size exclusion chromatography (SE-HPLC), visible particle counting, analytical ultracentrifugation (AUC), dynamic light scattering (DLS), static light scattering (SLS), elastic light scattering, OD320 / OD280 measurement, OD500 measurement, and turbidimetry.

[0203] In some embodiments, the amount of HMW species after expression of a polypeptide described herein (e.g., as measured by SE-HPLC) is less than 5%. In some embodiments, the amount of HMW species after expression of a polypeptide described herein (e.g., as measured by SE-HPLC) is reduced compared to the amount of HMW species after expression of A041600087 (SEQ ID NO: 83). In some embodiments, the amount of HMW species after expression of a polypeptide described herein (e.g., as measured by SE-HPLC) is reduced compared to the amount of HMW species after expression of A041600085 (SEQ ID NO: 137).

[0204] In some embodiments, the amount of HMW species of a polypeptide described herein after a first purification step by Protein A chromatography (e.g., as measured by SE-HPLC) is less than 5%. In some embodiments, the amount of HMW species of a polypeptide described herein after a first purification step by Protein A chromatography (e.g., as measured by SE-HPLC) is less than the amount of HMW species of A041600087 (SEQ ID NO: 83) after the first purification step by Protein A chromatography. In some embodiments, the amount of HMW species of a polypeptide described herein after a first purification step by Protein A chromatography (e.g., as measured by SE-HPLC) is less than the amount of HMW species of A041600085 (SEQ ID NO: 137) after the first purification step by Protein A chromatography. In some embodiments, the amount of HMW species of a polypeptide described herein after a polishing step (e.g., an ion exchange chromatography step) (e.g., as measured by SE-HPLC) is less than 1%.

[0205] In some embodiments, for a polypeptide described herein, the amount of variants with missing disulfide bridges at the end of the expression process (e.g., as measured by RP-UHPLC) is less than 5%. In some embodiments, the amount of variants with missing disulfide bridges at the end of the expression process (e.g., as measured by RP-UHPLC) is lower for a polypeptide described herein compared to the amount of variants with missing disulfide bridges at the end of the expression process for A041600087 (SEQ ID NO: 83). In some embodiments, the amount of variants with missing disulfide bridges at the end of the expression process (e.g., as measured by RP-UHPLC) is lower for a polypeptide described herein compared to the amount of variants with missing disulfide bridges at the end of the expression process for A041600085 (SEQ ID NO: 137). In one aspect, "amount of variants with missing disulfide bridges" refers to a measurement of the subpopulation of polypeptide variants with missing disulfide bridges compared to the total population of polypeptides.

[0206] In some embodiments, for a polypeptide described herein, the amount of variants with missing disulfide bridges after Cu treatment at the end of the expression process (e.g., as measured by RP-UHPLC) is less than 5%. In some embodiments, the amount of variants with missing disulfide bridges after Cu treatment at the end of the expression process (e.g., as measured by RP-UHPLC) is lower for a polypeptide described herein compared to the amount of variants with missing disulfide bridges at the end of the expression process of A041600087 (SEQ ID NO: 83). In some embodiments, the amount of variants with missing disulfide bridges after Cu treatment at the end of the expression process (e.g., as measured by RP-UHPLC) is lower for a polypeptide described herein compared to the amount of variants with missing disulfide bridges at the end of the expression process of A041600085 (SEQ ID NO: 137).

[0207] In some embodiments, the total % of hexoses on a polypeptide described herein after the expression process (as determined by MS-ID) is less than 15%. In some embodiments, the total % of hexoses on a polypeptide described herein after the expression process (as determined by MS-ID) is lower compared to the total % of hexoses on A041600087 (SEQ ID NO: 83) after the expression process. In some embodiments, the total % of hexoses on a polypeptide described herein after the expression process (as determined by MS-ID) is lower compared to the total % of hexoses on A041600085 (SEQ ID NO: 137) after the expression process.

[0208] In some embodiments, the amount of LMW species after expression of a polypeptide described herein (e.g., as measured by CGE) is less than 5%. In some embodiments, the amount of LMW species after expression of a polypeptide described herein (e.g., as measured by CGE) is less than the amount of LMW species after expression of A041600087 (SEQ ID NO: 83). In some embodiments, the amount of LMW species after expression of a polypeptide described herein (e.g., as measured by CGE) is less than the amount of LMW species after expression of A041600085 (SEQ ID NO: 137).

[0209] In some embodiments, the amount of LMW species of a polypeptide described herein after a first purification step by Protein A chromatography (e.g., as measured by CGE) is less than 5%. In some embodiments, the amount of LMW species of a polypeptide described herein after a first purification step by Protein A chromatography (e.g., as measured by SE-HPLC) is reduced compared to the amount of LMW species of A041600087 (SEQ ID NO: 83) after the first purification step by Protein A chromatography. In some embodiments, the amount of LMW species of a polypeptide described herein after a first purification step by Protein A chromatography (e.g., as measured by CGE) is reduced compared to the amount of LMW species of A041600085 (SEQ ID NO: 137) after the first purification step by Protein A chromatography. In some embodiments, the amount of LMW species of a polypeptide described herein after a polishing step (e.g., an ion exchange chromatography step) (e.g., as measured by CGE) is less than 1%.

[0210] In some embodiments, the downstream (DS) capture recovery rate of the polypeptides described herein is greater than 90%. In some embodiments, the DS capture recovery rate is higher for the polypeptides described herein compared to the DS capture recovery rate of A041600087 (SEQ ID NO: 83). In some embodiments, the DS capture recovery rate is higher for the polypeptides described herein compared to the DS capture recovery rate of A041600085 (SEQ ID NO: 137).

[0211] In some embodiments, downstream (DS) capture binding by the polypeptides described herein is greater than 18 mg / mL. In some embodiments, DS capture binding is greater for the polypeptides described herein compared to DS capture binding by A041600087 (SEQ ID NO: 83). In some embodiments, DS capture binding is greater for the polypeptides described herein compared to DS capture binding by A041600085 (SEQ ID NO: 137).

[0212] In some embodiments, the pH-adjusted recovery after downstream (DS) capture of the polypeptides described herein is greater than 90%. In some embodiments, the pH-adjusted recovery after DS capture is greater for the polypeptides described herein compared to the DS capture pH-adjusted recovery of A041600087 (SEQ ID NO: 83). In some embodiments, the pH-adjusted recovery after DS capture is greater for the polypeptides described herein compared to the DS capture pH-adjusted recovery of A041600085 (SEQ ID NO: 137).

[0213] In some embodiments, the downstream UF / DF / UF recovery of the polypeptides described herein is greater than 90%. In some embodiments, the DS UF / DF / UF recovery is higher for the polypeptides described herein compared to the DS UF / DF / UF recovery of A041600087 (SEQ ID NO: 83). In some embodiments, the DS UF / DF / UF recovery is higher for the polypeptides described herein compared to the DS UF / DF / UF recovery of A041600085 (SEQ ID NO: 137).

[0214] In some embodiments, the turbidity (as measured by OD500) after a polishing step (e.g., an ion exchange chromatography step) was lower for the polypeptides described herein compared to the turbidity of A041600087 (SEQ ID NO: 83). In some embodiments, the turbidity (as measured by OD500) was lower for the polypeptides described herein compared to the turbidity of A041600085 (SEQ ID NO: 137).

[0215] In some embodiments, for the proteins described herein, the amount of host cell protein (HCP) (as measured by immunoenzymatic assay) after a polishing step (e.g., an ion exchange chromatography step) was less than 30 ppm. In some embodiments, the amount of host cell protein (HCP) (as measured by immunoenzymatic assay) was less for the polypeptides described herein compared to the amount of host cell protein (HCP) of A041600087 (SEQ ID NO: 83). In some embodiments, the amount of host cell protein (HCP) (as measured by immunoenzymatic assay) was less for the polypeptides described herein compared to the amount of host cell protein (HCP) of A041600085 (SEQ ID NO: 137).

[0216] 5.6 Composition The present technology also provides a composition comprising at least one polypeptide of the present technology, at least one polynucleotide encoding the polypeptide of the present technology, or at least one vector comprising 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 may also comprise one or more additional pharmaceutically active polypeptides and / or compounds.

[0217] Thus, for pharmaceutical use, the polypeptides of the present technology may be formulated as a pharmaceutical preparation 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. Thus, according to a further aspect, the present technology relates to a pharmaceutical composition or preparation 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.

[0218] 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 the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0219] 5.7 Uses of Polypeptides The polypeptides may be further optimized for in vitro or in vivo administration (e.g., therapeutic uses) that may be assessed by modulation of CX3CR1 activity in cells, tissues, organs, or organisms. For example, the polypeptides may be for use in inhibiting the binding of CX3CR1 to fractalkine in mammalian cells.

[0220] In one aspect, the present technology provides a polypeptide 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 in treating a disease or disorder associated with the fractalkine receptor CX3CR1 (or a single nucleotide polymorphism (SNP) variant of CX3CR1).

[0221] The terms "therapeutic agent," "pharmaceutical agent," and "drug" refer to a compound (e.g., a polypeptide containing an ISVD) used to prevent or treat a disease or disorder or to improve the health of a subject.

[0222] The term "disease" or "disorder" refers to an alteration in a cell, tissue, organ, or organism compared to a normal (healthy) cell, tissue, organ, or organism. In some cases, physiological functions associated with natural organ function, homeostasis, aging, or regeneration may be altered, such as in abnormal organ development, inflammatory diseases, autoimmune diseases, chronic diseases, infectious diseases, or cancer.

[0223] In one aspect, the disease or disorder is associated with a change in the amount or activity of the fractalkine receptor CX3CR1 (or a single nucleotide polymorphism (SNP) variant of CX3CR1) in a cell, tissue, organ or organism, for example CX3CR1. For example, the disease, disorder or condition is selected from inflammatory diseases, cardiac 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-Schönlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft versus host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative and demyelinating disorders, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, and cancer, including ovarian cancer.

[0224] The term "subject" as used herein may refer to any animal, such as a mammal. Among mammals, a distinction can be made between humans and non-human mammals. Non-human mammals may be, for example, companion animals (e.g., dogs, cats), livestock (e.g., bovine, equine, ovine, caprine, or porcine animals), or mammals commonly used for research purposes and / or to produce antibodies (e.g., mice, rats, rabbits, cats, dogs, caprines, ovine, equines, porcines, non-human primates, such as cynomolgus monkeys, or camelids, such as llamas or alpacas). In the context of prophylactic and / or therapeutic purposes, the subject may be any animal, more particularly any mammal, such as a human subject. As will be clear to those skilled in the art, the subject to be treated is in particular one who is suffering from or at risk of suffering from the diseases, disorders, or conditions mentioned herein.

[0225] The term "administered," as used herein, refers to giving a substance (e.g., a composition comprising a polypeptide) to a subject for the diagnosis, treatment, or prevention of disease. Methods of administration include parenteral administration (e.g., intravenous (IV), intraperitoneal, subcutaneous, intramuscular, oral, intraarterial, or intramedullary administration) or oral administration. In some cases, the route of administration may be different or the same for administration of a polypeptide, polynucleotide, or composition. When a polypeptide, polynucleotide, or composition is intended for administration to a subject (e.g., for prophylactic, therapeutic, and / or diagnostic purposes), it may contain immunoglobulin sequences that do not naturally occur in said subject.

[0226] One or more doses may be administered. When two or more doses are administered, the doses may be administered at appropriate intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.

[0227] The term "(pharmaceutically or therapeutically or prophylactically) effective amount or dose" of a substance or composition, as used herein, refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect.

[0228] The polypeptides or polynucleotides of the present technology may be used for the prevention, treatment, amelioration, and / or diagnosis of CX3CR1-related diseases, disorders, or conditions, particularly inflammatory diseases, cardiac and cerebrovascular atherosclerosis, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schönlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative and demyelinating disorders, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, or cancer including ovarian cancer.

[0229] In another aspect, the present technology provides a polypeptide described herein or a pharmaceutical composition comprising said polypeptide for use in the treatment or prevention of atherosclerosis.

[0230] In another aspect, the present technology provides a polypeptide as described herein or a pharmaceutical composition comprising said polypeptide for use in the treatment or prevention of 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.

[0231] In another aspect, the present technology provides a polypeptide as described herein or a pharmaceutical composition comprising said polypeptide for use in the treatment or prevention of atherosclerosis by altering the composition of plaques to reduce the risk of plaque rupture and atherothrombotic events.

[0232] In one aspect, the technology may be used to prevent, treat, mitigate and / or diagnose inflammatory diseases, cardiac and cerebrovascular atherosclerosis, peripheral arterial disease, myocardial infarction, restenosis, diabetic nephropathy.

[0013] Provided are methods of treating or reducing the risk of glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schönlein purpura and Wegener's granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative and demyelinating disorders, multiple sclerosis (MS), Alzheimer's disease, lung diseases such as COPD, asthma, neuroinflammation, neuropathic pain, inflammatory pain, or cancer, including ovarian cancer, in a person suffering from or at risk of said disease or condition, comprising administering to the person a therapeutically effective amount of a polypeptide as described herein, or a pharmaceutical composition comprising said polypeptide.

[0233] In one aspect, the present technology also provides a method of treating or reducing the risk of atherosclerosis in a person suffering from or at risk of said disease or condition, comprising administering to the person a therapeutically effective amount of a polypeptide described herein or a pharmaceutical composition comprising said polypeptide.

[0234] In one aspect, the present technology provides a method of treating or reducing the risk of 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 in a person suffering from or at risk of said disease or condition, comprising administering to the person a therapeutically effective amount of a polypeptide described herein or a pharmaceutical composition comprising said polypeptide.

[0235] In one aspect, the present technology also provides a method of treating or reducing the risk of atherosclerosis by altering the composition of plaque to reduce the risk of plaque rupture and atherothrombotic events in a person suffering from or at risk of said disease or condition, comprising administering to the person a therapeutically effective amount of a polypeptide described herein or a pharmaceutical composition comprising said polypeptide.

[0236] In one aspect, the polypeptides described herein are indicated for use in the treatment or prevention of diseases or conditions in which modulation of activity at the CX3CR1 receptor is desirable. In one aspect, the present technology also provides a method for treating or reducing the risk of a disease or condition in which antagonism of the CX3CR1 receptor is beneficial, comprising administering a polypeptide described herein to a person suffering from or at risk of said disease or condition.

[0237] Prevention is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at high risk of, the disease or condition in question. Persons at risk of developing a particular disease or condition generally include those who have a family history of the disease or condition, or those who have been identified by genetic testing or screening as being particularly likely to develop the disease or condition.

[0238] It will also be apparent to one skilled in the art that the above methods of treating a disease include the preparation of a medicament for the treatment of said disease.

[0239] Furthermore, it is clear that the polypeptides of the present technology can be used as an active ingredient in medicaments or pharmaceutical compositions intended to treat the above-mentioned diseases. Thus, the present technology also relates to the use of the polypeptides of the present technology in the preparation of pharmaceutical compositions for the prevention, treatment, and / or alleviation of any of the above-mentioned diseases, disorders, or conditions. The present technology further relates to polypeptides of the present technology for therapeutic or prophylactic use, particularly for the prevention, treatment, and / or alleviation of any of the above-mentioned diseases, disorders, or conditions. The present technology further relates to pharmaceutical compositions for use in the prevention, treatment, and / or alleviation of the above-mentioned diseases, disorders, or conditions, comprising at least one polypeptide of the present technology.

[0240] The polypeptides of the present technology and / or compositions comprising same can be administered to a patient in need thereof in any suitable manner, depending on the particular pharmaceutical formulation or composition used.

[0241] The polypeptides and / or compositions comprising the polypeptides described herein are administered according to a treatment regimen suitable for preventing, treating, and / or alleviating the disease, disorder, or condition to be prevented, treated, or alleviated. A clinician can generally determine a suitable treatment regimen depending on factors such as the disease, disorder, or condition to be prevented, treated, or alleviated, the severity of the disease, the severity of its symptoms, the particular polypeptide of the present technology used, the particular route of administration and pharmaceutical formulation or composition used, the age, sex, weight, diet, general condition of the patient, and similar factors well known to clinicians. Generally, a treatment regimen involves the administration of an effective amount or dose of one or more polypeptides of the present technology, or one or more compositions comprising same.

[0242] The efficacy of the polypeptides of the present technology and compositions comprising same can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se, or any combination thereof, depending on the particular disease involved. Suitable assays and animal models will be apparent to those skilled in the art.

[0243] The term "half-life" as used herein refers to the time required for the serum concentration of a polypeptide to decrease by 50% in vivo, for example, due to degradation of the polypeptide and / or clearance or sequestration of the polypeptide by natural mechanisms. The in vivo half-life of a polypeptide can be determined by any method known per se, such as pharmacokinetic analysis. Half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta, and area under the curve (AUC). Reference is made to standard handbooks, 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 Rev. edition). The terms "increased half-life" or "increased half-life" refer to an increase in t1 / 2-beta, either with or without an increase in t1 / 2-alpha and / or AUC, or both.

[0244] In some embodiments, the polypeptides of the present technology exhibit a half-life (e.g., blood half-life) in the treated subject that is sufficiently long so that successive treatments can be conveniently spaced.

[0245] In one aspect, the polypeptides described herein, when administered to a mammal, such as a cynomolgus monkey or a human, are characterized by a pharmacokinetic (PK) profile that differs from the PK profile observed for a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83 or SEQ ID NO: 137. In particular, the polypeptides may exhibit longer serum persistence (i.e., longer half-life) compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83. This effect may be particularly pronounced at low doses (Figure 18). In one aspect, the PK profile of the polypeptides described herein does not exhibit target-mediated drug deposition (TMDD). In one aspect, the polypeptides described herein, when administered to a mammal, such as a cynomolgus monkey or a human, are characterized by a PK profile with reduced target-mediated drug deposition (TMDD) compared to a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83. The lack of TMDD provides improved selectivity and, consequently, a better safety profile for the polypeptides described herein.

[0246] In one aspect, when a polypeptide described herein is compared to a reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83, the polypeptide has a higher exposure in a subject over a longer period of time after administration of a similar effective dose of the polypeptide (e.g., 0.1 mg / kg or 1 mg / kg). In one aspect, the polypeptide has the same exposure as the reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83 after administration of a lower dose compared to said reference polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 83.

[0247] According to yet another embodiment, there is provided a method for diagnosing a disease, disorder, or condition mediated by CX3CR1 dysfunction, comprising: a) obtaining a sample from a subject; b) contacting the sample in vitro with a polypeptide of the present technology as defined above; c) detecting binding of said polypeptide to said sample; d) comparing the binding detected in step (c) with a standard, wherein a difference in binding compared to the sample is diagnostic of a disease, disorder, or condition characterized by CX3CR1 dysfunction; and A method is provided, comprising:

[0248] According to another embodiment, there is provided a method of diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction, comprising: a) obtaining a sample from a subject; b) contacting the sample with a polypeptide of the present technology as defined above; c) determining the amount of CX3CR1 in the sample; d) comparing the amount determined in step (c) with a standard, wherein a difference in the amount compared to the sample is diagnostic of a disease, disorder, or condition characterized by CX3CR1 dysfunction; and A method is provided, comprising:

[0249] The above diagnostic methods can also be used to monitor the effectiveness of a subject's therapeutic treatment.

[0250] According to another embodiment, a kit for diagnosing diseases, disorders or conditions mediated by CX3CR1 dysfunction and / or for monitoring the effectiveness of therapeutic treatment is provided, and the kit is used in the above-defined method.Such kit may comprise at least one polypeptide, polynucleotide or composition of the present technology, and optionally one or more culture 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.

[0251] The present technology further provides kits comprising at least one polypeptide, polynucleotide, or composition of the present technology, and one or more other components selected from the group consisting of other drugs used in the treatment of the above diseases and disorders, and the above devices. [Example]

[0252] Hereinafter, the present technology will be specifically described in more detail with reference to examples, but is not intended to limit the scope of the present technology.

[0253] Example 1: Sequence optimization of monovalent anti-CX3CR1 ISVD The anti-CX3CR1 ISVD components 66B02 (SEQ ID NO: 4) and 54A12 (SEQ ID NO: 1) are described in WO 2013 / 130381 (SEQ ID NOs: 1 and 2 in WO 2013 / 130381). These ISVD components were sequence-optimized to 66B02_SO (SEQ ID NO: 5) and 54A12_SO (SEQ ID NO: 2) to make them more human-like (humanized), reduce their binding by existing antibodies, reduce antigenicity (removal of T cell epitopes), and remove post-translational modification (PTM) sites. An alignment of the ISVD sequences described in WO 2013 / 130381 and exemplary ISVDs sequence-optimized using the present technology is shown in Figure 1.

[0254] Example 2: Characterization of sequence-optimized monovalent ISVDs The sequence-optimized ISVD variants 66B02_SO (SEQ ID NO: 5) and 54A12_SO (SEQ ID NO: 2) were characterized for biophysical properties and potency.

[0255] 2.1 Thermal shift assay Thermal shift assays (TSA) were performed in 96-well plates on a qPCR machine (LightCycler 480II, Roche). One ISVD protein per row was analyzed over the following pH ranges: 4, 5, 6, 7, 8, and 9.

[0256] Per well, 5 μL of ISVD protein sample (0.8 mg / mL in D-PBS) was added to 5 μL of Sypro Orange (40x in MilliQ water; Invitrogen, catalog no. S6551) and 10 μL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate, and 115 mM NaCl, pH range 4–9). A temperature gradient (37–99°C at a rate of 0.03°C / s) was applied, which induced unfolding of the ISVD protein and thus exposure of hydrophobic patches. Binding of Sypro Orange to these hydrophobic patches caused an increase in fluorescence intensity, which was measured (Ex / Em = 465 / 580 nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 served as a measure of the melting temperature (Tm).

[0257] The Tm (°C) values obtained for the sequence-optimized variants are shown in Table B-1. All ISVDs showed acceptable Tm values.

[0258] 2.2 Oligomerization assay The oligomerization tendency of monovalent ISVD proteins under stress conditions (45°C for 1 week) was investigated by analytical size-exclusion chromatography (SE-HPLC). For this purpose, 3xFLAG-His6-tagged ISVD proteins produced in E. coli were purified by IMAC followed by preparative SEC, filtered (0.22 μm), and used at a concentration of 1 mg / mL (PBS). SE-HPLC profiles of two 100 μL aliquots were compared: one sample was incubated at -20°C (TO) for 1 week, and the other at 45°C for 1 week. The samples were clarified by centrifugation at 20,000 RCF for 5 min and then 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 difference in relative pre-peak area between stressed (+45°C) and unstressed (-20°C) samples was calculated and reported as Δ%HMW (=%HMW 1W 45°C - %HMW T0).

[0259] The Δ% of HMW values obtained for the sequence-optimized variants are shown in Table B-1. The % of oligomerization was acceptable for all ISVDs.

[0260] 2.3 Initiation of aggregation temperature determination (Tagg assay) The temperature at which ISVD proteins begin to aggregate (= aggregation onset temperature = Tagg) was determined by dynamic light scattering (DLS) using a DynaPro plate reader (Wyatt). For this purpose, ISVD proteins bearing a 3xFLAG-His6 tag produced in E. coli were purified by IMAC followed by preparative SEC, filtered (0.22 μm), and used at a concentration of 1 mg / mL (D-PBS). After thawing, the samples were filtered through a 0.1 μm membrane and centrifuged at 14,000 rpm for 5 min. A 30 μL sample (four replicates) was heated from 40 to 80 °C at a constant rate of 0.25 °C / min with continuous recording of light scattering intensity. The hydrodynamic radius derived from the measured intensity was plotted against temperature to determine the temperature at which the radius began to increase (= Tagg, °C).

[0261] The Tagg values obtained for the sequence-optimized variants are shown in Table B-1. All ISVDs had acceptable Tagg values.

[0262] 2.4 Inhibition of CX3CL1 / Fraktalkine Ligand Binding to Human and Cyno CX3CR1 The ability of purified ISVD proteins to inhibit the interaction between human CX3CL1 / Fraktalkine expressed on the cell surface of CHO K1 cells and human and cynomolgus monkey (cyno) CX3CR1 was assessed by flow cytometry. CHO cells overexpressing human or cyno 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 into 384-well Bio-One V-bottom plates (Greiner, catalog number 781280) with a total of 2E04 cells seeded per well. After washing, ISVD protein (250 nM in assay buffer, 3-fold dilutions, starting at 11 points) was premixed with 30 μM HSA (Sigma-Aldrich, catalog no. A8763) and AF647-labeled human CX3CL1 (R&D Systems, catalog no. 365-FR; labeled in-house) at a final concentration of 2E-10 M (approximately EC30) and added to the cells for 4 h at 4 °C. The plates were washed three times, and the cells were resuspended in PI (Miltenyi Biotec, catalog no. 130-093-233, diluted 1:1000 in assay buffer). The cell suspension was analyzed using iQue Screener PLUS or iQue 3 (Intellicyt). IC50s were estimated by dose-response modeling. Curves were fitted using four-parameter logistic regression in GraphPad (GraphPad Software Inc.).

[0263] The IC50 values obtained for the sequence-optimized variants are shown in Table B-1. The sequence-optimized components retained acceptable Tm values for use in further development.

[0264] [Table 7]

[0265] Example 3: Preparation of multivalent ISVD constructs A panel of 24 formats was created containing sequence-optimized anti-CX3CR1 ISVD components, 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 component (ALB23002; SEQ ID NO: 37). The components were fused head-to-tail with a 9GS (SEQ ID NO: 69), 20GS (SEQ ID NO: 73), or 35GS (SEQ ID NO: 76) linker (Tables A-7). In this panel, ALB23002 was located at the second or third position. All 24 formats also had an alanine (A) introduced at the C-terminus. Two formats (A041600085 and A041600087) were generated using building blocks 307 (SEQ ID NO: 7) (building block 307 is SEQ ID NO: 223 in WO 2013 / 130381), 307(D1E) (SEQ ID NO: 8), and ALB23002 (SEQ ID NO: 37) or ALB11 (SEQ ID NO: 42), respectively. In A041600085, a C-terminal alanine (A) was introduced (see Table B-2).

[0266] [Table 8]

[0267] Example 4: Characterization of multivalent ISVD constructs by expression profiling Twenty-four different polyvalent ISVD constructs were characterized for their expression levels in P. pastoris (Komagataella phaffii). Briefly, different ISVD formats were transformed into P. pastoris, strain NRRLY-11430 (ATC76273), and plated on YPDS plates (2% peptone (w / v), 1% yeast extract (w / v), 18.2% sorbitol (w / v), 1.5% select agar (w / v), 2% glucose (w / v)) containing 1000 μg / ml Zeocin®. After agar selection, 20 clones per polyvalent ISVD construct were picked and used for expression profiling in 96-well deep-well plates. For this purpose, Pichia clones were cultured in 100 μL of BGCM medium (10% citric acid, 1 M NaCl, 100 μL HCl) in a master 96-well deep-well plate. The cultures were inoculated into a pH 6.0 medium containing 0.1% glycerol (v / v), 13.4 g / L yeast nitrogen base (no amino acids) (w / v), 0.00004% (w / v) biotin, 2.6% peptone (w / v), and 1.4% yeast extract (w / v). After 24 hours of incubation (30°C, 200 rpm), 5 μL of the preculture was used to inoculate 100 μL of BGCM medium into a second 96-well deep-well plate. After 24 hours of growth, the cultures in the expression plate were induced by adding 75 μL of MeOH (0.66% v / v) to each well. An additional 75 μL of MeOH (0.66% v / v) was added to each culture to maintain induction throughout the fermentation. After 24 hours of induction, cells were harvested and the supernatants were analyzed. The amount of protein expressed in the medium was measured using the ProtA biosensor with Octet. After normalization to the reference, relative normalized expression was plotted for each clone within each format to determine the average normalized expression per format. Formats were then ordered according to their ISVD components and linker length.

[0268] An overview of the expression profiling can be found in Figure 2. Within each format, the expression levels of the clones showed considerable variation. Based on the normalized average expression (Table B-3), several trends were identified. For formats containing the 54A12_SO component, higher expression was obtained compared to formats containing the 66B02_SO component. Higher titers could be obtained when the ALB32002 component was located at the C-terminus. As a linker between the first and second components, the 9GS linker was preferred over the 20GS and 35GS linkers. As a linker between the second and third components, the 20GS linker was preferred over the 9GS and 35GS linkers.

[0269] [Table 9]

[0270] [Table 10]

[0271] Based on the ranking obtained after expression profiling, seven formats were evaluated in fed-batch fermentation. Most formats expressed >2 g / L. Ten ISVD formats were selected (out of 24 formats) for further characterization (see Table B-4).

[0272] Formats A041600085 and A041600087 were not included in the expression profiling experiment but were included for further characterization.

[0273] Example 5: In vitro characterization of multivalent ISVD constructs 5.1 Characterization of multivalent ISVD constructs in CX3CR1 binding assays Selected ISVD formats and A041600085 were characterized for binding to CHO K1 human and cyno CX3CR1 cells using flow cytometry in the presence of 30 μM HSA and compared to A041600087 (SEQ ID NO: 83).

[0274] CHO cells overexpressing human or cynomolgus monkey CX3CR1 were generated using techniques known in the art. Cells were harvested, resuspended in assay buffer (PBS, 2% FBS, 0.05% NaN3), and seeded separately into 384-well Bio-One V-bottom plates (Greiner, catalog no. 781280) with a total of 2E04 cells seeded per well. After washing, serial dilutions of multivalent ISVD constructs (starting at 250 nM, 11-fold, 3-fold dilutions in assay buffer) were added and incubated for 4 hours at 4°C. Binding was performed in the presence of 30 μM HSA (Sigma-Aldrich, catalog no. A8763). Plates were then washed three times, and cells were incubated with anti-VHH mouse antibody (diluted 1:3300 in assay buffer) for 30 minutes at 4°C. The plates were then washed three times, and the cells were incubated in goat anti-mouse Fc APC (Jackson ImmunoResearch, Cat. No. 115-135-164 / 115-116-071) (1:100 dilution in assay buffer) for 30 minutes at 4°C. Finally, the plates were washed three times, and the cells were resuspended in PI (Miltenyi Biotec, Cat. No. 130-093-233, 1:1000 dilution in assay buffer). The cell suspension was analyzed using iQue Screener PLUS (Intellicyt). EC50 was estimated by dose-response modeling. Curves were fitted using four-parameter logistic regression in GraphPad (GraphPad Software Inc.). Purified anti-human CX3CR1 antibody [K0124E1] (BioLegend, Cat. No. 355701 / 355702) was used as a positive control to detect CX3CR1 expression on the cell surface.

[0275] The values obtained are reported in Table B-4. All formats showed binding to human CX3CR1 with an EC50 between 0.1 and 0.6 nM and cross-reactivity with cyno CX3CR1 (up to a 3.3-fold difference in EC50). Binding curves for selected formats are shown in Figure 3.

[0276] [Table 11]

[0277] 5.2 Characterization of multivalent ISVD constructs in the CX3CL1 / fractalkine ligand competition assay Ten selected multivalent ISVD constructs, formats A041600085 and A041600087 and "KAND-567", were further characterized in competition assays with AF-647-labeled soluble CX3CL1 / Fraktalkine ligand at an EC30 concentration in the presence of 30 μM HSA, as described in Example 2.4.

[0278] The values obtained are reported in Table B-5. All formats showed very similar IC50 values ranging from 0.7 to 7.7 nM, with up to an 8-fold difference for cyno CX3CR1. Competition curves for five selected multivalent ISVD constructs are shown in Figure 4.

[0279] [Table 12]

[0280] 5.3 Characterization of multivalent ISVD constructs for inhibition of CX3CL1 / Fraktalkine-induced chemotaxis of BA / F3 cells expressing human CX3CR1 Ten selected multivalent ISVD constructs, formats A041600085 and A041600087, and "KAND-567" were also tested in a chemotaxis assay in which Ba / F3-huCX3CR1 cells were stimulated with soluble CX3CL1 / Fraktalkine in the presence of 30 μM HSA. Ba / F3 cells overexpressing human CX3CR1 were generated using techniques known in the art. The ability of the purified ISVD constructs to inhibit CX3CL1-induced migration of BA / F3 cells expressing human CX3CR1 was evaluated in a Boyden chamber-based method using NeuroProbe Chemotax plates (catalog number 106 / 5). Briefly, the bottom wells of the chambers were filled with 0.5, 1, or 5 nM human CX3CL1 (R&D Systems, catalog number 365-FR) in 30 μL of assay buffer (RPMI medium containing 30 μM HSA (Sigma, catalog number A8763)). BA / F3 cells were preincubated with the ISVD construct or inhibitor in RPMI medium containing 30 μM HSA (Sigma, catalog number SLCD9951) for 30 min at 37 °C. Next, 50 μL of the ISVD construct or inhibitor / cell suspension (containing 1E05 cells) mix was added to the membrane, and the plate was incubated for 3 h at 37 °C with 5% CO2. Migrated cells were quantified using CellTiter Glo, which measures cellular ATP content. For this purpose, 30 μL of the remaining cell suspension was transferred to a white Costar 96-well plate (#3917), and the wells were washed with 20 μL of assay medium. Next, 50 μL of CellTiter Glo reagent (Promega, Cat. No. G7571) was added to each well, and the contents were mixed on an orbital shaker at 1000 RPM for 5 minutes to induce cell lysis. After a 10-minute incubation at RT, the signal was measured on an Envision 2 (RLU-1 sec).

[0281] All multivalent ISVD constructs tested blocked huCX3CL1-induced chemotaxis of Ba / F3-huCX3CR1 cells with IC50s ranging from 1.6 to 3.6 nM (using 1 nM CX3CL1). Assays were performed multiple times, and the geometric means of the different IC50s are shown in Table B-6. A graph of chemotaxis inhibition for five multivalent ISVD constructs selected for further characterization is shown in Figure 5 and compared with A041600087.

[0282] [Table 13]

[0283] Example 6: In vitro characterization of selected multivalent ISVD constructs Based on potency, high titer, biophysical properties and component diversity, multivalent ISVD constructs A041600025, A041600034, A041600041, A041600035 and A041600085 were selected for further characterization.

[0284] 6.1 Characterization of multivalent ISVD constructs for binding to different huCX3CR1 variants Two significant SNPs have been described in huCX3CR1, namely V249I (Rs3732379) and T280M (Rs3732378), which may affect receptor activity and binding by the ISVD. Both SNPs are prevalent in the global population. Different combinations of these SNPs were introduced into human CX3CR1, and binding by five selected multivalent ISVD constructs was assessed by flow cytometry after transient transfection of the huCX3CR1 variants in HEK293T cells.

[0285] Briefly, the CX3CR1 construct was cloned into pcDNA3.1 (ThermoFisher Scientific, Catalog No. V79020), and plasmid DNA was prepared from Escherichia coli TOP10 cells. HEK293T cells were seeded at a density of 1.5E06 cells per T75 flask and incubated overnight at 37°C in DMEM medium (Gibco, Catalog No. 31966) supplemented with 10% FBS (Sigma, Catalog No. F7524). The medium was then replaced with Opti-MEM medium (Gibco, Catalog No. 31985). 9 μg of plasmid DNA in a final volume of 1 mL of Opti-MEM was incubated at room temperature for 15 minutes and then added to the cells. After 3 hours of incubation at 37°C, 10 mL of DMEM supplemented with 20% FBS was added, and incubation was continued. After 48 hours, the cells were washed with PBS and resuspended with 4 mL of trypsin-EDTA (Gibco, Cat. No. 25200-056), followed by the addition of 6 mL of DMEM medium supplemented with 10% FBS. Binding assays were performed as described in Example 5.1. In addition to ISVD binding, binding of AF-647-labeled soluble CX3CL1 / Fraktalkine ligand to different receptor variants was also included as a positive control for CX3CR1 expression. The tested multivalent ISVD constructs showed similar binding to the different huCX3CR1 variants and did not bind to parental HEK293T cells (Table B-7 and Figure 6).

[0286] [Table 14]

[0287] 6.2 Binding by existing antibodies The five selected multivalent ISVD constructs were tested for binding by pre-existing antibodies present in 96 serum samples from healthy volunteers using ProteOn XPR36 (Bio-Rad Laboratories, Inc.) and compared to A041600087.

[0288] The results show that binding by existing antibodies is very low for the five multivalent ISVD constructs.

[0289] 6.3 Specificity of multivalent ISVD constructs 6.3.1 Specificity for CCR2 and CCR5 The specificity of the five selected multivalent ISVD constructs for the huCX3CR1 receptor was evaluated by performing 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. Binding of the multivalent ISVD constructs to huCR2 or huCCR5 expressed on the cell surface was examined by flow cytometry as described in Example 5.1.

[0290] No binding to huCCR2 or huCCR5 was detectable. Binding curves are shown in Figures 7 and 8. Purified anti-human CCR2 [K036C2] (BioLegend, catalog no. 357201 / 357202) or anti-human CCR5 [3A9] (BD Biosciences catalog no. 556041) were used as positive controls to detect CCR2 or CCR5 expression on the cell surface.

[0291] 6.3.2 Specificity in human membrane protein arrays To profile specificity and target selectivity, the binding of A041600035 and A041600087 was assessed on a human membrane protein array (MPA).

[0292] MPA is a protein library composed of 6,000 distinct human membrane protein clones, each overexpressed in live cells from an expression plasmid. Each clone was individually transfected into 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 were arrayed in duplicate in a matrix format for high-throughput screening.

[0293] Prior to screening with MPA, the optimal A041600035 and A041600087 concentration of 20 μg / mL for screening was determined on HEK-293T cells expressing positive (CX3CR1 or membrane-tethered protein A) and negative (mock-transfected) binding controls, followed by detection by flow cytometry using a fluorescently labeled secondary antibody that binds to the ISVD.

[0294] A041600035 and A041600087 ligands were added to the MPA at a predetermined concentration of 20 μg / mL, and binding of the entire protein library was measured on the Intellicyt iQue using fluorescently labeled secondary antibodies on unfixed cells. Each array plate contained both positive (Fc binding) and negative (empty vector) controls to ensure plate-to-plate reproducibility. Nonspecific fluorescence was determined to be any value less than three standard deviations below the mean background value.

[0295] Test ligand interactions with any targets identified by MPA screening were confirmed in a second flow cytometry experiment using serial dilutions of the test antibody in buffer containing 10% human serum albumin, and target identity was re-verified by sequencing. The dose response at the two highest concentrations tested and validated targets showing an MFI ≥2-fold above background are shown in Figure 17A for A041600035 and Figure 17B for A041600087.

[0296] For A041600035, specific binding to CX3CR1 was observed, but binding to other human membrane proteins was not observed. Specific binding to CX3CR1 and MCAM (alternative name: MUC18; https: / / www.uniprot.org / uniprot / P43121 ) was observed for A041600087.

[0297] Example 7: Manufacturability assessment of selected multivalent ISVD constructs Based on potency, high titer, biophysical properties and component diversity, multivalent ISVD constructs A041600085, A041600025, A041600034, A041600041 and A041600035 were selected for further manufacturability evaluation.

[0298] Manufacturability studies focused on product potency and purity during upstream processing (USP) and post-downstream processing (DSP) purification recovery, purity, and DSP platform suitability.

[0299] 7.1. Product Potency and Purity During Upstream Processing (USP) The titer was determined in the supernatant and cell broth at the end of the fermentation.

[0300] Other criteria measured were related to product purity and were assessed using different methods. ISVD-associated HMW species at the end of fermentation were quantified as the sum of the relative areas of the pre-peaks in the SE-HPLC chromatogram after Protein A clarification of the fermentation sample. At the start of process development, less than 5% HMW species are preferred.

[0301] The area after the peak in 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 exceptionally low molecular weight variants, are also observed as post-peaks, but their identification requires additional characterization (e.g., LC-MS). Therefore, the area after the peak is considered to represent only variants with missing disulfide bridges. Preferably, the amount of variants with missing disulfide bridges after copper treatment (WO 2010 / 125187) is less than 5%.

[0302] There is no numerical score or threshold for the SDS-PAGE banding pattern. The major band should be clearly visible at the expected molecular weight, and there should be no or only limited amounts of degradation observed on the SDS-PAGE gel. The degree of O-glycosylation of the format was determined by MS-ID. Ideally, the total % of hexoses is less than 15%.

[0303] The USP manufacturability of exemplary multivalent ISVD constructs is shown in Table B-8. A041600025 demonstrated very good titer and product quality at the end of fermentation. Most USP manufacturability criteria achieved high quality scores, except for LMW species and the post-peak percentage of RP-UHPLC before copper treatment (just above the threshold).

[0304] A041600034 exhibited lower titers and poor product quality at the end of fermentation. A high percentage of LMW species was observed, with post-peak percentages on RP-UHPLC before and after copper treatment (aCu) just above the threshold. Lower titers can negatively impact development timelines, fermentation yields, and cost of goods.

[0305] A041600035 had the second highest titer and the best quality at the end of fermentation. Apart from the high percentage of LMW, the product quality was very good.

[0306] A041600041 showed good potency. Only one USP manufacturability criterion had a high quality score (percent post peak on RP-UHPLC after copper treatment).

[0307] A041600085 showed good potency. Only one USP manufacturability criterion had a high quality score (percent post peak on RP-UHPLC after copper treatment).

[0308] [Table 15]

[0309] 7.2 Downstream Processing (DSP) Manufacturability Assessment The DSP manufacturability assessment aims to evaluate the suitability of the multivalent ISVD construct for purification. Qualitative and quantitative assessments, 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 preservation of molecular integrity during UF / DF / UF, were performed during larger-scale purification.

[0310] The results of the DSP manufacturability assessment are summarized in Table B-9. The target capture resin binding capacity was set to "greater than 18 mg / mL" to account for the trivalent format of the multivalent ISVD constructs. Of the five multivalent ISVD constructs tested, two candidates, A041600085 and A041600041, had binding capacities to the capture resin below this predetermined limit. This lower capture resin binding capacity resulted in lower recoveries during capture for these two multivalent ISVD constructs at a resin load of 20 mg ISVD / mL. While limited protein loss was measured during pH adjustment for A041600035, A041600025, and A041600041, a more significant decrease was observed for A041600085 and A041600034, with recoveries below 95%. Finally, all five multivalent ISVD constructs had high recovery rates during the final UF / DF / UF step without affecting their molecular integrity.

[0311] [Table 16]

[0312] DSP manufacturability assessment of 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-adjusted recovery (>95%), UF / DF / UF recovery (>85%), and preservation of molecular integrity during UF / DF / UF (Table B-9).

[0313] 7.3 Molecular Profiling of Exemplary Multivalent ISVD Constructs Molecular profiling was assessed for the five selected multivalent ISVD constructs to identify and characterize the biochemical properties and structure-related responsibilities of the five selected multivalent ISVD constructs.

[0314] 7.3.1 Determination of high molecular weight products The levels of high molecular weight products (HMW) were determined by size exclusion high performance liquid chromatography (SE-HPLC), and the results are shown in Figure 9.

[0315] The total HMW after capture was low for A041600034 (1.5% HMW) compared to the other four candidates. However, HMW decreased to less than 1% after polishing chromatography for all five selected multivalent ISVD constructs. Additionally, this level of HMW remained stable at less than 1% during UF / DF / UF and through the final formulation steps.

[0316] 7.3.2 Determination of low molecular weight products The levels of low molecular weight products (LMW) were determined by capillary gel electrophoresis (CGE). The polishing step by cation exchange chromatography (CEX) significantly reduced the low molecular weight products (LMW), as shown in Figure 10, but not to different levels. A041600041 did not decrease below 1% after the polishing step. The levels of LMW remain stable during UF / DF / UF until the final step of formulation in all five multivalent ISVD constructs.

[0317] 7.3.3 Visual Inspection - Turbidity Visual inspection was performed by directly observing the bulk final product (Figure 11). Results were classified as "clear," "slightly cloudy," or "turbid." At the intermediate concentration of 50 mg / mL, all multivalent ISVD constructs tested were "clear." At the target concentration of 100 mg / mL, four of the five multivalent ISVD constructs were found to be "slightly cloudy," with significant turbidity and / or opalescence observed for A041600025, which was therefore classified as "turbid."

[0318] Further analysis of this turbidity and / or opalescence was performed using OD500 (Figure 12) and by comparison to a turbidity standard curve, both of which confirm direct observation of the sample. While A041600025 is confirmed as "turbid," the other multivalent ISVD constructs are considered "slightly turbid" at 100 mg / mL. Additionally, the final products of the five multivalent ISVD constructs were found to be slightly colored (yellow) and free of particles by direct visualization.

[0319] 7.3.4 Host Cell Protein Measurement Host cell proteins (HCPs) were measured by immunoenzymatic assay of five multivalent ISVD constructs after different DSP steps. The HCP content in A04160035 was less than 30 ppm. The four other multivalent ISVD constructs had HCPs in the range of 30-60 ppm.

[0320] 7.3.5 Molecular Profiling Conclusions Table B-10 summarizes the results of the molecular profiling data.

[0321] [Table 17]

[0322] Example 8: Further characterization of selected multivalent ISVD constructs 8.1 Characterization of multivalent ISVD constructs to block hu CX3CL1-induced ERK phosphorylation in BA / F3 cells expressing human CX3CR1 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 evaluated in an HTRF-based assay using the Advanced phospho-ERK (Thr202 / Tyr204) cell kit from Cisbio Bioassays (catalog 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 for 10 minutes at room temperature. The plate was centrifuged at 300 g for 2 minutes, and the assay medium was removed by inverting 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 minutes with shaking at 900 RPM, followed by an additional 40 minutes of incubation at room temperature on the bench. Prior to reading, the plate was shaken again at 900 RPM for 30 seconds, and 16 μL of the lysate (without pipetting up and down) was transferred to an HTRF-compatible 384-well plate (Perkin Elmer; ProxiPlate-384 Plus, White 384 Shallow-Well Microplate; #6008280). The donor and acceptor antibodies were mixed in a 1:1 ratio in detection buffer, and 4 μL of this mixture was added to each well and incubated for 3 hours in the dark at room temperature. Signals were determined using a TECAN F200 and ratios were determined by dividing the TRF signal at 665 nm by the TRF signal at 620 nm and multiplying by 1E04.

[0323] The obtained values are reported in Table B-11. The multivalent ISVD constructs block huCX3CL1-induced ERK phosphorylation in cells expressing human CX3CR1 with IC50s ranging from 2.7 nM to 5.4 nM (see Figure 13). The assay was performed multiple times and the geometric means of the different IC50s are shown in Table B-11.

[0324] [Table 18]

[0325] 8.2 Characterization of Multivalent ISVD A041600035 to Block Human or Mouse CX3CL1-Induced ERK Phosphorylation in CHO K1 Cells Expressing Human CX3CR1 The pERK assay was repeated for A041600035 using CHO K1 cells expressing human CX3CR1 and stimulated with human or mouse CX3CL1. A surrogate antibody recognizing mouse CX3CR1 (AB5715, Biolegend catalog no. 149002) was used as a positive control to block CX3CL1-induced ERK phosphorylation in CHOK1 mouse CX3CR1 cells.

[0326] A041600035 can equally block the activity of both ligands on human CX3CR1 with an IC50 of 6 nM (see Table B-12 and Figure 14). These experiments also show that mouse CX3CL1 is approximately 10-40 times less potent at inducing ERK phosphorylation compared to human CX3CL1 (both human CX3CR1 and mouse CX3CR1). A surrogate antibody recognizing mouse CX3CR1 (AB5715, Biolegend catalog number 149002) blocked CX3CL1-induced ERK phosphorylation with an IC50 of 0.8-1.6 nM in CHOK1 mouse CX3CR1 cells (see Table B-12 and Figure 14).

[0327] [Table 19]

[0328] 8.3 Affinity Determination The solution affinity of A041600035 for human and cyno CX3CR1 cells was determined in the presence of 30 μM HSA via the solution equilibrium kinetic exclusion KD measurement method MSD-SET (Meso Scale Discovery - Solution Equilibrium Titration). Affinity was determined on three independent assays.

[0329] The geometric means of three independent experiments are shown in Table B-13. There is no difference in affinity between human CX3CR1 and cyno CX3CR1.

[0330] [Table 20]

[0331] 8.4 Characterization of A041600035 in blocking fractalkine-mediated monocyte chemotaxis from healthy and lupus nephritis patients. Pan monocytes from a healthy donor (HemaCare) were thawed at 37°C, washed with RPMI containing 10% FBS, and resuspended in RPMI 1640 containing 10% FBS and 25 μg / mL DNAse I (Sigma-Aldrich) at 37°C and 5% CO for 1 hour. Monocytes were spun down and stained with anti-human CD14-APC (Biolegend) and anti-human CD16-PE (Biolegend) at a 1:100 dilution in RPMI 1640 containing 10% FBS for 10 minutes at room temperature. Monocytes were then washed and resuspended in RPMI 1640 containing 1% FBS at a concentration of 5 x 10E6 / mL. The monocyte suspension was incubated with either A041600035, A041600087, or a negative control (IRR00163; see VHH) or different concentrations of medium in a 1:1 volume ratio at room temperature for 10 minutes. Full-length recombinant fractalkine (R&D systems) was diluted to 50 ng / mL in RPMI 1640 containing 1% FBS, and 500 μL of the diluted fractalkine was added to the lower chamber of a transwell (24-well, Corning). Then, 100 μL of the monocyte suspension was gently added to the upper chamber of the transwell. The transwell plate was placed in an incubator at 37°C and 5% CO2 for 3 hours. The upper chamber was then removed, and the lower chamber was gently mixed by pipetting. Flow cytometry analysis was performed to count monocyte subsets (classical: CD14+CD16-, intermediate: CD14-CD16+, and non-classical: CD14-CD16+) in 100 μL of suspension from the lower chamber. An arbitrary chemotaxis index was expressed as the fold change relative to the medium control wells: (number of cells) / (number of cells in the medium control) (Table B-14; Figure 15).

[0332] [Table 21]

[0333] Blood samples from patients with lupus nephritis (LN disease classes 2–5) were provided by Dr. Ian R. Rifkin at Boston Medical Center, MA. PBMCs were isolated from fresh blood using SepMate (Stemcell), and pan-monocytes were enriched using a pan-monocyte enrichment kit (Miltenyi Biotech). Isolated monocytes were used in triplicate transwell assays, and arbitrary chemotaxis indices were calculated as described above (Table B-15 and Figure 16).

[0334] [Table 22]

[0335] Example 9: Evaluation of selected multivalent ISVD constructs in a non-accelerated model of NTS-induced glomerulonephritis in knock-in hCX3CR1 transgenic mice 9.1 Nephrotoxic Nephritis (NTN) Model The non-accelerated NTN model in mice has been described in the literature as a model of acute glomerulonephritis in which nephritis is initiated by administration of anti-glomerular serum (i.e., nephrotoxic antiserum [NTS]). NTS binds to and deposits in the glomeruli of the kidney, impairing the glomerular filtration barrier and resulting in proteinuria and inflammation (Ougaard et al. 2018, Int. J. Nephrol. 2018:8424502:1-12). Given the lack of mouse cross-reactivity of multivalent ISVD constructs, 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 an observation period of 7 days (Example 9.4) or 21 days (Example 9.5) after NTS administration.

[0336] 9.2 Immunohistochemical procedures Tissue injury mediated by glomerular infiltrating cells is a key early pathogenic event expected to be detectable in this model (Ougaard et al. 2018, Int. J. Nephrol. ID 8424502:1-12). The efficacy of treatment with multivalent ISVD constructs on compartment-specific monocyte / macrophage and T cell infiltration in the renal cortex was analyzed using 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). Treatment with dexamethasone (daily administration at specific doses) and an irrelevant VHH (IRR00163) that does not bind to any mouse protein were used as controls to assess the effect of treatment.

[0337] Using CD11b and Mac-2 antibodies, we differentially detected both infiltrating blood-borne monocytes and macrophages, which were distinct from the F4 / 80-positive resident macrophages and more mature macrophages found in the interstitial and periglomerular 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, galectin-3 protein (recognized by Mac-2 antibody) expression in renal tissue and serum galectin-3 levels were elevated in patients with LN compared with healthy controls and have been identified as a possible biomarker of disease activity in LN (Kang et al. 2009, Lupus 18:22-28). Additional CD4 and CD8 immunostaining was used to detect subpopulations of lymphocytic T cells that may also be important in LN pathogenesis (Couzi et al. 2007, Arthritis Rheum. 56:2362-2370).

[0338] 9.2.1 FFPE kidney tissue preparation On days 7 and / or 21 after animal necropsy, the left kidneys reserved for immunohistochemistry studies were divided into two pieces along their longitudinal axis (a horizontal section through the middle of the kidney) at their midsection. The two halves were placed in 4% w / v formaldehyde buffered in RS solution (Sigma-Aldrich) at pH 6.9 at room temperature for 2 days. The fixation step was stopped by rinsing the kidney pieces, kept in appropriate cassettes, for 2 x 5 min in PBS buffer (1x PBS Gibco pH 7.4, catalog no. 10010-031), followed by dehydration and embedding in paraffin.

[0339] Paraffin-embedded tissue blocks containing the kidneys of hCX3CR1 KI mice were sectioned at 3 μm thickness on a microtome, and the sections were transferred to glass slides suitable for IHC. Immunostaining was performed on the dewaxed slides using either a Ventana Discovery XT or Ventana ULTRA automated system according to the manufacturer's instructions (Ventana Medical Systems, Inc., USA).

[0340] 9.2.2 Immunostaining Different primary antibodies were used to detect myeloid cell types on mouse FFPE kidney tissue (i.e., anti-F4 / 80, anti-CD11b, and anti-Mac-2 / Galectin-3 antibodies) in different IHC assays performed on either the Ventana Discovery XT or Ventana ULTRA automated systems. Anti-CD4 and / or anti-CD8 antibodies were used to detect lymphoid T cells. Different amplification detection systems (Ventana Medical System Inc.) were used depending on the primary antibody used.

[0341] The F4 / 80(D2S9R)XP® antibody, a rabbit monoclonal immunoglobulin G (IgG) (Cell Signaling Technology, ref. 70076), is typically used to detect macrophage cell types. The detection system was Biotin-free Discovery anti-rabbit UltraMap™ horseradish peroxidase (HRP) conjugate (760-4315, Ventana Medical Systems, Inc., USA).

[0342] To detect monocyte / macrophage cell types, a rat monoclonal IgG2a antibody (clone M3 / 38, Cedarlane, ref. CL8942AP) is typically used, along with a secondary antibody (linker) corresponding to Rabbit@Rat IgG (clone R18-2, Abcam, ref. ab125900) and a biotin-free Discovery anti-Rabbit OmniMap horseradish peroxidase (HRP) conjugate (760-4311, Ventana Medical Systems, Inc., USA).

[0343] CD11b [EPR1344] rabbit antibody, a rabbit monoclonal IgG antibody (Abcam, reference ab133357), is typically used to detect monocytic cell types. The detection system was Discovery anti-rabbit HQ (760-4815, Ventana Medical Systems, Inc., USA) and anti-HQ HRP multimer (760-4820, Ventana Medical Systems, Inc., USA).

[0344] A CD4 rat monoclonal IgG1 antibody (Clone 4SM95, Invitrogen, Reference 14-9766-82) was used to detect cell surface receptors on a subpopulation of lymphocyte T cells. The detection system consisted of Rabbit@Rat IgG (Clone R18-2, Abcam, Reference ab125900), Discovery anti-Rabbit HQ (760-4815, Ventana Medical Systems, Inc., USA), and the corresponding secondary antibody (linker) Discovery anti-HQ HRP multimer (760-4820, Ventana Medical Systems, Inc., USA).

[0345] The CD8a rat monoclonal IgG2a antibody (Clone 4SM15, Invitrogen, Reference 14-0808-82) was used to detect cell surface receptors on a subpopulation of lymphocyte T cells. The detection system consisted of Rabbit@Rat IgG (Clone R18-2, Abcam, Reference ab125900), Discovery anti-Rabbit HQ (760-4815, Ventana Medical Systems, Inc., USA), and the corresponding secondary antibody (linker) Discovery anti-HQ HRP multimer (760-4820, Ventana Medical Systems, Inc., USA).

[0346] Immunostaining was finalized using a universal 3,3'-diaminobenzidine (DAB) chromogenic detection kit (760-159, Ventana Medical Systems, Inc., USA) for CD11b, F4 / 80, and Mac-2 / galectin-3 markers. For CD4 and CD8 markers, a Discovery purple kit (760-229) was used. A counterstaining step was performed using hematoxylin II (790-2208, Ventana Medical Systems, Inc., USA), and a bluing reagent was applied (760-2037, Ventana Medical Systems, Inc., USA). Stained slides were dehydrated and covered with cytoplasmic XYL (8312-4, Richard-Allan Scientific, USA).

[0347] 9.3 Data Analysis 9.3.1 Image Analysis Quantitative image analysis was performed on an image analysis platform (HALO, Indica Labs) using a Multiplex IHC module based on automated cell counting. Analysis was performed on either the entire cortex (in this case, the mean density of positive cells per treatment group was analyzed) or glomeruli only (in this case, the mean number of positive cells in the analyzed glomeruli) considered as two distinct regions of interest. For CD11b / CD4 double staining, glomeruli were automatically segmented using the HALO AI Dense Net neural network (a range of 130–214 glomeruli were detected per animal at 7 days, and 143–284 glomeruli were detected per animal at 21 days for analysis). For single Mac-2 / Galectin-3 (day 7) or dual CD8 and Mac-2 / Galectin-3 (day 21) staining, 80 manually defined glomeruli throughout the cortex (7-day study) or 100 glomeruli (21-day study) were analyzed for each animal.

[0348] 9.3.2 Statistical analysis One-way ANOVAs were fitted for each IHC parameter to log-transformed responses to mitigate variance heterogeneity. Due to the log transformation, effect interpretation is in terms of GM ratios (fold changes). Dunnett's correction (both p-values and CI boundaries) was applied to each IHC parameter for 2-day and 4-day treatment effect comparisons to control for family-wise error rates. Analysis results are expressed according to the IHC parameter, including fold reduction and significance scoring.

[0349] Analyses were performed using SAS (©) software version 9.4 for Windows 10 Enterprise 64-bit in SAS Enterprise Guide v 8.2.0. Fold reduction and significance scoring were scripted in RStudio v 1.1.453 with R v 4.1.3 using the packages tidyverse, rstatix, ggplot2, ggpubr, plotly, and EnvStats.

[0350] 9.4 Renal immunohistochemical evaluation of 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 9.4.1 Research design 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 administration route of NTS were based on 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, each of which initially contained 10 female hCX3CR1 KI mice injected with 240 μl of NTS on day 1. The protocol for the experimental in vivo study design is outlined in Figure 19.

[0351] 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.

[0352] [Table 23]

[0353] 9.4.2 Microscopic observation Compared with two naive, untreated hCX3CR1 KI mice, all hCX3CR1 KI mice in the NTS / IRR00163 group showed significant changes in the distribution pattern and density of F4 / 80-, CD11b-, and Mac-2 / Galectin-3-positive cells on kidney tissues observed microscopically (Figure 20). As expected, NTS injection induced a significant overall accumulation of F4 / 80-positive cells throughout the cortical region, almost exclusively in the periglomerular and tubulointerstitial regions, at day 7. In contrast, the increase in CD11b- and Mac-2 / Galectin-3-positive cells at day 7 was primarily detectable in more localized subregions of the glomerulus and tubulointerstitium compared with F4 / 80. Of note, Mac-2 / galectin-3 protein expression was also significantly increased in tubular epithelium within the renal interstitium, as previously reported in cases of renal pathology (Henderson et al. 2008, Am. J. Pathol. 172:288-298).

[0354] 9.4.3 Quantitative analysis Five IHC marker / location variables and (hereafter referred to as IHC parameters) were analyzed: 1) “mean density of F4 / 80+ cells in the whole cortex (mm 2 2) "mean density of CD11b+ cells in the whole cortex (mm 2 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".

[0355] IHC analysis of tissues from the study revealed a dose-dependent significant decrease in the number of monocytes / macrophages (CD11b+; Mac-2 / Galectin-3+) per glomerulus in response to A041600035 treatment compared to mice receiving the negative control, with a fold reduction (FD) of greater than 2.0 (Table B-17). In comparison, daily dexamethasone administration at 0.3 mg / kg daily IP for 7 consecutive days induced a more limited protective effect on the mean number of positive cells per cortical glomerulus for both CD11b (estimated mean of 1.35 FD, p=0.0105) and Mac-2 / Galectin-3 (estimated mean of 1.50 FD, p=0.0058) markers.

[0356] No change in the mean density of CD4+ cells in the cortex could be detected with any of the treatments.

[0357] [Table 24]

[0358] Based on IHC evaluation of CD11b, Mac-2 / Galectin-3, and F4 / 80 immune cell markers, this study suggests that A041600035 can induce a dose-dependent decrease in the number of monocyte / macrophage cell types in kidney tissue from a non-accelerated NTN model in hCX3CR1 KI mice at day 7 after NTN injection. The fold reductions observed for CD11b and Mac-2 / Galectin-3 IHC markers for the two highest concentrations of A041600035 were more pronounced compared to the dexamethasone tested dose.

[0359] 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 9.4.1 Research design 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 administration route of NTS were based on 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 an optimization study. This study contained 11 treatment groups, each of which included seven female hCX3CR1 KI mice injected with 240 μl of NTS on day 1, except for the naive group (no NTS injection), which contained two animals. For all treatment groups, dosing began on day 0, and necropsies were performed on a subset of animals (corresponding to groups 1-6) on day 7, and then on day 21 (corresponding to groups 7-11) after the initial injection of NTS. The protocol for the experimental in vivo study design is outlined in Figure 21. Treatments were administered as detailed in Table B-18.

[0360] [Table 25]

[0361] 9.4.2 Microscopic observation As previously described in Example 9.3, significant changes in the distribution pattern and density of F4 / 80, CD11b, Mac-2 / Galectin-3, and CD4-positive cells on the kidney tissue were observed microscopically in all hCX3CR1 KI mice treated with NTS and IRR00163 Nb at the early time point of 7 days after the individual injection of NTS, compared with two naive non-NTS-treated hCX3CR1 KI mice. 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 kidney cortex of hCX3CR1 KI mice treated with NTS and IRR00163 (Figure 22). A significant overall accumulation of F4 / 80-positive cells throughout the cortical region, almost exclusively in the periglomerular and tubulointerstitial regions, was still clearly detectable at 21 days. The density of CD11b- and Mac-2 / Galectin-3-positive cells increased in more focal (compared to F4 / 80) subregions of the glomerulus and tubulointerstitium. Notably, Mac-2 / Galectin-3 protein expression remained elevated in tubular epithelium within the renal interstitium at day 21, as previously reported in cases of renal pathology (Henderson et al. 2008, Am. J. Pathol. 172:288-298). In contrast to other markers at day 21, the overall changes in CD8 marker expression induced at day 21 after NTS injection were limited.

[0362] 9.4.3 Quantitative analysis Various IHC markers / location variables (hereafter referred to as IHC parameters) were selected for analysis at day 7 and 21 after the first IP injection or oral gavage, considering the five treatment groups. For analysis at day 7, the following six were selected: "mean density of F4 / 80+ cells in the whole cortex (mm 2 ) and "mean density of CD4+ cells in the whole cortex (mm 2 ) and "mean density of CD11b+ cells in the whole 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.” Analysis on day 21 included the “mean density of CD8+ cells in the whole cortex (mm 2 Additional IHC markers were included: "CD8+ cell count per glomerulus" and "mean number of CD8+ cells per glomerulus in the cortex." Each of these parameters was assessed by comparing the dexamethasone (0.5 mg / kg), cyclophosphamide (30 mg / kg), mycophenolate mofetil (100 mg / kg), and NTS / A041600035 treatment groups with the NTS / IRR00163 (30 mg / kg) control group.

[0363] IHC analysis of tissues from days 7 and 21 post-NTS administration revealed a clear pharmacological effect for A041600035, showing a marked and significant reduction in CD11b+ and Mac-2 / Galectin-3 monocytes / macrophages per glomerulus. In comparison, dexamethasone and, to a lesser extent, mycophenolate mofetil reduced CD4+ lymphocyte T-cell populations (Table B-19).

[0364] [Table 26]

[0365] Overall, these results demonstrate a significant effect of A041600035 on CD11b+ and Mac-2 / Galectin-3+ infiltrating blood-borne monocytes and macrophages, with significantly more infiltrating blood-borne monocytes and macrophages in the kidney cortex and glomerular region of NTN animals at 7 and 21 days after NTS injection. A less pronounced but significant effect was observed only on F4 / 80+ macrophages in the 7-day study, possibly reflecting activity against blood-borne subsets of this mixed population of infiltrating and tissue-resident macrophages. No effect of A041600035 could be detected on CD4 and CD8 T cell populations.

[0366] The effect of A041600035 was particularly distinct from dexamethasone 21 days after NTS injection. In comparison, dexamethasone treatment induced a significant decrease in both CD4 and CD8 T cell populations 21 days after NTS injection, but did not affect monocyte and macrophage markers. In the same model, only mycophenolate mofetil treatment had a significant effect on the CD4 T cell population 21 days after NTS injection and when measured in the entire kidney cortex.

[0367] No significant effects were detected for any of the selected IHC markers following cyclophosphamide treatment in this model.

[0368] Example 10: Determination of Pharmacokinetic (PK) Profile A041600035 or A041600087 was administered as a single dose to cynomolgus monkeys by intravenous infusion. After administration, blood samples were collected at different time points. The concentrations of A041600035 or A041600087 were measured by a ligand binding assay. The concentration-time profiles were plotted (Figure 18).

[0369] The different pK profiles observed for A041600087 at high doses compared to low doses may be indicative of target-mediated drug deposition (TMDD). This TMDD is not observed for A041600035. Furthermore, at similar doses, exposure to A041600035 was higher compared to exposure to A041600087.

[0370] Industrial Applicability The present technology can be used in industry, and industrial applications and practical applications can be derived from this specification by the general knowledge of a person skilled in the art.

[0371] Sequence Listing The term "ID" in the table below (last column) refers to "SEQ ID NO." as used herein.

[0372] [Table 27]

[0373] Table 28

[0374] Table 29

[0375] Table 30

[0376] Table 31

[0377] Table 32

[0378]

Table 33

[0379] Table 34

[0380] Table 35

[0381] Table 36

[0382] Table 37

[0383] Table 38

[0384] Table 39

[0385] Table 40

[0386] Table 41

[0387] Table 42

[0388] Table 43

[0389] Table 44

[0390] Table 45

[0391] Table 46

[0392] 9. References: ·Imai et al.,Cell 1997 Nov 14;91(4):521-30,PMID:9390561 ·Umehara et al.,Arterioscler Thromb Vasc Biol.2004 Jan;24(1):34-40;PMID:12969992 ·Faure et al.,Science 2000 Mar 24;287(5461):2274-7;PMID:10731151 ·McDermott et al.Circ Res.2001 Aug 31;89(5):401-7,PMID:11532900 ·Moatti et al.Blood 2001 Apr 1;97(7):1925-8,PMID:11264153 ·McDermott,2003;J.Clin.Invest.111:1241,PMID:12697743 ·Ghilardi,2004,Stroke 35:1276,PMID:15118174 ·Combadiere,2003;Circulation,107:1009,PMID:12600915 ·Lesnik,2003;J.Clin.Invest.111:333,PMID:12569158 ·Wakita et al.2017,Mol.Pharmacol.92:502,PMID:28842393 ·Tanaka et al.2021,Modern Rheumatol 31 783,PMID:33427546 International Publication No. 2013 / 130381 Brochure ·Low et al.2020,Mabs,12(1):1709322,PMID:31924119 ·Hamers-Casterman et al.Nature 363:446-448,1993 Review article by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001) International Publication No. 94 / 04678 Brochure Ward et al. (see, e.g., WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996) Conrath et al., J. Biol. Chem., Vol. 276, 10, 7346-7350, 2001, as well as WO 96 / 34103 and WO 99 / 23221. International Publication No. 2008 / 020079 Brochure ·Davies and Riechman(1994 and 1996) ·Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91) ·Riechmann and Muyldermans,2000(J.Immunol.Methods 240(1-2):185-195 ·Kontermann and Duebel(Eds.2010,Antibody Engineering,vol 2,Springer Verlag Heidelberg Berlin,Martin,Chapter 3,pp.33-51) WO 94 / 25591, Gasser et al. Biotechnol. Bioeng. 94:535, 2006; Gasser et al. Appl. Environ. Microbiol. 73:6499, 2007; or Damasceno et al. Microbiol. Biotechnol. 74:381, 2007 ·Estep P.et al.2013 MAbs.5(2):270-8.PMID:23575269

Claims

1. A polypeptide comprising or comprising at least three immunoglobulin monovariable domains (ISVDs), each of the at least three ISVDs having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions and CDR1 to CDR3 refer to complementarity-determining regions; the polypeptide, (a) Specifically binds to the fractalkine receptor CX3CR1 and / or single nucleotide polymorphism (SNP) variants of CX3CR1, (according to AbM numbering) The amino acid sequence of Sequence ID No. 10 is CDR1; The amino acid sequence of SEQ ID NO: 12 is CDR2; and The amino acid sequence of SEQ ID NO: 14 is CDR3 The first ISVD including; and (b) Specifically binds to the fractalkine receptor CX3CR1 and / or single nucleotide polymorphism (SNP) variants of CX3CR1 (according to AbM numbering) The amino acid sequence of Sequence ID No. 10 is CDR1; The amino acid sequence of SEQ ID NO: 12 is CDR2; and The amino acid sequence of SEQ ID NO: 14 is CDR3 The second ISVD including; and (c) Specifically binds to albumin, (according to AbM numbering) CDR1 is an amino acid sequence of SEQ ID NO: 53, or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 53; CDR2 is an amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having two or one amino acid difference from SEQ ID NO: 55; and CDR3 is an amino acid sequence of SEQ ID NO: 57, or an amino acid sequence that differs from SEQ ID NO: 57 by two or one amino acid. The third ISVD, including Including; and (a), (b), and (c) are polypeptides linked directly or via a peptide linker.

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

3. The polypeptide according to claim 1, wherein the order of (a), (b), and (c) indicates the relative positions of elements within the polypeptide, taking into consideration the N-terminus to the C-terminus of the polypeptide.

4. The polypeptide according to claim 1, wherein the ISVD in (a), (b), and (c) is selected from the VH domain and the VHH domain.

5. The polypeptide according to claim 4, wherein the ISVD in (a), (b), and (c) is selected from a camelid VH domain, a humanized VHH domain, a domain antibody, and a dAb.

6. The polypeptide according to claim 1, wherein in (a) and (b), the ISVD comprises one or more selected from 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 claim 1, wherein in (a) and (b), the ISVD comprises one or more selected from serine (S) at amino acid position 11 according to Kabat numbering and glutamine (Q) at position 44 according to Kabat numbering.

8. The polypeptide according to claim 1, wherein in (a) and (b), the ISVD comprises one or more selected from threonine (T) at amino acid position 32 according to Kabat numbering, serine (S) at amino acid position 52 according to Kabat numbering, and threonine (T) at amino acid position 53 according to Kabat numbering.

9. The polypeptide according to claim 1, wherein in (a) and (b), the ISVD comprises one or more selected from 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 claim 1, wherein (a) the ISVD consists of the amino acid sequence shown in SEQ ID NO: 3, or amino acids having at least 90% sequence identity with SEQ ID NO: 3, and (b) the ISVD consists of the amino acid sequence shown in SEQ ID NO: 2, or amino acids having at least 90% sequence identity with SEQ ID NO: 2, and the amino acid sequence in the CDR region is ignored in order to determine the percentage of sequence identity.

11. The polypeptide according to claim 1, wherein (a) the ISVD consists of the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having a difference of two or one amino acids from SEQ ID NO: 2, and (b) the ISVD consists of the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having a difference of two or one amino acids from SEQ ID NO:

2.

12. The polypeptide according to claim 1, wherein in (a), the ISVD consists of the amino acid sequence shown in SEQ ID NO: 3, and in (b), the ISVD consists of the amino acid sequence shown in SEQ ID NO:

2.

13. The polypeptide according to claim 1, wherein in (a), the ISVD consists of the amino acid sequence shown in SEQ ID NO: 3, and in (b), the ISVD consists of the amino acid sequence shown in SEQ ID NO: 2, and the order of (a), (b), and (c) indicates the relative positions of each other within the polypeptide, taking into consideration the N-terminus to the C-terminus of the polypeptide.

14. (c) The polypeptide according to claim 1, wherein the ISVD specifically binds to human serum albumin.

15. (c) In this case, the ISVD specifically binds to human serum albumin (according to AbM numbering) The amino acid sequence of SEQ ID NO: 53 is CDR1; The amino acid sequence of Sequence ID No. 55 is CDR2; and The amino acid sequence of SEQ ID NO: 57 is CDR3 The polypeptide according to claim 1, comprising:

16. (c) The polypeptide according to claim 1, wherein the ISVD comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having more than 90% or more 95% sequence identity with SEQ ID NO:

37.

17. (c) The polypeptide according to claim 1, wherein the ISVD comprises or consists of the amino acid sequence shown in SEQ ID NO:

37.

18. The polypeptide according to claim 1, wherein in (a) and (b), the ISVD consists of the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having a difference of two or one amino acid from SEQ ID NO: 2, and in (c), the ISVD consists of the amino acid sequence shown in SEQ ID NO:

37.

19. The polypeptide according to claim 18, wherein the C-terminal extension of FR4 is a single Ala residue.

20. It comprises two peptide linkers, each peptide linker located between two ISVDs within the polypeptide; The polypeptide according to claim 1, wherein the two peptide linkers optionally include or consist of the amino acid sequence shown in SEQ ID NO:

69.

21. The polypeptide according to claim 1, comprising an amino acid sequence having more than 90% identity or more than 95% identity with SEQ ID NO:

82.

22. The polypeptide according to claim 21, comprising or consisting of the amino acid sequence shown in SEQ ID NO:

82.

23. The polypeptide according to claim 1, wherein the single nucleotide polymorphism (SNP) variant of CX3CR1 comprises at least one SNP selected from the group consisting of SNPs rs938203, rs2669849, rs1050592, rs3732379, and rs3732378.

24. Compared to the reference polypeptide consisting of the amino acid sequence shown in Sequence ID No. 83, yeast The polypeptide according to claim 1, characterized by an increased yield after expression in Pichia pastoris.

25. Characterized by at least one improved biophysical property compared to the reference polypeptide consisting of the amino acid sequence shown in Sequence ID No. 83; The aforementioned biophysical properties are (a) Improvement of the stability of the polypeptide after purification, for example, when measured by turbidity / opalescence of a polypeptide at a concentration of 100 mg / mL; (b) Reduction in the amount of low molecular weight species after expression of the polypeptide in a suitable host, for example, as measured by pre-peaking in reverse-phase chromatography (e.g., RP-UPLC); (c) For example, reduction in the amount of low molecular weight species after purification of the polypeptide as measured by capillary gel electrophoresis (CGE); (d) For example, reduction in the amount of high molecular weight species after expression of the polypeptide in a suitable host, as measured by size exclusion high-performance liquid chromatography (SE-HPLC); (e) reduction in the amount of high molecular weight species of the polypeptide after purification, as measured by, for example, size exclusion high-performance liquid chromatography (SE-HPLC); and (f) A combination of two or more of the biophysical properties mentioned above. A polypeptide according to claim 1, selected from the following.

26. The purification process is characterized by a higher recovery rate compared to the recovery rate of the reference polypeptide consisting of the amino acid sequence shown in Sequence ID No. 83, and the purification process is characterized by a higher recovery rate after the purification step. i. Capture process on resin; ii. pH adjustment step after the capture step on the resin; iii. Polishing process; iv. Ultrafiltration / dialysis filtration / ultrafiltration process; v. A combination of two or more of the above purification steps A polypeptide according to claim 1, selected from the following.

27. The polypeptide according to claim 1, wherein, compared to a reference polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 83, the amount of defective disulfide crosslinks is reduced after expression of the polypeptide in a suitable host, and the amount of polypeptide with reduced defective disulfide crosslinks is measured by reverse-phase high-performance liquid chromatography (RP-HPLC).

28. The polypeptide according to claim 1, which binds to the cell surface glycoprotein MUC18 with lower affinity compared to binding by a reference polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

83.

29. A polynucleotide encoding a polypeptide according to any one of claims 1 to 28.

30. A host or host cell comprising the polynucleotide described in claim 29.

31. A composition comprising, optionally, a pharmaceutical composition; and further optionally, at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, comprising a polypeptide according to any one of claims 1 to 28 or a polynucleotide encoding a polypeptide according to any one of claims 1 to 28.

32. - A step of expressing the polypeptide in a suitable host cell or host organism or another suitable expression system; Furthermore, optionally, - A step of isolating and / or purifying the polypeptide. A method for producing the polypeptide according to any one of claims 1 to 28, including the method described in any one of claims 1 to 28.

33. A polypeptide according to any one of claims 1 to 28, for use in inhibiting the binding of CX3CR1 to fractalkine in mammalian cells.

34. A polypeptide according to any one of claims 1 to 28, for use as a pharmaceutical.

35. A polypeptide, polynucleotide, or composition according to any one of claims 1 to 28, for use in the prevention, treatment, and diagnosis of a disease or disorder, wherein the disease or disorder is optionally related to the fractalkine receptor CX3CR1 or a single nucleotide polymorphism (SNP) variant of CX3CR1.

36. Polypeptides, polynucleotides, or compositions for use according to claim 35, wherein the disease or disorder is selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral artery disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schönlein purpura and Wegener granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders 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 fractalkine in mammalian cells, comprising administering the polypeptide according to any one of claims 1 to 28 to the cells, thereby inhibiting the fractalkine-mediated signaling, and comprising a pharmaceutical composition for use in the method, comprising the polypeptide according to any one of claims 1 to 28.

38. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 28 for use in a method of treating a disease or disorder, wherein the disease or disorder is selected from inflammatory diseases, cardiovascular and cerebrovascular atherosclerotic disorders, peripheral artery disease, myocardial infarction, restenosis, diabetic nephropathy, glomerulonephritis, human crescent glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schönlein purpura and Wegener granulomatosis, rheumatoid arthritis, graft-versus-host disease, atopic dermatitis, inflammatory bowel disease, Crohn's disease, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders 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, and the method comprises administering a pharmaceutically active amount of the polypeptide according to any one of claims 1 to 28 to a subject in need thereof.

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