Polypeptides comprising immunoglobulin single variable domains targeting il-6 and tnf-α

Abstract

This article provides a novel medicine for treating subjects with inflammatory diseases and / or autoimmune diseases, particularly rheumatoid arthritis. Specifically, this article provides a polypeptide comprising at least three immunoglobulin single variable domains (ISVDs), characterized in that at least one ISVD binds to TNF-α and at least two ISVDs bind to IL-6. This article also provides nucleic acids, vectors, and components.

Description

Technical Field

[0001] This article relates to peptides targeting interleukin-6 (IL-6) and TNF-α. It also relates to nucleic acid molecules encoding said peptides and carriers containing said nucleic acids, as well as compositions containing said peptides, nucleic acids, or carriers. Furthermore, this article relates to these products in methods for treating subjects with inflammatory diseases and / or autoimmune diseases. Additionally, this article relates to a method for producing these products. Prior Technology

[0002] Rheumatoid arthritis is a serious autoimmune disease affecting approximately 25 million patients worldwide (GBD 2015, Lancet. 2016 Oct 8;388(10053):1545-1602). The main symptoms of rheumatoid arthritis are joint pain and swelling. This is caused by joint inflammation, which involves inflammation of the synovial cavity of the joint. In rheumatoid arthritis, this inflamed synovial cavity is characterized by immune cell infiltration and stromal cell initiation (Klareskog, Catrina et al., Lancet. 2009 Feb 21;373(9664):659-72; Smolen, Aletaha et al., Nat Rev Dis Primers. 2018 Feb 8;4:18001). A specialized cell type, fibroblast-like synoviocytes (FLS), is considered a key player in this process (Bartok and Firestein, Immunol Rev. 2010 Jan;233(1):233-55). FLS, along with macrophage-like synoviocytes, form the inner lining of the synovium. In rheumatoid arthritis (RA), FLS proliferation and the accumulation of immune cells trigger inflammation and lead to synovial thickening, known as synovial hyperplasia—one of the main symptoms of RA. As a key mediator of joint inflammation in RA, FLS represents an attractive target cell type for RA treatment.

[0003] IL-6 is a pleiotropic cytokine secreted by various cell types, including T cells and B cells, monocytes, fibroblasts, and synovial cells.

[0004] IL-6 is a protein initially identified as a B-cell differentiation factor (Hirano et al., 1985, Proc. Natl. Acad. Sci. USA, 82: 5490-4; EP 0257406), which, together with IL-6R (Yamasaki et al., 1988, Science, 241: 825-8; EP 0325474), leads to the formation of the IL-6 / IL-6R complex. This complex binds to gp130, a receptor protein that transmits various physiological functions of IL-6 (Taga et al., 1989, Cell, 58: 573-81; EP 0411946). IL-6 is currently known to participate in the regulation of immune responses, hematopoiesis, acute phase responses, bone metabolism, angiogenesis, and inflammation, among other things. Dysregulation of IL-6 production is associated with symptoms of certain autoimmune and chronic inflammatory proliferative diseases (Ishihara and Hirano, 2002, Biochim. Biophys. Acta, 1592: 281-96). Peptides that specifically bind to IL-6 (Klein et al., 1991, Blood, 78: 1198-204; EP 0312996), IL-6R (EP 0409607), or gp130 (Saito et al., 1993, J. Immunol. Methods, 163: 217-223; EP 0572118) have been shown to effectively inhibit IL-6 function.

[0005] The prior art describes antibodies and antibody fragments targeting human IL-6, human IL-6R, and human gp130 protein for the prevention and treatment of IL-6-related disorders. Examples include tocilizumab (see Woo et al., 2005, Arthritis Res. Ther. 7: 1281-8; Nishimoto et al., 2005, Blood 106: 2627-32; Ito et al., 2004, Gastroenterology, 126: 989-96; Choy et al., 2002, Arthritis Rheum. 46: 3143-50) and BE8 (see Bataille et al., 1995, Blood 86: 685-91; Emilie et al., 1994, Blood 84: 2472-9; Beck et al., 1994, N. Engl. J. Med. 330: 602-5; Wendling et al., 1993, J. Rheumatol. 20: 259-62) and Centocor's CNTO-328 (see Journal of Clinical Oncology, 2004, 22 / 14S: 2560; Journal of Clinical Oncology, 2004, 22 / 14S: 2608; Int. J. Cancer, 2004, 111:592-5). Another known active ingredient for the prevention and treatment of IL-6-related disorders is the Fc fusion of soluble gp130 (see Becker et al. 2004, Immunity, 21: 491-501; Doganci et al. 2005, J. Clin. Invest. 115: 313-25; Nowell et al. 2003, J. Immunol. 171: 3202-9; Atreya et al. 2000, Nat. Med. 6: 583-8). The amino acid sequences and nano-antibodies against IL-6R, as well as the peptides containing them, are described in WO 08 / 020079.

[0006] Tumor necrosis factor-alpha (TNF-α; TNF-alpha) is a homotrimeric cytokine primarily produced by monocytes and macrophages, but also known to be secreted by CD4+ and CD8+ peripheral blood T lymphocytes. TNF-α can exist in a soluble form or as a transmembrane protein. The main function of TNF-α is to regulate immune cells. TNF-α acts as an endogenous pyrogen, and abnormal regulation of its production is associated with a variety of human diseases, including inflammatory bowel disease and other inflammatory diseases such as rheumatoid arthritis (RA).

[0007] Currently approved FDA treatments for rheumatoid arthritis include anti-TNF-α biologics such as Simponi® (golimumab), Enbrel® (etanercept), Remicade® (infliximab), and Humira® (adalimumab). However, these anti-TNF-α treatments have only shown complete disease remission in a small percentage of patients, and a large proportion remain unresponsive. Therefore, to date, no biologic has demonstrated sufficient efficacy in achieving disease remission in the majority of patients with rheumatoid arthritis, and the lack or loss of response remains a concern.

[0008] Targeting multiple disease factors can be achieved, for example, through the co-administration or combination of two separate biologics (e.g., antibodies binding to different therapeutic targets). However, from both a practical and commercial perspective, co-administering or combining separate biologics can be challenging. For instance, two injections of a single product present patients with a more inconvenient and painful treatment regimen, which can negatively impact adherence. Regarding a single injection of two separate products, providing a formulation that allows for acceptable viscosity and suitable stability of both products at the desired concentration may be difficult or impossible. Furthermore, co-administration and co-formulation require the production of two separate drugs, which can increase overall costs.

[0009] Bispecific antibodies capable of binding to two different antigens have been proposed as a strategy to address such limitations associated with the co-administration or combination of individual biologics (such as antibodies).

[0010] Various forms of bispecific antibody constructs have been proposed. For example, bispecific antibody forms can involve the chemical conjugation of two antibodies or fragments thereof (Brennan, M et al., Science, 1985. 229(4708): 81-83; Glennie, MJ et al., J Immunol, 1987. 139(7): 2367-2375).

[0011] However, the drawbacks of this type of bispecific antibody include: high viscosity at high concentrations making subcutaneous administration challenging, for example; and the requirement for two variable domains to interact for specific high-affinity binding per binding unit, which impacts peptide stability and production efficiency. This type of bispecific antibody may also potentially lead to CMC (chemistry, manufacturing, and control) problems related to light chain mismatch or heavy chain mismatch.

[0012] To date, no multispecific (e.g., bispecific) antibody constructs targeting TNF-α and IL-6 have entered clinical trials. Summary of the Invention

[0013] Because patients with rheumatoid arthritis (RA) still do not respond adequately to the available standard care treatments, there remains an unmet medical need for improved medications to treat RA.

[0014] The inventors of this case have developed novel and improved agents for the treatment of inflammatory and / or autoimmune diseases, such as rheumatoid arthritis (RA) in particular. These agents target two or more disease factors, including IL-6 and TNF-α, which mediate the biological mechanisms associated with inflammatory diseases, particularly RA.

[0015] The inventors made a surprising discovery that dual targeting of IL-6 and TNF-α with a single agent has the potential to provide effective treatment in patients with rheumatoid arthritis, where single-agent monospecific therapy for the same indication may not be effective enough.

[0016] The inventors of this invention have discovered that bispecific or multispecific peptides (e.g., immunoglobulin single variable domain (ISVD) constructs) that simultaneously and specifically target IL-6 and TNF-α have increased efficiency in modulating rheumatoid arthritis symptoms compared to monospecific anti-TNF-α or monospecific anti-IL-6 peptides. Such peptides (e.g., ISVD constructs) can be efficiently produced (e.g., in a microbial host) and conveniently administered. Furthermore, it has been demonstrated that such peptides (e.g., ISVD constructs) exhibit limited reactivity to antibodies pre-existing in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with the antibody construct). In some embodiments, such peptides (e.g., ISVD constructs) exhibit a sufficiently long half-life in the subject to be treated, allowing for limitation of the number of consecutive treatments and thus sufficient temporal spacing.

[0017] The polypeptides described herein (e.g., immunoglobulin single variable domain (ISVD) constructs) comprise or consist of at least three immunoglobulin single variable domains (ISVDs), wherein at least one ISVD specifically binds to TNF-α, and at least two ISVDs specifically bind to IL-6. According to some embodiments, the at least one ISVD specifically binding to TNF-α binds to human TNF-α (hTNF-α), and the at least two ISVDs specifically binding to IL-6 bind to human IL-6 (hIL-6).

[0018] According to some preferred embodiments, the polypeptides herein also include one or more other groups, residues, portions, or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without said one or more other groups, residues, portions, or binding units. For example, the binding unit may be an ISVD that binds to a serum protein, such as a human serum protein like human serum albumin.

[0019] Also provided are nucleic acid molecules capable of representing the polypeptides described herein, nucleic acids or carriers comprising said nucleic acids, and compositions comprising said polypeptides, said nucleic acids, or said carriers. In some embodiments, said compositions are pharmaceutical compositions.

[0020] It also provides a (non-human) host or host cell containing a nucleic acid or vector encoding a polypeptide according to this article.

[0021] A method for generating a polypeptide according to the present invention is also provided, the method comprising at least the following steps: a. Expressing nucleic acids in a suitable host cell or (non-human) host organism or another suitable (e.g., cell-free) expression system; optionally, proceeding thereafter: b. Isolate and / or purify the polypeptide according to this document.

[0022] Furthermore, this document provides the peptide, a composition comprising the peptide, or a composition comprising a nucleic acid or carrier containing a nucleotide sequence encoding the peptide, the peptide or composition being used as a drug. In some embodiments, the peptide or composition is used to treat inflammatory and / or autoimmune diseases, such as RA.

[0023] Additionally, a method for treating inflammatory diseases such as rheumatoid arthritis (RA) is provided, wherein the method includes administering a pharmaceutically active amount of a polypeptide or composition according to this document to a subject in need. In some embodiments, the method further includes administering one or more additional therapeutic agents.

[0024] The use of the peptides or compositions described herein in the preparation of medicaments (such as pharmaceutical compositions) for the treatment of inflammatory diseases and / or autoimmune diseases such as RA is also provided.

[0025] Specifically, the following embodiments are provided in this document:

[0026] Example 1. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid containing a nucleotide sequence encoding said polypeptide, said polypeptide or composition being used as a drug, wherein said polypeptide comprises or is composed of at least three immunoglobulin single variable domains (ISVDs), wherein each said ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein: a) The first ISVD contains i. An amino acid sequence having the same as SEQ ID NO: 6 or a CDR1 differing from SEQ ID NO: 6 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 10 or a CDR2 differing from SEQ ID NO: 10 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 14 or a CDR3 differing from SEQ ID NO: 14 by 2 or 1 amino acids; b) The second ISVD contains iv. An amino acid sequence having the same as SEQ ID NO: 8 or a CDR1 differing from SEQ ID NO: 8 by 2 or 1 amino acids; v. Having the amino acid sequence of SEQ ID NO: 12 or CDR2 differing from SEQ ID NO: 12 by 2 or 1 amino acid; and vi. Having the amino acid sequence of SEQ ID NO: 16 or CDR3 differing from SEQ ID NO: 16 by 2 or 1 amino acid; and c) The third ISVD contains vii. An amino acid sequence having the same as SEQ ID NO: 9 or a CDR1 differing from SEQ ID NO: 9 by 2 or 1 amino acids; viii. A CDR2 having the amino acid sequence of SEQ ID NO: 13 or differing from SEQ ID NO: 13 by 2 or 1 amino acid; and ix. An amino acid sequence having the same as SEQ ID NO: 17 or a CDR3 differing from SEQ ID NO: 17 by 2 or 1 amino acids. The first ISVD, the second ISVD, and the third ISVD are optionally included in an order starting from the N end.

[0027] Example 2. A composition for the intended use according to Example 1, wherein the composition is a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more other pharmaceutically active peptides and / or compounds.

[0028] Example 3. A polypeptide or composition for the stated purpose according to Example 1 or 2, wherein: a) The first ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 10, and CDR3 having the amino acid sequence of SEQ ID NO: 14; b) The second ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 8, CDR2 having the amino acid sequence of SEQ ID NO: 12, and CDR3 having the amino acid sequence of SEQ ID NO: 16; and c) The third ISVD comprises CDR1 having an amino acid sequence of SEQ ID NO: 9, CDR2 having an amino acid sequence of SEQ ID NO: 13, and CDR3 having an amino acid sequence of SEQ ID NO: 17.

[0029] Example 4. A polypeptide or composition for the purpose according to any one of Examples 1 to 3, wherein: a) The amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO: 2; b) The amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO: 4; and c) The amino acid sequence of the third ISVD has a sequence identity with SEQ ID NO: 5 greater than 90%.

[0030] Example 5. A polypeptide or composition for the stated purpose according to any one of Examples 1 to 4, wherein: a) The first ISVD has the amino acid sequence of SEQ ID NO: 2; b) The second ISVD has the amino acid sequence of SEQ ID NO: 4; and c) The third ISVD has the amino acid sequence of SEQ ID NO: 5.

[0031] Example 6. A polypeptide or composition for the purpose according to any one of Examples 1 to 5, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0032] Example 7. A polypeptide or composition for the purpose according to Example 6, wherein the one or more other groups, residues, portions or binding units that provide an increased half-life for the polypeptide are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

[0033] Example 8. A polypeptide or composition for the purpose according to any one of Examples 6 to 7, wherein the one or more other groups, residues, portions or binding units that provide an increased half-life for the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0034] Example 9. A polypeptide or composition for the purpose according to Example 8, wherein the binding unit that provides an increased half-life for the polypeptide is an ISVD that can bind to human serum albumin.

[0035] Example 10. A polypeptide or composition for the stated purpose according to Example 9, wherein the ISVD bound to human serum albumin comprises i. An amino acid sequence having the same as SEQ ID NO: 7 or a CDR1 differing from SEQ ID NO: 7 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 11 or a CDR2 differing from SEQ ID NO: 11 by 2 or 1 amino acids; and iii. Having the amino acid sequence of SEQ ID NO: 15 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO: 15.

[0036] Example 11. A polypeptide or composition for the purpose according to any one of Examples 9 to 10, wherein the ISVD that binds to human serum albumin comprises CDR1 having an amino acid sequence of SEQ ID NO: 7, CDR2 having an amino acid sequence of SEQ ID NO: 11, and CDR3 having an amino acid sequence of SEQ ID NO: 15.

[0037] Example 12. A polypeptide or composition for the purpose according to any one of Examples 9 to 11, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity with SEQ ID NO: 3.

[0038] Example 13. A polypeptide or composition for the purpose according to any one of Examples 9 to 12, wherein the ISVD that binds to human serum albumin has the amino acid sequence of SEQ ID NO: 3.

[0039] Example 14. A polypeptide or composition for the purpose according to any one of Examples 1 to 13, wherein the polypeptide has an extension of 1 to 5 amino acid residues at its C-terminus, preferably an extension of a single amino acid residue, wherein the amino acid residues are independently selected from naturally occurring amino acids, preferably independently selected from glycine or alanine, leucine, isoleucine and valine, more preferably alanine and glycine, and most preferably alanine.

[0040] Example 15. A polypeptide or composition for the purpose according to any one of Examples 1 to 14, wherein the amino acid sequence of the polypeptide comprises or is composed of the following amino acid sequences, the amino acid sequences having greater than 90% sequence identity with SEQ ID NO: 1.

[0041] Example 16. A polypeptide or composition for the purpose according to any one of Examples 1 to 15, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0042] Example 17. A polypeptide or composition according to any one of Examples 1-16 for the purpose described herein, said polypeptide or composition for the treatment of inflammatory and / or autoimmune diseases, such as rheumatoid arthritis.

[0043] Example 18. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), wherein each ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein: a) The first ISVD is combined with IL-6 and contains i. An amino acid sequence having the same as SEQ ID NO: 6 or a CDR1 differing from SEQ ID NO: 6 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 10 or a CDR2 differing from SEQ ID NO: 10 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 14 or a CDR3 differing from SEQ ID NO: 14 by 2 or 1 amino acids; b) The second ISVD is combined with IL-6 and contains iv. An amino acid sequence having the same as SEQ ID NO: 8 or a CDR1 differing from SEQ ID NO: 8 by 2 or 1 amino acids; v. Having the amino acid sequence of SEQ ID NO: 12 or CDR2 differing from SEQ ID NO: 12 by 2 or 1 amino acid; and vi. Having the amino acid sequence of SEQ ID NO: 16 or CDR3 differing from SEQ ID NO: 16 by 2 or 1 amino acid; and c) The third ISVD binds to TNF-α and contains vii. An amino acid sequence having the same as SEQ ID NO: 9 or a CDR1 differing from SEQ ID NO: 9 by 2 or 1 amino acids; viii. A CDR2 having the amino acid sequence of SEQ ID NO: 13 or differing from SEQ ID NO: 13 by 2 or 1 amino acid; and ix. An amino acid sequence having the same as SEQ ID NO: 17 or a CDR3 differing from SEQ ID NO: 17 by 2 or 1 amino acids. The ISVDs are optionally included in order starting from the N end.

[0044] Example 19. The polypeptide according to Example 18, wherein: a) The first ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 10, and CDR3 having the amino acid sequence of SEQ ID NO: 14; b) The second ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 8, CDR2 having the amino acid sequence of SEQ ID NO: 12, and CDR3 having the amino acid sequence of SEQ ID NO: 16; and c) The third ISVD comprises CDR1 having an amino acid sequence of SEQ ID NO: 9, CDR2 having an amino acid sequence of SEQ ID NO: 13, and CDR3 having an amino acid sequence of SEQ ID NO: 17.

[0045] Example 20. The polypeptide according to any one of Examples 18 or 19, wherein: a) The amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO: 2; b) The amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO: 4; and c) The amino acid sequence of the third ISVD has a sequence identity with SEQ ID NO: 5 greater than 90%.

[0046] Example 21. The polypeptide according to any one of Examples 18 to 20, wherein: a) The first ISVD has the amino acid sequence of SEQ ID NO: 2; b) The second ISVD has the amino acid sequence of SEQ ID NO: 4; and c) The third ISVD has the amino acid sequence of SEQ ID NO: 5.

[0047] Example 22. A polypeptide according to any one of Examples 18 to 21, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0048] Example 23. The polypeptide according to Example 22, wherein the one or more other groups, residues, portions or binding units providing the increased half-life of the polypeptide are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

[0049] Example 24. A polypeptide according to any one of Examples 22 to 23, wherein the one or more other groups, residues, portions or binding units that provide an increased half-life for the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0050] Example 25. The polypeptide according to Example 24, wherein the binding unit providing the increased half-life of the polypeptide is an ISVD that can bind to human serum albumin.

[0051] Example 26. The polypeptide according to Example 25, wherein the ISVD bound to human serum albumin comprises: i. An amino acid sequence having the same as SEQ ID NO: 7 or a CDR1 differing from SEQ ID NO: 7 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 11 or a CDR2 differing from SEQ ID NO: 11 by 2 or 1 amino acids; and iii. Having the amino acid sequence of SEQ ID NO: 15 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO: 15.

[0052] Example 27. The polypeptide according to any one of Examples 25 to 26, wherein the ISVD that binds to human serum albumin comprises CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 15.

[0053] Example 28. The polypeptide according to any one of Examples 25 to 27, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity with SEQ ID NO: 3.

[0054] Example 29. The polypeptide according to any one of Examples 25 to 28, wherein the ISVD that binds to human serum albumin has the amino acid sequence of SEQ ID NO: 3.

[0055] Example 30. A polypeptide according to any one of Examples 18 to 29, wherein the polypeptide has an extension of 1 to 5 amino acid residues at its C-terminus, preferably an extension of a single amino acid residue, wherein the amino acid residues are independently selected from naturally occurring amino acids, preferably independently selected from glycine or alanine, leucine, isoleucine and valine, more preferably alanine and glycine, and most preferably alanine.

[0056] Example 31. A polypeptide according to any one of Examples 18 to 30, wherein the amino acid sequence of the polypeptide comprises or is composed of the following amino acid sequence, the amino acid sequence having greater than 90% sequence identity with SEQ ID NO: 1.

[0057] Example 32. A polypeptide according to any one of Examples 18 to 31, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0058] Example 33. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of Examples 18 to 32.

[0059] Example 34. A host or host cell comprising the nucleic acid described in Example 33.

[0060] Example 35. A method for generating a polypeptide according to any one of Examples 18 to 32, the method comprising at least the following steps: a) Expressing the nucleic acid as described in Example 33 in a suitable host cell or host organism or another suitable expression system; optionally, proceeding thereafter: b) Isolate and / or purify the polypeptide according to any one of Examples 18 to 32.

[0061] Example 36. A composition comprising at least one polypeptide according to any one of Examples 18 to 32 or a nucleic acid according to Example 33.

[0062] Example 37. The composition according to Example 36 is a pharmaceutical composition, which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprises one or more other pharmaceutically active peptides and / or compounds.

[0063] Example 38. A method for treating inflammatory diseases and / or autoimmune diseases such as rheumatoid arthritis, wherein the method comprises administering to a subject in need a pharmaceutically active amount of a polypeptide according to any one of Examples 18 to 32 or a composition according to any one of Examples 36 to 37.

[0064] Example 39. The method according to claim 37, wherein the inflammatory disease and / or autoimmune disease is rheumatoid arthritis.

[0065] Example 40. Use of the polypeptide according to any one of Examples 18 to 32 or the composition according to any one of Examples 36 to 37 in the preparation of pharmaceutical compositions for treating inflammatory diseases and / or autoimmune diseases such as rheumatoid arthritis.

[0066] Example 41. Use of the polypeptide or composition according to claim 40, wherein the inflammatory disease and / or autoimmune disease is rheumatoid arthritis.

[0067] Example 42. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), wherein each ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein: a) The first ISVD contains i. An amino acid sequence having the same as SEQ ID NO: 6 or a CDR1 differing from SEQ ID NO: 6 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 10 or a CDR2 differing from SEQ ID NO: 10 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 14 or a CDR3 differing from SEQ ID NO: 14 by 2 or 1 amino acids; b) The second ISVD contains iv. An amino acid sequence having the same as SEQ ID NO: 8 or a CDR1 differing from SEQ ID NO: 8 by 2 or 1 amino acids; v. Having the amino acid sequence of SEQ ID NO: 12 or CDR2 differing from SEQ ID NO: 12 by 2 or 1 amino acid; and vi. Having the amino acid sequence of SEQ ID NO: 16 or CDR3 differing from SEQ ID NO: 16 by 2 or 1 amino acid; and c) The third ISVD contains vii. An amino acid sequence having the same as SEQ ID NO: 9 or a CDR1 differing from SEQ ID NO: 9 by 2 or 1 amino acids; viii. A CDR2 having the amino acid sequence of SEQ ID NO: 13 or differing from SEQ ID NO: 13 by 2 or 1 amino acid; and ix. An amino acid sequence having the same as SEQ ID NO: 17 or a CDR3 differing from SEQ ID NO: 17 by 2 or 1 amino acids. The ISVDs are optionally included in order starting from the N end.

[0068] Example 43. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid containing a nucleotide sequence encoding said polypeptide, said polypeptide or composition being used as a drug, wherein said polypeptide comprises or is composed of at least three immunoglobulin single variable domains (ISVDs), wherein each said ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein: a) The first ISVD contains i. An amino acid sequence having the same as SEQ ID NO: 9 or a CDR1 differing from SEQ ID NO: 9 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 13 or a CDR2 differing from SEQ ID NO: 13 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 17 or a CDR3 differing from SEQ ID NO: 17 by 2 or 1 amino acids; b) The second ISVD contains iv. An amino acid sequence having the same as SEQ ID NO: 150 or a CDR1 differing from SEQ ID NO: 150 by 2 or 1 amino acids; v. Having the amino acid sequence of SEQ ID NO: 151 or a CDR2 differing from SEQ ID NO: 151 by 2 or 1 amino acid; and vi. An amino acid sequence having the same as SEQ ID NO: 152 or a CDR3 differing from SEQ ID NO: 152 by 2 or 1 amino acid; and c) The third ISVD contains vii. An amino acid sequence having the same as SEQ ID NO: 153 or a CDR1 differing from SEQ ID NO: 153 by 2 or 1 amino acids; viii. An amino acid sequence having the same as SEQ ID NO: 154 or a CDR2 differing from SEQ ID NO: 154 by 2 or 1 amino acid; and ix. An amino acid sequence having the same as SEQ ID NO: 155 or a CDR3 differing from SEQ ID NO: 155 by 2 or 1 amino acids. The first ISVD, the second ISVD, and the third ISVD are optionally included in an order starting from the N end.

[0069] Example 44. A polypeptide or composition for the stated purpose according to Example 43, wherein: a) The first ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 13, and CDR3 having the amino acid sequence of SEQ ID NO: 17; b) The second ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 150, CDR2 having the amino acid sequence of SEQ ID NO: 151, and CDR3 having the amino acid sequence of SEQ ID NO: 152; and c) The third ISVD comprises CDR1 having an amino acid sequence of SEQ ID NO: 153, CDR2 having an amino acid sequence of SEQ ID NO: 154, and CDR3 having an amino acid sequence of SEQ ID NO: 155.

[0070] Example 45. A polypeptide or composition for the said use according to any one of Examples 43 or 44, wherein: a) The amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO: 5; b) The amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO: 148; and c) The amino acid sequence of the third ISVD has a sequence identity greater than 90% with SEQ ID NO: 149.

[0071] Example 46. A polypeptide or composition for the said use according to any one of Examples 43 to 45, wherein: a) The first ISVD has the amino acid sequence of SEQ ID NO: 5; b) The second ISVD has the amino acid sequence of SEQ ID NO: 148; and c) The third ISVD has the amino acid sequence of SEQ ID NO: 149.

[0072] Example 47. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid containing a nucleotide sequence encoding said polypeptide, said polypeptide or composition being used as a drug, wherein said polypeptide comprises or is composed of at least three immunoglobulin single variable domains (ISVDs), wherein each said ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein: a) The first ISVD contains i. An amino acid sequence having the same as SEQ ID NO: 9 or a CDR1 differing from SEQ ID NO: 9 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 13 or a CDR2 differing from SEQ ID NO: 13 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 17 or a CDR3 differing from SEQ ID NO: 17 by 2 or 1 amino acids; b) The second ISVD contains i. An amino acid sequence having the same as SEQ ID NO: 160 or a CDR1 differing from SEQ ID NO: 160 by 2 or 1 amino acids; ii. An amino acid sequence having the same as SEQ ID NO: 161 or a CDR2 differing from SEQ ID NO: 161 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 162 or a CDR3 differing from SEQ ID NO: 162 by 2 or 1 amino acid; and c) The third ISVD contains i. An amino acid sequence having the same as SEQ ID NO: 150 or a CDR1 differing from SEQ ID NO: 150 by 2 or 1 amino acids; ii. An amino acid sequence having the same as SEQ ID NO: 151 or a CDR2 differing from SEQ ID NO: 151 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 152 or a CDR3 differing from SEQ ID NO: 152 by 2 or 1 amino acids. The first ISVD, the second ISVD, and the third ISVD are optionally included in an order starting from the N end.

[0073] Example 48. A polypeptide or composition for the stated purpose according to Example 47, wherein: a) The first ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 13, and CDR3 having the amino acid sequence of SEQ ID NO: 17; b) The second ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 160, CDR2 having the amino acid sequence of SEQ ID NO: 161, and CDR3 having the amino acid sequence of SEQ ID NO: 162; and c) The third ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO: 150, CDR2 having the amino acid sequence of SEQ ID NO: 151, and CDR3 having the amino acid sequence of SEQ ID NO: 152.

[0074] Example 49. A polypeptide or composition for the said use according to any one of Examples 47 or 48, wherein: a) The amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO: 5; b) The amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO: 159; and c) The amino acid sequence of the third ISVD has a sequence identity greater than 90% with SEQ ID NO: 148.

[0075] Example 50. A polypeptide or composition for the said use according to any one of Examples 47 to 49, wherein: a) The first ISVD has the amino acid sequence of SEQ ID NO: 5; b) The second ISVD has the amino acid sequence of SEQ ID NO: 159; and c) The third ISVD has the amino acid sequence of SEQ ID NO: 148.

[0076] Example 51. A polypeptide or composition for the purpose according to any one of Examples 43 to 50, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0077] Example 52. A polypeptide or composition for the purpose according to any one of Examples 51, wherein the one or more other groups, residues, portions or binding units that provide an increased half-life to the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0078] Example 53. A polypeptide or composition for the stated purpose according to Example 52, wherein the binding unit providing the increased half-life of the polypeptide is an ISVD that can bind to human serum albumin.

[0079] Example 54. A polypeptide or composition for the stated purpose according to Example 53, wherein the ISVD bound to human serum albumin comprises i. An amino acid sequence having the same as SEQ ID NO: 7 or a CDR1 differing from SEQ ID NO: 7 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 11 or a CDR2 differing from SEQ ID NO: 11 by 2 or 1 amino acids; and iii. Having the amino acid sequence of SEQ ID NO: 15 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO: 15.

[0080] Example 55. A polypeptide or composition for the purpose according to any one of Examples 53 or 54, wherein the ISVD that binds to human serum albumin comprises CDR1 having an amino acid sequence of SEQ ID NO: 7, CDR2 having an amino acid sequence of SEQ ID NO: 11, and CDR3 having an amino acid sequence of SEQ ID NO: 15.

[0081] Example 56. A polypeptide or composition for the purpose according to any one of Examples 53 to 55, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity with SEQ ID NO: 3.

[0082] Example 57. A polypeptide or composition for the purpose according to any one of Examples 53 to 56, wherein the ISVD that binds to human serum albumin has the amino acid sequence of SEQ ID NO: 3.

[0083] Example 58. A polypeptide or composition for the purpose according to any one of Examples 43 to 57, wherein the polypeptide has an extension of 1 to 5 amino acid residues at its C-terminus, preferably an extension of a single amino acid residue, wherein the amino acid residues are independently selected from naturally occurring amino acids, preferably independently selected from glycine or alanine, leucine, isoleucine and valine, more preferably alanine and glycine, and most preferably alanine.

[0084] Example 59. A polypeptide or composition for the purpose according to any one of Examples 43 to 46, wherein the amino acid sequence of the polypeptide comprises or is composed of the following amino acid sequence, the amino acid sequence having greater than 90% sequence identity with SEQ ID NO: 147.

[0085] Example 60. A polypeptide or composition for the purpose according to any one of Examples 43 to 46, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 147.

[0086] Example 61. A polypeptide or composition for the purpose according to any one of Examples 47 to 50, wherein the amino acid sequence of the polypeptide comprises or is composed of the following amino acid sequence, the amino acid sequence having greater than 90% sequence identity with SEQ ID NO: 158.

[0087] Example 62. A polypeptide or composition for the purpose according to any one of Examples 47 to 50, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 158.

[0088] Example 63. A polypeptide or composition according to any one of Examples 43-62 for the purpose described herein, said polypeptide or composition for treating inflammatory and / or autoimmune diseases, such as rheumatoid arthritis.

[0089] Example 64. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of Examples 43 to 63.

[0090] Example 65. A host or host cell comprising the nucleic acid as described in Example 64.

[0091] Example 66. A method for generating a polypeptide according to any one of Examples 43 to 63, the method comprising at least the following steps: a) Expressing the nucleic acid as described in Example 64 in a suitable host cell or host organism or another suitable expression system; optionally, proceeding thereafter: b) Isolate and / or purify the polypeptide according to any one of Examples 43 to 63.

[0092] Example 67. A composition comprising at least one polypeptide according to any one of Examples 43 to 63 or a nucleic acid according to Example 64.

[0093] Example 68. The composition according to Example 67 is a pharmaceutical composition, which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprises one or more other pharmaceutically active peptides and / or compounds.

[0094] Example 69. A method for treating inflammatory diseases and / or autoimmune diseases such as rheumatoid arthritis, wherein the method comprises administering to a subject in need a pharmaceutically active amount of a polypeptide according to any one of Examples 43 to 63 or a composition according to any one of Examples 67 or 68.

[0095] Example 70. The method according to Example 69, wherein the inflammatory disease and / or autoimmune disease is rheumatoid arthritis.

[0096] Example 71. Use of the polypeptide according to any one of Examples 43 to 63 or the composition according to any one of Examples 67 or 68 in the preparation of a pharmaceutical composition for treating inflammatory diseases and / or autoimmune diseases such as rheumatoid arthritis.

[0097] Example 72. Use of the polypeptide or composition according to claim 71, wherein the inflammatory disease and / or autoimmune disease is rheumatoid arthritis. Simple Explanation of the Diagram

[0098] [picture] [1]: A sensory map showing the simultaneous binding of recombinant soluble hTNF-α and hIL-6 to F027201062 captured via HSA.

[0099] [picture] [2]: The representative figure (experiment n3 in Table 11) shows the inhibition of soluble human and cynomolgus monkey TNF-α by ISVD F027201062 and the reference anti-TNF-α mAb IRR00096, which served as a negative control ISVD, in the Glo response™ HEK293_NFκB-NLucP reporter assay.

[0100] [picture] [3] [:] represents Figure n3 in Table 12, which shows the inhibition of soluble human and cynomolgus monkey IL-6 by ISVD F027201062 and the reference anti-IL-6 mAb1 and mAb2 (IRR00096) as negative controls in the IL-6-induced TF-1 proliferation assay. LCI = lower confidence interval, UCI = upper confidence interval.

[0101] [picture] [4] [:] Box plot, which shows the binding of pre-existing antibodies present in 96 human serum samples to anti-IL-6 / anti-TNF-α bispecific ISVD F027201062 and control ISVD F027301186.

[0102] [picture] [5] [:] Progressive arthritis in a collagen-induced arthritis model. N=13 male DBA / 1 mice were immunized twice with 100 μg of adjuvant collagen II on days 0 and 21. Starting on day 22, mice were treated twice weekly with the injected compound via intraperitoneal injection. Treatment was discontinued after day 56. Clinical signs and symptom scores of joint inflammation were assessed in the mice. Mean ± SEM is shown. Statistical analysis was performed using a two-way ANOVA, which compared the number of days treated throughout the disease course with the negative control.

[0103] [picture] [6] The area under the curve of arthritis score over time in a collagen-induced arthritis model. AUCs were calculated for days 21–56 of treatment, days 57–91 of treatment-free period, and the entire study duration. Individual values ​​and mean ± SEM are shown. Statistical analysis was performed using a one-way ANOVA with comparisons between treatment and negative controls.

[0104] [picture] [7] Anti-collagen II antibodies in plasma on day 91 of a collagen-induced arthritis model. Collagen II-specific titers were determined by ELISA in collagen II-coated plates. Individual values ​​and mean ± SEM are shown. Statistical analysis was a one-way analysis of variance with comparisons between treatment and negative controls.

[0105] [picture] [8] [:] Histological scores of the metatarsophalangeal joints of the hindfoot on day 91 of a collagen-induced arthritis model. Histological processing of the hindfoot was performed, and sections were stained with hematoxylin and eosin, as well as safranin-O, for visualization of chondroitin proteoglycans. Scores were assessed individually by two blinded examiners. Mean ± SEM is shown. Statistical analysis was a two-way analysis of variance, comparing all four aspects of the histological assessment between treatment and negative controls.

[0106] [picture] [9] [:] RNAseq from the paw pads of mice with collagen-induced arthritis treated with anti-IL-6, anti-TNF, and a combination of both. a) Bian plot of significantly dysregulated transcripts (FC>1.2) on day 91 in isotype-treated control mice. b) Major upregulated and downregulated pathways in CIA (left) and after treatment with the combination of anti-TNF and anti-IL-6 (right).

[0107] [picture]

[10] : A quantitative systems pharmacology model for rheumatoid arthritis (RA). The model was analogous to the RA disease score DAS28-CRP after treatment with anti-TNF-α and anti-IL-6 comparators and showed improved clinical efficacy (based on DAS28-CRP), even at a lower dose of F027201062. [picture]

[11] [:] Co-culture of fibroblast-like synovial cells (FLS) from rheumatoid arthritis (RA) patients with T cells from healthy donors. Co-culture and stimulation with IL-17A, sIL-6R, and anti-CD3 induced TNF-α and IL-6 levels similar to those published in human joints from RA patients.

[0108] [picture]

[12] [:] MMP1 secretion was cumulatively inhibited in RA-FLS / T cell co-cultures using F027201062. An IgG1 isotype control was used as a negative control for the comparative antibody. An ISVD isotype control was a negative control for multispecific ISVD. Comparative antibodies and combinations thereof were used as positive controls. Only donors responsive to anti-TNF-α treatment were selected. Combinations reflected full-dose combinations (e.g., 200 nM anti-human TNF-α + 200 nM anti-human IL-6). F027201062 and F027200926 are anti-TNF-α / IL-6 ISVD constructs. Figures shown are mean ± SEM, 8 different T cell donors, and 3 technical replicates.

[0109] [picture]

[13] [:] G-CSF secretion was cumulatively inhibited in RA-FLS / T cell co-cultures using F027201062. An IgG1 isotype control was used as a negative control for the comparative antibody. An ISVD isotype control was a negative control for multispecific ISVD. Comparative antibodies and their combinations were used as positive controls. Combinations reflect full-dose combinations (e.g., 200 nM anti-human TNF-α + 200 nM anti-human IL-6). F027201062 and F027200926 are anti-TNF-α / IL-6 ISVD constructs. Only donors responsive to anti-TNF-α treatment were selected. The results are shown as mean ± SEM, with 8 different T cell donors and 3 technical replicates.

[0110] [picture]

[14] [:] CXCL13 was cumulatively inhibited in human adenoid cultures using F027201062. An IgG1 isotype control was used as a negative control for the comparative antibody. An ISVD isotype control was a negative control for F027201062 (anti-TNF-α / IL-6 ISVD construct). Comparative antibodies and combinations thereof were used as positive controls. Combinations reflect full-dose combinations (e.g., 200 nM anti-human TNF-α + 200 nM anti-human IL-6). Mean ± SEM is shown, with 4 to 7 different donors and 2 technical replicates.

[0111] [picture]

[15] [:] CXCL13 was cumulatively inhibited in human adenoid cultures using F027201062 at 200 nM. An IgG1 isotype control was used as a negative control for the comparative antibody. An ISVD isotype control was used as a negative control for F027201062 (anti-TNF-α / IL-6 ISVD construct). Comparative antibodies and their combinations were used as positive controls. Combinations reflect the full-dose combination (200 nM anti-human TNF-α + 200 nM anti-human IL-6). Mean ± SEM is shown for 7 different donors and 2 technical replicates. Statistical analysis was performed using one-way ANOVA and Tukey multiple comparison test. ** p < 0.01. *** p < 0.001.

[0112] [picture]

[16] [:] The TNF-α-dependent efficacy of the anti-TNF-α / IL-6 ISVD construct in human whole blood assays is shown. The IC50 of MCP-1 inhibition in SEB-stimulated whole blood is presented. An IgG1 isotype control was used as a negative control for the comparative antibody. The ISVD isotype control was a negative control for the anti-TNF-α / IL-6 ISVD construct. An anti-hTNF-α comparative antibody was used as a positive control. The following anti-TNF-α / IL-6 ISVDs were evaluated: F027200926, F027201029, F027201060, F027201061, and F027201062. Mean ± SEM values ​​are shown for seven different donors.

[0113] [picture]

[17] [:] The TNF-α-dependent efficacy of the anti-TNF / IL-6 ISVD construct in human whole blood assays is shown. A dose-dependent inhibition of CCL4 in SEB-stimulated whole blood is demonstrated. An IgG1 isotype control was used as a negative control for the comparative antibody. The ISVD isotype control is a negative control for the anti-TNF-α / IL-6 ISVD construct. An anti-hTNF-α comparative antibody was used as a positive control. F027201062 is a multispecific anti-TNF-α / IL-6 ISVD. Mean ± SEM is shown for 7 different donors.

[0114] [picture]

[18] [:] TNF-α-dependent efficacy of the anti-TNF / IL-6 ISVD construct in human whole blood assays. IC50 of CCL4 inhibition in SEB-stimulated whole blood is shown. IgG1 isotype control was used as a negative control for the comparative antibody. ISVD isotype control was a negative control for the anti-TNF-α / IL-6 ISVD construct. Anti-hTNF-α comparative antibody was used as a positive control. F027201062 is a multispecific anti-TNF-α / IL-6 ISVD. Mean ± SEM is shown for 7 different donors.

[0115] [picture]

[19] : IL-6-dependent efficacy of the anti-TNF-α / IL-6 ISVD construct in human RA-FLS (fibroblast-like synovial cells from patients with rheumatoid arthritis). Inhibition of VEGF-A secretion against isotype controls is shown. The IgG1 isotype control was used as a negative control for the comparative antibody. The ISVD isotype control was a negative control for the anti-TNF-α / IL-6 ISVD construct. Dose-dependent inhibition was achieved using the anti-hIL-6 comparative antibody (positive control) and the following anti-TNF-α / IL-6 ISVDs: F027200926, F027201029, F027201060, F027201061, and F027201062. Mean ± SEM values ​​were shown at two different stages, for three different rheumatoid arthritis donors.

[0116] [picture]

[20] : IL-6-dependent efficacy of the anti-TNF-α / IL-6 ISVD construct in human RA-FLS (fibroblast-like synovial cells from patients with rheumatoid arthritis). IgG1 isotype control was used as a negative control for the comparative antibody. ISVD isotype control was a negative control for the anti-TNF-α / IL-6 ISVD construct. Anti-hIL-6 comparative antibody was used as a positive control. IC50 for VEGF-A is shown for the anti-hIL-6 comparative antibody (positive control) and the following anti-TNF-α / IL-6 ISVDs: F027200926, F027201029, F027201060, F027201061, F027201062. Mean ± SEM is shown for testing in two different phases with three different rheumatoid arthritis donors.

[0117] [picture] [twenty one] Efficacy of [:]F027201062 in a Tg197 hTNF-α-driven arthritis model. Arthritis score over time. Eight Tg197 mice (4 males and 4 females) were treated twice weekly by intraperitoneal injection of the indicator compound for 5 weeks. Arthritis scores were monitored weekly. Mean ± SEM is shown. Statistical analysis was a two-way analysis of variance weekly, with comparisons between treatment and negative controls.

[0118] [picture] [twenty two] Efficacy of [:]F027201062 in a Tg197 hTNF-α-driven arthritis model. Area under the curve of arthritis score over time. Individual values ​​and mean ± SEM are shown. Statistical analysis is a one-way analysis of variance with comparisons between treatment and negative control.

[0119] [picture] [twenty three] [:]F027201062 Power of F027201062 on ankle joint histological scores in a Tg197 hTNF-α-driven arthritis model (n = 2 per animal). After sacrifice at the end of the study, both hind paws were histologically processed, and ankle joint sections were stained with hematoxylin and eosin. Slides were read and scored in a blinded manner. Individual values ​​and mean ± SEM are shown. Statistical analysis was a one-way ANOVA with comparisons between treatment and negative controls.

[0120] [picture] [twenty four] [:]IL-6-induced haptoglobin secretion. Indicator compounds were injected into N = 8 female BALB / c mice. Eight hours later, if indicated, 25 μg / kg recombinant human IL-6 was injected intraperitoneally into the mice. Sixteen hours later, the mice were exsanguinated, and haptoglobin levels in the plasma were analyzed using fluorescent bead assays. Individual values ​​and mean ± SEM are shown. Statistical analysis was a one-way ANOVA with comparisons between treatment and negative controls.

[0121] [picture]

[25] Splenomegaly in hIL-6 transgenic mice. N = 6–7 male and female hemizygous C.B6-Tg(H2-L-IL6)1 Kish / J mice aged 57–71 days were treated with intraperitoneal injection of the indicator compound three times weekly for 2 weeks. After sacrifice, the spleen was removed and its weight recorded. Wild-type littermates served as controls. Individual values ​​and mean ± SEM are shown. Statistical analysis was a one-way analysis of variance with comparisons between treatment and negative controls.

[0122] [picture]

[26] Hypergammaglobulinemia in hIL-6 transgenic mice. N = 6–7 male and female hemizygous C.B6-Tg(H2-L-IL6)1 Kish / J mice aged 57–71 days were treated with an indicator compound via intraperitoneal injection three times weekly for 2 weeks. After sacrifice, the mice were exsanguinated, and IgG1 and IgG2a isotype immunoglobulins were measured by chemiluminescent bead assay. Plasma from wild-type littermates served as a control. Individual values ​​and mean ± SEM are shown. Statistical analysis was a one-way ANOVA with comparisons between treatment and negative controls.

[0123] [picture]

[27] Single-dose pharmacokinetics of F027201062 in non-human primates. Serum concentration curves of F027201062 after single-dose administration at indicated concentrations and routes of administration (n = 3 male, untreated cynomolgus monkeys per group). The red dashed line indicates the presence of ADA in all three groups.

[0124] [picture]

[28] : F027201062 amino acid sequence (SEQ ID NO: 1). Implementation

[0125] This article presents a novel drug for the treatment of inflammatory diseases and / or autoimmune diseases such as rheumatoid arthritis (RA).

[0126] The inventors of this invention have discovered that peptides simultaneously targeting TNF-α and IL-6 are more efficient at modulating the symptoms of rheumatoid arthritis in vitro and / or in vivo compared to single-specific anti-TNF-α or anti-IL-6 peptides. These peptides can be produced efficiently (e.g., in a microbial host). Furthermore, limited reactivity of such peptides to antibodies pre-existing in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with the antibody construct) has been demonstrated. In some embodiments, such peptides can be conveniently administered and exhibit a sufficiently long half-life in the subject to be treated to maintain a limited number of consecutive treatments and thus these treatments can be conveniently spaced out over time.

[0127] The polypeptide is at least bispecific, but may also be, for example, trispecific, tetraspecific, or pentaspecific. Furthermore, the polypeptide is at least trivalent, but may also be, for example, tetravalent, pentavalent, or hexavalent, preferably tetravalent.

[0128] The terms "bispecific," "trispecific," "quadrispecific," or "pentaspecific" all fall under the term "multispecific," and respectively refer to binding to two, three, four, or five different target molecules. The terms "divalent," "trivalent," "quadrivalent," "pentavalent," or "hexavalent" all fall under the term "multivalent," and respectively indicate the presence of two, three, four, or five binding units (e.g., ISVDs). For example, the polypeptide may be trispecific and tetravalent, such as a polypeptide containing four ISVDs or composed of them, one ISVD binding to human TNF-α, two ISVDs binding to human IL-6, and one ISVD binding to human serum albumin (e.g., ISVD construct F027201062). Such a polypeptide may also be doubly complementary, for example, in the case where two ISVDs bind to two different epitopes on human IL-6. The term "doubly complementary" refers to binding to two different parts (e.g., epitopes) of the same target molecule.

[0129] As used herein, the terms "first ISVD," "second ISVD," "third ISVD," etc., indicate only the presence of one, two, or three ISVDs, but also preferably indicate the relative positions of the ISVDs to each other, with the numbering starting from the N-terminus of the polypeptide herein. Thus, "first ISVD" is preferably closer to the N-terminus than "second ISVD," and "second ISVD" is closer to the N-terminus than "third ISVD," and so on. Therefore, when considered from the C-terminus, the ISVD arrangement is reversed. Since the numbering is not absolute and may only indicate the relative positions of the at least three ISVDs, the presence of other binding units / building blocks, such as additional ISVDs binding to TNF-α or IL-6, or ISVDs binding to another target, is not excluded. Furthermore, the possibility of placing other binding units / building blocks (such as ISVDs) in between is not excluded. For example, as further described below (see Section 5.3 "(In vivo) half-life extension"), the polypeptide may also contain another ISVD that binds to human serum albumin, which may even be located between, for example, "first ISVD" and "second ISVD".

[0130] In view of the above, this article provides a polypeptide comprising at least three ISVDs or thereof, wherein at least one ISVD specifically binds to TNF-α and at least two ISVDs specifically bind to IL-6.

[0131] The components of the polypeptide, such as ISVD, can be linked together by one or more suitable linkers (such as peptide linkers).

[0132] The use of linkers to connect two or more (multi)peptides is well known in the industry. Exemplary peptide linkers are shown in Table A-5. One commonly used class of peptide linkers is called "Gly-Ser" or "GS" linkers. These are linkers that consist essentially of glycine (G) and serine (S) residues and typically contain one or more repeats of a peptide motif, such as the GGGGS (SEQ ID NO: 65) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7, or greater). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 68), the 15GS linker (n = 3), and the 35GS linker (n = 7). For example, see Chen et al., Adv. Drug Deliv. Rev. 2013 Oct 15; 65(10): 1357–1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27 (10): 325-330.

[0133] In some embodiments of the peptides described herein, 9GS linkers may be used to link the peptide components to each other.

[0134] In one embodiment, the ISVD that specifically binds to TNF-α is located at the C-terminus of the peptide. The inventors have surprisingly discovered that such a configuration can significantly increase the potency of the compound and improve certain characteristics important for optimal compound production, such as solubility and performance levels.

[0135] In another embodiment, an ISVD that specifically binds to IL-6 is located at the C-terminus or N-terminus of the polypeptide, preferably at the N-terminus.

[0136] Therefore, in some embodiments, the polypeptide comprises or consists of the following items in sequence, starting from the N-terminus of the polypeptide: a first ISVD that specifically binds to IL-6, as defined herein, an optional binding unit that provides an increased half-life for the polypeptide; a second ISVD that specifically binds to IL-6; and a third ISVD that specifically binds to TNF-α. In a preferred embodiment, the binding unit that provides an increased half-life for the polypeptide is an ISVD.

[0137] In some embodiments, the polypeptide comprises, or consists of, the following items in sequence starting from the N-terminus of the polypeptide: a first ISVD specifically binding to IL-6, a linker, an ISVD specifically binding to human serum albumin (HSA), a linker, a second ISVD specifically binding to IL-6, a linker, and an ISVD specifically binding to TNF-α. In some embodiments, the linker is a 9GS linker.

[0138] Such a configuration of the polypeptide can provide increased product yield, good CMC characteristics, and optimized functionality and potency in regulating immune responses.

[0139] Therefore, in some embodiments, the polypeptide exhibits a solubility of at least 120 mg / ml, such as at least 130 mg / ml, such as at least 140 mg / ml, preferably at least 145 mg / ml.

[0140] In some embodiments, the peptides described herein exhibit reduced binding to pre-existing antibodies in human serum. For this purpose, in one embodiment, the peptide has valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD (preferably at least one ISVD located at the C-terminus of the peptide) or in each ISVD. In another embodiment, the peptide has an extension of 1 to 5 amino acids (naturally occurring, non-naturally occurring, or mixtures thereof) at the C-terminus of the C-terminal ISVD, such as a single alanine (A) extension. The C-terminus of the ISVD may be VTVSS (SEQ ID NO: 81). In another embodiment, the peptide has lysine (K) or glutamic acid (Q) at position 110 (according to Kabat numbering) in at least one ISVD (preferably at least one ISVD located at the C-terminus of the peptide) or in each ISVD. In another embodiment, the ISVD contains lysine (K) or glutamic acid (Q) at position 112 (according to Kabat numbering) in at least one ISVD (preferably at least one ISVD located at the C-terminus of the polypeptide) or in each ISVD. In some embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 82), VQVSS (SEQ ID NO: 83), VTVKS (SEQ ID NO: 84), VTVQS (SEQ ID NO: 85), VKVKS (SEQ ID NO: 86), VKVQS (SEQ ID NO: 87), VQVKS (SEQ ID NO: 88), or VQVQS (SEQ ID NO: 89), such that after the addition of a single alanine, the C-terminus of the polypeptide has, for example, the sequence VTVSSA (SEQ ID NO: 90), VKVSSA (SEQ ID NO: 91), VQVSSA (SEQ ID NO: 92), VTVKSA (SEQ ID NO: 93), VTVQSA (SEQ ID NO: 94), VKVKSA (SEQ ID NO: 95), VKVQSA (SEQ ID NO: 96), or VQVKSA (SEQ ID NO: 89). 97) or VQVQSA (SEQ ID NO: 98). In one embodiment, the sequence is VKVSSA (SEQ ID NO: 91).In another embodiment, the polypeptide has valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD, optionally lysine (K) or glutamic acid (Q), preferably K, at amino acid position 110 (according to Kabat numbering) in at least one ISVD (preferably at least in the ISVD located at the C-terminus of the polypeptide), and the C-terminus of the C-terminal ISVD has an extension of 1 to 5 amino acids (natural, non-natural, or mixtures thereof), such as a single alanine (A) extension (such that the C-terminus of the polypeptide has, for example, the sequence VTVSSA (SEQ ID NO: 90), VKVSSA (SEQ ID NO: 91), or VQVSSA (SEQ ID NO: 92)). For further information in this regard, see, for example, WO 2012 / 175741 and WO 2015 / 173325, which are incorporated herein by reference in their entirety. The amino acid residues used for the extension are preferably selected independently from glycine, alanine, valine, leucine, or isoleucine, more preferably glycine and alanine, and most preferably alanine. Preferably, the extension consists of a single amino acid residue.

[0141] In another embodiment, the polypeptide herein comprises or is composed of an amino acid sequence having greater than 90%, such as greater than 95% or greater than 99%, sequence identity with SEQ ID NO: 1, wherein the CDRs of the four ISVDs are defined as items A to D (or A' to D', if Kabat definitions are used) as shown in the following sections “5.1 Immunoglobulin Single Variable Domain” and “5.3 (In Vivo) Half-Life Extension”, wherein in particular: • The first ISVD that specifically binds to IL-6 has a CDR1 containing the amino acid sequence of SEQ ID NO: 6, a CDR2 containing the amino acid sequence of SEQ ID NO: 10, and a CDR3 containing the amino acid sequence of SEQ ID NO: 14; • The second ISVD that specifically binds to IL-6 has a CDR1 containing the amino acid sequence of SEQ ID NO: 8, a CDR2 containing the amino acid sequence of SEQ ID NO: 12, and a CDR3 containing the amino acid sequence of SEQ ID NO: 16; • The ISVD that specifically binds to TNF-α has a CDR1 containing the amino acid sequence of SEQ ID NO: 9, a CDR2 containing the amino acid sequence of SEQ ID NO: 13, and a CDR3 containing the amino acid sequence of SEQ ID NO: 17; and • The ISVD that binds to human serum albumin has CDR1 containing the amino acid sequence of SEQ ID NO: 7, CDR2 containing the amino acid sequence of SEQ ID NO: 11, and CDR3 containing the amino acid sequence of SEQ ID NO: 15. Alternatively, if Kabat is used as the definition: • The first ISVD that specifically binds to IL-6 has a CDR1 containing the amino acid sequence of SEQ ID NO: 33, a CDR2 containing the amino acid sequence of SEQ ID NO: 37, and a CDR3 containing the amino acid sequence of SEQ ID NO: 14; • The second ISVD that specifically binds to IL-6 has a CDR1 containing the amino acid sequence of SEQ ID NO: 35, a CDR2 containing the amino acid sequence of SEQ ID NO: 39, and a CDR3 containing the amino acid sequence of SEQ ID NO: 16; • The ISVD that specifically binds to TNF-α has a CDR1 containing the amino acid sequence of SEQ ID NO: 36, a CDR2 containing the amino acid sequence of SEQ ID NO: 40, and a CDR3 containing the amino acid sequence of SEQ ID NO: 17; and • The ISVD that binds to human serum albumin has CDR1 containing the amino acid sequence of SEQ ID NO: 34, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR3 containing the amino acid sequence of SEQ ID NO: 15.

[0142] In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 1.

[0143] In some embodiments, the peptides described herein have at least half, at least the same, or even higher binding affinity for human TNF-α and human IL-6 compared to the peptides composed of the amino acids of SEQ ID NO: 1, wherein the binding affinity is measured using the same method (e.g., SPR). [5.1] [Immunoglobulin single variable domain]

[0144] The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen-binding site is located on a single immunoglobulin domain and formed by it. This distinguishes the immunoglobulin ISVD from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, discFv), where 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 VH and VL will contribute to the antigen-binding site; that is, a total of six CDRs will participate in the formation of the antigen-binding site.

[0145] Given the above definitions, the antigen-binding domain of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the industry) or Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or biantibodies (all known in the industry) derived from such conventional four-chain antibodies will generally not be considered as immunoglobulin monovariable domains. This is because in these cases, it is not a single immunoglobulin domain that binds to the corresponding antigenic epitope, but rather a pair of associated immunoglobulin domains (such as light chain and heavy chain variable domains), i.e., the V HV L pair of immunoglobulin domains, that bind to the corresponding antigenic epitope, which bind together to the corresponding antigenic epitope.

[0146] In contrast, immunoglobulin monovariable domains (MMUs) can specifically bind to antigenic epitopes without pairing with other immunoglobulin MMUs. The binding site of an immunoglobulin MMU is formed by a single VH, a single VHH, or a single VL domain.

[0147] Therefore, the single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0148] Immunoglobulin single variable domains (ISVDs) can be, for example, heavy chain ISVDs, such as VH, VHH, including camel-like VH or humanized VHH. According to some embodiments, the immunoglobulin single variable domain (ISVD) is VHH, including camel-like VH or humanized VHH. Heavy chain ISVDs can be derived from conventional four-chain antibodies or heavy chain antibodies.

[0149] For example, an immunoglobulin monovariable domain can be a single-domain antibody (or an amino acid sequence suitable for use as a single-domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) or Nanobody® (as defined herein, and including but not limited to V HH); other monovariable domains, or any suitable fragment of any of them.

[0150] Specifically, the immunoglobulin single variable domain can be Nanobody® (such as V HH, including humanized V HH or camel-like V H) or a suitable fragment thereof. Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx NV.

[0151] The "VHH domain," also known as VHH, VHH antibody fragment, and VHH antibody, was originally described as a variable domain of antigen-binding immunoglobulin of "heavy chain antibodies" (i.e., "without light chain antibodies"; Hamers-Casterman et al., Nature 363: 446-448, 1993). The term "VHH domain" was chosen to distinguish these variable domains from the heavy chain variable domains (referred to herein as "VH domains") present in conventional 4-chain antibodies and the light chain variable domains (referred herein as "VL domains") present in conventional 4-chain antibodies. For a further description of V HH, reference is made to Muyldermans' review article (Reviews in Molecular Biotechnology 74: 277-302, 2001), and the following patent applications mentioned as general background: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of the Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; and WO 94 / 95 / 65057 of the Vlaams Instituut voor Biotechnologie (VIB). 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531 of Algonomics NV and Ablynx NV; WO 01 / 90190 of the National Research Council of Canada; WO 03 / 025020 (= EP 1433793) of the Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, each of which is incorporated herein by reference in its entirety.

[0152] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for desired specificity. Alternatively, immunoglobulins can be generated by screening immature or synthetic libraries, for example, through phage display.

[0153] The generation of immunoglobulin sequences (such as Nanobodies®) has been extensively described in various publications (of which WO 94 / 04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001) can be cited as examples, which are incorporated herein by reference in their entirety). In these methods, camelids are immunized with a target antigen to induce an immune response against the target antigen. The Nanobodies library obtained from the immunization is further screened for Nanobodies that bind to the target antigen.

[0154] In these cases, antibody production requires purified antigens for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production.

[0155] Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0156] Immunoglobulin sequences from various sources can be used, including mouse, rat, rabbit, donkey, human, and camel immunoglobulin sequences. This document also includes fully human, humanized, or chimeric sequences. For example, this document includes camel immunoglobulin sequences and humanized camel immunoglobulin sequences or camel-like domain antibodies, such as camel-like dAbs described by Ward et al. (see, for example, WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996, each of which is incorporated herein by reference in its entirety). In addition, this article also uses fused immunoglobulin sequences, such as those forming multivalent and / or multispecific constructs (for multivalent and multispecific polypeptides and their formulations containing one or more VHH domains, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001 and, for example, WO 96 / 34103 and WO 99 / 23221, each of which is incorporated herein by reference in its entirety) and immunoglobulin sequences containing markers or other functional parts (e.g., toxins, labels, radiochemicals, etc., which may be derived from the immunoglobulin sequences described herein), etc.

[0157] "Humanized VHH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain but has been "humanized," i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and particularly the structural sequence) with one or more amino acid residues present at corresponding positions in the VH domain of a conventional human 4-chain antibody (e.g., as shown above). This can be done in a manner known per se, which is clear to those skilled in the art, for example, based on further description herein and in the literature (e.g., WO 2008 / 020079). Furthermore, it should be noted that such humanized VHH can be obtained in any suitable manner known per se, and is therefore not strictly limited to peptides already obtained using peptides containing naturally occurring VHH domains as starting materials.

[0158] "Camelization of VH" comprises an amino acid sequence that corresponds to the naturally occurring VH domain but has been "camelized," i.e., camelized by replacing one or more amino acid residues in the naturally occurring VH domain of a conventional 4-chain antibody with one or more amino acid residues present at corresponding positions in the VHH domain of a heavy chain antibody. This can be done in a manner known per se, which will be clear to those skilled in the art, for example, based on further description herein and literature (e.g., WO 2008 / 020079). As defined herein, such "camelization" substitutions can be inserted at amino acid positions forming and / or present at the VHVL interface and / or at so-called cameloid marker residues (see, for example, WO 94 / 04678 and Davies and Riechmann, 1994 and 1996, ibid.). In some embodiments, the VH sequence used as the starting material or starting point for generating or designing camel-like VHs is a VH sequence from mammals or humans, such as the VH3 sequence. However, it should be noted that such camel-like VHs can be obtained in any suitable manner known per se, and are therefore not strictly limited to peptides obtained using peptides containing naturally occurring VH domains as starting materials.

[0159] It should be noted that one or more immunoglobulin sequences can be linked together and / or linked to other amino acid sequences (e.g., via disulfide bridges) to provide potentially useful peptide constructs (e.g., Fab' fragments, F(ab')2 fragments, scFv constructs, "double antibodies," and other multispecific constructs). See, for example, the review by Holliger and Hudson, Nat Biotechnol. Sep 2005;23(9):1126-36. Typically, when a peptide is intended for administration to a subject (e.g., for prophylactic, therapeutic, and / or diagnostic purposes), it may contain immunoglobulin sequences that are not naturally present in said subject.

[0160] Non-limiting examples of the structure of an immunoglobulin single variable domain sequence can be considered to consist of four architecture regions (“FR”), referred to in the art and herein as “architecture region 1” (“FR1”); “architecture region 2” (“FR2”); “architecture region 3” (“FR3”); and “architecture region 4” (“FR4”); said architecture regions are interrupted by three complementarity-determining regions (“CDR”), referred to in the art and herein as “complementarity-determining region 1” (“CDR1”); “complementarity-determining region 2” (“CDR2”); and “complementarity-determining region 3” (“CDR3”).

[0161] As further described in paragraphs q) on pages 58 and 59 of WO 08 / 020079 (incorporated hereby by reference), the amino acid residues of the immunoglobulin single variable domain can be numbered according to the general numbering of the VH domain given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), such as the VHH domain from camel in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see, for example, Figure 2 of that publication). It should be noted that, as is well known in the industry, the total number of amino acid residues in each CDR for the VH and VHH domains can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number). This means that, generally, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. The total number of amino acid residues in the VH and VHH domains is typically between 110 and 120, often in the range of 112 to 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0162] In this application, unless otherwise stated, the CDR sequence is determined according to the AbM numbering described in Kontermann and Dübel (2010 edition, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues at positions 1 to 25, CDR1 contains amino acid residues at positions 26 to 35, FR2 contains amino acid residues at positions 36 to 49, CDR2 contains amino acid residues at positions 50 to 58, FR3 contains amino acid residues at positions 59 to 94, CDR3 contains amino acid residues at positions 95 to 102, and FR4 contains amino acid residues at positions 103 to 113.

[0163] The determination of the CDR region can also be performed using different methods. According to Kabat's CDR determination, FR1 of the immunoglobulin monovariable domain (IMDV) contains amino acid residues at positions 1 to 30; CDR1 of the IMDV contains amino acid residues at positions 31 to 35; FR2 of the IMDV contains amino acid residues at positions 36 to 49; CDR2 of the IMDV contains amino acid residues at positions 50 to 65; FR3 of the IMDV contains amino acid residues at positions 66 to 94; CDR3 of the IMDV contains amino acid residues at positions 95 to 102; and FR4 of the IMDV contains amino acid residues at positions 103 to 113.

[0164] In such immunoglobulin sequences, the architecture sequence can be any suitable architecture sequence, and examples of suitable architecture sequences will be clear to those skilled in the art, for example based on standard manuals and further disclosures and prior art mentioned herein.

[0165] The architecture sequence can be an immunoglobulin architecture sequence or a suitable combination of architecture sequences derived from immunoglobulin architecture sequences (e.g., through humanization or camelification). For example, the architecture sequence can be an architecture sequence derived from a light chain variable domain (e.g., a VL sequence) and / or a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In one embodiment, the architecture sequence is an architecture sequence derived from a VHH sequence, which may optionally be partially or fully humanized; or a conventional VH sequence that has been camelified (as defined herein).

[0166] In particular, the architectural sequences present in the ISVD sequences disclosed herein may contain one or more marker residues (as defined herein) such that the ISVD sequence is a Nanobody® (e.g., VHH, including humanized VHH or camelified VH). Some non-limiting examples of such architectural sequences (suitable combinations) will become clear from further disclosure herein.

[0167] Similarly, as described herein with respect to the general description of immunoglobulin sequences, any suitable fragment (or combination of fragments) mentioned above may be used, such as a fragment containing one or more CDR sequences, which are appropriately side-joined with one or more architecture sequences and / or linked via one or more architecture sequences (e.g., appearing in the full-size immunoglobulin sequence from which the fragment is derived in the same order as these CDR and architecture sequences).

[0168] However, it should be noted that this document is not limited to the origin of the ISVD sequence (or the origin of the nucleotide sequence used to represent the ISVD sequence), nor to the manner in which the ISVD sequence or nucleotide sequence was generated or obtained (or has been generated or obtained). Therefore, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In certain but not limiting respects, 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" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and particularly partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR transplantation, faceting, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineered immunoglobulin sequences known to those skilled in the art; or any suitable combination of the foregoing.

[0169] Similarly, the nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated from a suitable naturally occurring template by PCR (e.g., DNA or RNA isolated from cells), a nucleotide sequence that has been isolated from a library (and in particular, an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0170] As stated above, ISVD can be Nanobody® or a suitable fragment thereof. For a general description of Nanobodies® (Nanobody® and Nanobodies® are registered trademarks of Ablynx NV (Sanofi Company),) refer to the following further description and the prior art cited herein. However, in this respect, it should be noted that this description and prior art primarily describe Nanobodies® of the so-called "V H3 class" (i.e., Nanobodies® with high sequence homology to human phylogenetic sequences of the V H3 class such as DP-47, DP-51, or DP-29). However, it should be noted that this document may generally use any type of Nanobody® in its broadest sense, and, for example, also uses Nanobodies® belonging to the so-called "V H4 class" (i.e., Nanobodies® with high sequence homology to human phylogenetic sequences of the V H4 class such as DP-78), with examples described in WO 2007 / 118670, which is incorporated herein by reference in its entirety.

[0171] Typically, Nanobodies® (especially VHH sequences, including (partially) humanized VHH sequences and camel-like VH sequences) are characterized by the presence of one or more "marker residues" (as described herein) in one or more structural sequences (again, as further described herein). Therefore, Nanobody® can generally be defined as an immunoglobulin sequence with a (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more of the marker residues are as further defined herein.

[0172] Specifically, Nanobody® can be an immunoglobulin sequence with a (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the architecture sequence is as further defined herein.

[0173] More specifically, Nanobody® can be an immunoglobulin sequence with a (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to architecture regions 1 to 4, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively.

[0174] According to the Kabat number, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 are selected from the marker residues mentioned in Table X below.

[0175] The FRs shown herein may suitably be selected from the FRs shown in Table A-2 (or Table A-2.1, with regard to Kabat numbering), preferably from the same clone (i.e., the FRs shown in the same row), and optionally have the amino acids as described herein at a particular position.

[0176] [surface] [X] [:] [Nanobodies®] [Marker residues in] [Location] [Human] [V, H , 3 ] [Marker residues] 11 L, V; mainly L L,S,V,M,W,F,T,Q,E,A,R,G,K,Y,N,P,I 37 V, I, F; usually V F (1), Y, V, L, A, H, S, I, W, C, N, G, D, T, P 44 (8) G E (3), Q (3), G (2), D, A, K, R, L, P, S, V, H, T, N, W, M, I 45 (8) L L (2), R (3), P, H, F, G, Q, S, E, T, Y, C, I, D, V 47 (8) W, Y F (1), L (1) or W (2) G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D 83 R or K; usually R R, K (5), T, E (5), Q, N, S, I, V, G, M, L, A, D, Y, H 84 A, T, D; mainly A P (5), S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E 103 W W (4), R (6), G, S, K, A, M, Y, L, F, T, N, V, Q, P (6), E, ​​C 104 G G,A,S,T,D,P,N,E,C,L 108 L, M, or T; primarily L Q, L (7), R, P, E, K, S, T, M, A, H [Notice:] (1) Specifically, but not exclusively, in combination with KERE or KQRE at positions 43 to 46. (2) Usually GLEW at position 44 to 47. (3) Typically, it is KERE or KQRE at positions 43 to 46, such as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW, or KQREG at positions 43 to 47. Alternatively, sequences such as TERE (e.g., TEREL), TQRE (e.g., TQREL), KECE (e.g., KECEL or KECER), KQCE (e.g., KQCEL), RERE (e.g., REREG), RQRE (e.g., RQREL, RQREF, or RQREW), QERE (e.g., QEREG), QQRE (e.g., QQREW, QQREL, or QQREF), KGRE (e.g., KGREG), and KDRE (e.g., KDREV). Some other possible sequences include, for example, DECKL and NVCEL. (4) Two GLEWs at positions 44 to 47 and KERE or KQRE at positions 43 to 46. (5) Usually the KP or EP at position 83 to 84 of the naturally occurring V HH domain. (6) Specifically, but not exclusively, in combination with GLEW at positions 44 to 47. (7) The premise is that when positions 44 to 47 are GLEW, position 108 in the (non-humanized) V HH sequence containing W at position 103 is always Q. (8) The GLEW group also contains GLEW-like sequences at positions 44 to 47, such as GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.

[0177] In some embodiments, the marker residue at position 11 is L. In some embodiments, the marker residue at position 37 is F(1) or Y. In some embodiments, the marker residue at position 44 is G(2) or Q(3). In some embodiments, the marker residue at position 45 is L(2) or R(3). In some embodiments, the marker residue at position 47 is F(1), L(1), or W(2). In some embodiments, the marker residue at position 83 is K. In some embodiments, the marker residue at position 84 is P. In some embodiments, the marker residue at position 103 is W. In some embodiments, the marker residue at position 104 is G. In some embodiments, the marker residue at position 108 is Q or L.

[0178] Furthermore, when the ISVD having an N-terminal glutamic acid (E) at position 1 is located at the N-terminus of the polypeptide, the glutamic acid is preferably substituted with aspartic acid (D). Therefore, for example, if SEQ ID NO: 3, 4, or 5 is at the N-terminus of the polypeptide, then E at position 1 can be changed to D. Conversely, if, for example, SEQ ID NO: 2 is not present at the N-terminus of the polypeptide, then D at position 1 can be changed to E.

[0179] This article specifically uses ISVDs that can specifically bind to TNF-α or IL-6. In the context of this article, "binding to a target molecule" has the common meaning understood in the industry, such as in the context of antibodies and their corresponding antigens.

[0180] The peptide described herein may contain one or more ISVDs that bind to TNF-α and two or more ISVDs that bind to IL-6. For example, the peptide may contain one ISVD that binds to TNF-α and two ISVDs that bind to IL-6.

[0181] In some embodiments, the at least one ISVD can functionally block its target molecule. For example, the targeting portion can block the interaction between TNF-α and TNFR (TNF receptor), or it can block the interaction between IL-6 and IL-6R (interleukin-6 receptor). Thus, in one embodiment, the polypeptide herein comprises at least one ISVD that specifically binds to TNF-α and inhibits its interaction with TNFR, and two ISVDs that specifically bind to IL-6 and functionally block its interaction with IL-6R. Thus, in a preferred embodiment, the polypeptide herein comprises two ISVDs that specifically bind to IL-6, one of which functionally blocks the interaction between IL-6 and IL-6R.

[0182] The ISVDs used in this article form part of the polypeptide described herein, which contains or consists of at least three ISVDs such that the polypeptide can specifically bind to TNF-α and IL-6.

[0183] Therefore, the target molecules of the at least three ISVDs used in the peptides described herein are TNF-α and IL-6. Examples are mammalian TNF-α and IL-6. While human TNF-α (Uniprot accession number P01375) and human IL-6 (Uniprot accession number P05231) can be used, forms from other species are also applicable here, such as TNF-α and IL-6 from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (such as cynomolgus monkeys (also referred to herein as "cyno")) or camels (such as llamas or alpacas).

[0184] Specific examples of ISVDs that specifically bind to TNF-α or IL-6 and can be used in this article are described in items A through C below:

[0185] A. ISVDs that specifically bind to human IL-6 and include the following: i. An amino acid sequence having the same as SEQ ID NO: 6 or a CDR1 differing from SEQ ID NO: 6 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 10 or a CDR2 differing from SEQ ID NO: 10 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 14 or a CDR3 differing from SEQ ID NO: 14 by 2 or 1 amino acids. In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 6, CDR2 has the amino acid sequence of SEQ ID NO: 10, and CDR3 has the amino acid sequence of SEQ ID NO: 14.

[0186] Non-limiting examples of such ISVDs that specifically bind to human IL-6 have one or more or all of the architectural regions (and CDRs as defined in item A above) as shown in Table A-2 for construct 17C04, such as ISVDs having the complete amino acid sequence of construct 17C04 (SEQ ID NO: 2, see Tables A-1 and A-2).

[0187] Furthermore, in one embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-6 may have greater than 90%, such as greater than 95% or greater than 99%, sequence identity with SEQ ID NO: 2, wherein the CDR is optionally as defined in item A above. In some embodiments, the ISVD that specifically binds to IL-6 has the amino acid sequence of SEQ ID NO: 2.

[0188] When such an ISVD that specifically binds to IL-6 has a difference of 2 or 1 amino acids in at least one CDR relative to the corresponding reference CDR sequence (item A above), in some embodiments, the ISVD has at least half the binding affinity to human IL-6, preferably at least the same, or even higher binding affinity, compared to construct 17C04, wherein the binding affinity is measured using the same method (e.g., SPR).

[0189] B. ISVDs that specifically bind to human IL-6 and include the following: i. An amino acid sequence having the same as SEQ ID NO: 8 or a CDR1 differing from SEQ ID NO: 8 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 12 or a CDR2 differing from SEQ ID NO: 12 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 16 or a CDR3 differing from SEQ ID NO: 16 by 2 or 1 amino acids. In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 8, CDR2 has the amino acid sequence of SEQ ID NO: 12, and CDR3 has the amino acid sequence of SEQ ID NO: 16.

[0190] Non-limiting examples of such ISVDs that specifically bind to human IL-6 have one or more or all of the architectural regions (and CDRs as defined in item B above) as shown in Table A-2 for construct 6B12, such as ISVDs having the complete amino acid sequence of construct 6B12 (SEQ ID NO: 4, see Tables A-1 and A-2).

[0191] Furthermore, in one embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-6 may have greater than 90%, such as greater than 95% or greater than 99%, sequence identity with SEQ ID NO: 4, wherein the CDR is optionally as defined in item B above. In some embodiments, the ISVD that binds to IL-6 has the amino acid sequence of SEQ ID NO: 4.

[0192] When such an ISVD that binds to IL-6 has a difference of 2 or 1 amino acids in at least one CDR relative to the corresponding reference CDR sequence (item B above), in some embodiments, the ISVD has at least half, at least the same, or even higher binding affinity to human IL-6 compared to construct 6B12, wherein the binding affinity is measured using the same method (e.g., SPR).

[0193] C. The following ISVD specifically binds to human TNF-α and contains i. An amino acid sequence having the same as SEQ ID NO: 9 or a CDR1 differing from SEQ ID NO: 9 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 13 or a CDR2 differing from SEQ ID NO: 13 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 17 or a CDR3 differing from SEQ ID NO: 17 by 2 or 1 amino acids. In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 9, CDR2 has the amino acid sequence of SEQ ID NO: 13, and CDR3 has the amino acid sequence of SEQ ID NO: 17.

[0194] Non-limiting examples of such ISVDs that specifically bind to human TNF-α have one or more or all of the architectural regions (and CDRs as defined in item C above) as shown in Table A-2 for construct 6C11, such as ISVDs having the complete amino acid sequence of construct 6C11 (SEQ ID NO: 5, see Tables A-1 and A-2).

[0195] Furthermore, in one embodiment, the amino acid sequence of the ISVD that specifically binds to human TNF-α may have greater than 90%, such as greater than 95% or greater than 99%, sequence identity with SEQ ID NO: 5, wherein the CDR is optionally as defined in item C above. In some embodiments, the ISVD that binds to TNF-α has the amino acid sequence of SEQ ID NO: 5.

[0196] When such an ISVD that specifically binds to TNF-α has a difference of 2 or 1 amino acids in at least one CDR relative to the corresponding reference CDR sequence (item C above), the ISVD has at least half, at least the same, or even higher binding affinity for human TNF-α compared to construct 6C11, wherein the binding affinity is measured using the same method (e.g., SPR).

[0197] In some embodiments, each ISVD as defined in items A through C above is included in the polypeptide herein. In some embodiments, such a polypeptide of this article containing each ISVD as defined in items A through C above has at least half, at least the same, or even higher binding affinity for human TNF-α and human IL-6 compared to a polypeptide composed of the amino acids of SEQ ID NO: 1, wherein the binding affinity is measured using the same method (e.g., SPR).

[0198] The SEQ ID NO mentioned in items A through C above is based on the CDR definition defined according to AbM (see Table A-2). Note that the SEQ ID NO of the same CDR defined according to Kabat (see Table A-2.1) can also be used for items A through C above.

[0199] Therefore, the specific ISVD that binds specifically to TNF-α or IL-6 as used in this article, as defined by AbM as described above, can also be described using the Kabat definition as described in items A' through C' below:

[0200] A'. ISVD that specifically binds to human IL-6 and includes the following: i. An amino acid sequence having the same as SEQ ID NO: 33 or a CDR1 differing from SEQ ID NO: 33 by 2 or 1 amino acids; ii. An amino acid sequence having the same as SEQ ID NO: 37 or a CDR2 differing from SEQ ID NO: 37 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 14 or a CDR3 differing from SEQ ID NO: 14 by 2 or 1 amino acids. In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 33, CDR2 has the amino acid sequence of SEQ ID NO: 37, and CDR3 has the amino acid sequence of SEQ ID NO: 14.

[0201] Non-limiting examples of such ISVDs that specifically bind to human IL-6 have one or more or all of the architectural regions (and CDRs as defined in the aforementioned item A') as shown in Table A-2.1 for construct 17C04, such as ISVDs having the complete amino acid sequence of construct 17C04 (SEQ ID NO: 2, see Tables A-1 and A-2.1).

[0202] B'. ISVD that specifically binds to human IL-6 and includes the following: i. An amino acid sequence having the same as SEQ ID NO: 35 or a CDR1 differing from SEQ ID NO: 35 by 2 or 1 amino acids; ii. An amino acid sequence having the same as SEQ ID NO: 39 or a CDR2 differing from SEQ ID NO: 39 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 16 or a CDR3 differing from SEQ ID NO: 16 by 2 or 1 amino acids. In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 35, CDR2 has the amino acid sequence of SEQ ID NO: 39, and CDR3 has the amino acid sequence of SEQ ID NO: 16.

[0203] Non-limiting examples of such ISVDs that specifically bind to human IL-6 have one or more or all of the architectural regions (and CDRs as defined in item B above) as shown in Table A-2.1 for construct 6B12, such as ISVDs having the complete amino acid sequence of construct 6B12 (SEQ ID NO: 4, see Tables A-1 and A-2.1).

[0204] C'. The following ISVD specifically binds to human TNF-α and contains i. An amino acid sequence having the same as SEQ ID NO: 36 or a CDR1 differing from SEQ ID NO: 36 by 2 or 1 amino acids; ii. An amino acid sequence having the same as SEQ ID NO: 40 or a CDR2 differing from SEQ ID NO: 40 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 17 or a CDR3 differing from SEQ ID NO: 17 by 2 or 1 amino acids. In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 36, CDR2 has the amino acid sequence of SEQ ID NO: 40, and CDR3 has the amino acid sequence of SEQ ID NO: 17.

[0205] Non-limiting examples of such ISVDs that specifically bind to human TNF-α have one or more or all of the architectural regions (and CDRs as defined in item C' above) as shown in Table A-2.1 for construct 6C11, such as ISVDs having the complete amino acid sequence of construct 6C11 (SEQ ID NO: 5, see Tables A-1 and A-2.1).

[0206] The percentage of "sequence identity" between the first amino acid sequence and the second amino acid sequence can be calculated as follows: ([the number of amino acid residues in the first amino acid sequence that are identical to the corresponding amino acid residues in the second amino acid sequence] / [the total number of amino acid residues in the first amino acid sequence]) × 100%, where each deletion, insertion, substitution, or addition of amino acid residues in the second amino acid sequence compared to the first amino acid sequence is considered a difference at a single amino acid residue (i.e., a single position).

[0207] Typically, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the "first" amino acid sequence, and the other amino acid sequence is designated as the "second" amino acid sequence.

[0208] As used herein, "amino acid difference" refers to the deletion, insertion, or substitution of a single amino acid residue relative to a reference sequence. In some embodiments, the amino acid difference is a substitution. Generally, it is preferred to have fewer amino acid differences from a given reference sequence. For example, when the CDR has 2 or 1 amino acid differences from a given SEQ ID NO, 1 amino acid difference is preferred.

[0209] In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, such a conservative substitution is a substitution in which one amino acid from the following groups (a)-(e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or micropolar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) acetylamines: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0210] In some embodiments, the conservative substitutions are as follows: Ala becomes Gly or Ser; Arg becomes Lys; Asn becomes Gln or His; Asp becomes Glu; Cys becomes Ser; Gln becomes Asn; Glu becomes Asp; Gly becomes Ala or Pro; His becomes Asn or Gln; Ile becomes Leu or Val; Leu becomes Ile or Val; Lys becomes Arg, becomes Gln or Glu; Met becomes Leu, becomes Tyr or Ile; Phe becomes Met, becomes Leu or Tyr; Ser becomes Thr; Thr becomes Ser; Trp becomes Tyr; Tyr becomes Trp; and / or Phe becomes Val, becomes Ile or Leu. [5.2] [Specificity]

[0211] The terms "specific," "specifically binding," or "specifically 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 a sufficiently high affinity (see below). "Specific," "specifically binding," or "specifically binding" are used interchangeably herein with "selective," "selectively binding," or "selectively binding." According to some embodiments, the binding unit (such as an ISVD) binds specifically to its designated target.

[0212] The specificity / selectivity of binding units can be determined based on affinity. Affinity represents the strength or stability of molecular interactions. Affinity is usually given by KD or the dissociation constant, in moles per liter (or M). Affinity can also be expressed as the association constant KA, which is equal to 1 / KD and has units of (moles per liter)⁻¹ (or M⁻¹).

[0213] Affinity is a measure of the binding strength between a part of the target molecule and its binding site: the smaller the KD value, the stronger the binding strength between the target molecule and the target part.

[0214] Typically, the binding unit (e.g., ISVD) used in this paper will bind to its target (at room temperature) with a dissociation constant (KD) of 10⁻⁵ to 10⁻¹² mol / L or lower, such as 10⁻⁷ to 10⁻¹² mol / L or lower, and more particularly such as 10⁻⁸ to 10⁻¹² mol / L (i.e., an association constant (KA) of 10⁵ to 10¹² mol / L or higher, such as 10⁷ to 10¹² mol / L or higher, and more particularly such as 10⁸ to 10¹² mol / L).

[0215] Any KD value greater than 10⁻⁴ mol / L (or any KA value less than 10⁴ mol / L) is generally considered to indicate nonspecific binding.

[0216] KD values ​​that are considered to have specific biological interactions (such as the binding of immunoglobulin sequences to antigens) are typically in the range of 10⁻⁵ mol / L (10,000 nM or 10 µM) to 10⁻¹² mol / L (0.001 nM or 1 pM) or lower.

[0217] Therefore, specific / selective binding may mean that, using the same measurement method (e.g., SPR), the binding unit (or the peptide containing it) binds to TNF-α and / or IL-6 with a KD value of 10⁻⁵ to 10⁻¹² mol / L or lower, and to related cytokines with a KD value greater than 10⁻⁴ mol / L. Examples of TNF-α-related cytokines are members of the TNF superfamily, such as FASL, TNFβ, LIGHT, TL-1A, and RANKL. Examples of IL-6-related cytokines are members of the IL-6 family, such as IL-11, ciliary neurotrophic factor (CNTF), leukemia suppressor factor (LIF), oncogene M (OSM), cardiotrophin 1 (CT-1), cardiotrophin-like cytokines (CLC), and IL-27. Therefore, in the embodiments, at least one ISVD contained in the polypeptide binds to TNF-α with a KD value of 10⁻⁵ to 10⁻¹² mol / L or less and to FASL, TNFβ, LIGHT, TL-1A, and RANKL of the same species with a KD value greater than 10⁻⁴ mol / L, and at least two ISVDs contained in the polypeptide bind to IL-6 with a KD value of 10⁻⁵ to 10⁻¹² mol / L or less and to IL-11, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin 1 (CT-1), cardiotrophin-like cytokines (CLC), and IL-27 of the same species with a KD value greater than 10⁻⁴ mol / L.

[0218] Therefore, in some embodiments, the peptides described herein have at least half, at least the same, or even higher binding affinity for human TNF-α and human IL-6 compared to the peptides composed of the amino acids of SEQ ID NO: 1, wherein the binding affinity is measured using the same method (e.g., SPR).

[0219] Specific binding to a specific target from a particular species does not preclude the binding unit from specifically binding to similar targets from different species. For example, specific binding to human TNF-α does not preclude the binding unit (or a peptide containing said binding unit) from specifically binding to TNF-α from cynomolgus monkeys. Similarly, specific binding to human IL-6, for example, does not preclude the binding unit or a peptide containing said binding unit from specifically binding to IL-6 from cynomolgus monkeys.

[0220] The specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including but not limited to Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay, as well as different variants of them known in the art; and other techniques mentioned herein.

[0221] As will be apparent to those skilled in the art, the dissociation constant can be either actual or apparent. Methods for determining the dissociation constant will be clear to those skilled in the art and include, for example, the techniques mentioned below. In this regard, it will also be clear that dissociation constants greater than 10⁻⁴ mol / L or 10⁻³ mol / L (e.g., 10⁻² mol / L) may not be measurable. Optionally, as will also be clear to those skilled in the art, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) through the relationship [KD = 1 / KA].

[0222] The affinity of molecular interactions between two molecules can be measured using various techniques known in themselves, such as the well-known surface plasma resonance (SPR) biosensor technique (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559). As used herein, the term "surface plasma resonance" refers to an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration in a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule flows through the immobilized molecule under flow conditions, resulting in k-on, k-off measurements and thus KD (or KA) values. This can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscatave, New Jersey). For further details, please 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).

[0223] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biomembrane interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377: 209-217). As used herein, the term "biomembrane interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and an immobilized protein layer on the biosensor tip (signal beam). Variations in the number of molecules bound to the biosensor tip result in a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the biosensor tip. Because interactions can be measured instantaneously, association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Parker, USA).

[0224] Alternatively, affinity can be measured in a kinetic exclusion assay (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem., 328: 35-43), using the KinExA® platform (Sapidyne Instruments Inc., Boise, USA). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a column containing beads pre-coated with an antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is performed using a fluorescently labeled protein that binds the antibody (or antigen).

[0225] The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74). [5.3] [Prolonged half-life (in vivo)]

[0226] The polypeptide may also include one or more other groups, residues, portions, or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased (in vivo) half-life to the polypeptide compared to a corresponding polypeptide without said one or more other groups, residues, portions, or binding units. An extended in vivo half-life means, for example, that the polypeptide has an increased half-life in mammalian subjects such as humans after administration. Half-life may be expressed, for example, as t1 / 2β.

[0227] The types of groups, residues, parts or binding units are generally unrestricted and may be selected, for example, from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc parts and small proteins or peptides that can bind to serum proteins.

[0228] More specifically, the one or more other groups, residues, portions, or binding units that provide an increased half-life to the polypeptide may be selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG). In some embodiments, the binding unit may bind to human serum albumin. In some embodiments, the binding unit is an ISVD.

[0229] For example, WO 04 / 041865 (incorporated in its entirety by reference) describes Nanobodies® that bind to serum albumin (and particularly to human serum albumin) and can be linked to other proteins (such as one or more other Nanobodies® that bind to a desired target) to increase the half-life of said proteins.

[0230] International application WO 06 / 122787 (incorporated in its entirety by reference) describes several Nanobodies® targeting (human) serum albumin. These Nanobodies® include Nanobody® called Alb-1 (SEQ ID NO: 52 in WO 06 / 122787, in its entirety by reference) and its humanized variants such as Alb-8 (SEQ ID NO: 62 in WO 06 / 122787, in its entirety by reference). Similarly, these can be used to extend the half-life of therapeutic proteins and peptides, as well as other therapeutic entities or portions.

[0231] In addition, WO 2012 / 175400 (incorporated in its entirety by reference) describes a further improved form of Alb-1, referred to as Alb-23.

[0232] In one embodiment, the polypeptide comprises a serum albumin-binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10, and Alb-23. In some embodiments, the polypeptide comprises Alb-8 or Alb-23 or variants thereof as shown on pages 7-9 of WO 2012 / 175400, and albumin conjugates described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, and WO 2018 / 134234, each of which is incorporated herein by reference in its entirety. Some non-limiting examples of serum albumin conjugates are also shown in Tables A-4. In some embodiments, the polypeptides described herein comprise other components as described in item D:

[0233] D. ISVD that binds to human serum albumin and includes the following: i. An amino acid sequence having the same as SEQ ID NO: 7 or a CDR1 differing from SEQ ID NO: 7 by 2 or 1 amino acid; ii. An amino acid sequence having the same as SEQ ID NO: 11 or a CDR2 differing from SEQ ID NO: 11 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 15 or a CDR3 differing from SEQ ID NO: 15 by 2 or 1 amino acids; In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 7, CDR2 has the amino acid sequence of SEQ ID NO: 11, and CDR3 has the amino acid sequence of SEQ ID NO: 15.

[0234] Non-limiting examples of such ISVDs that bind to human serum albumin have one or more or all of the architectural regions (and CDRs as defined in item D above) as shown in Table A-2 for construct ALB23002, such as ISVDs having the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 3, see Tables A-1 and A-2).

[0235] Alternatively, you can use Kabat definitions to describe project D as follows:

[0236] D'. ISVD that binds to human serum albumin and contains the following: i. An amino acid sequence having the same as SEQ ID NO: 34 or a CDR1 differing from SEQ ID NO: 34 by 2 or 1 amino acids; ii. An amino acid sequence having the same as SEQ ID NO: 38 or a CDR2 differing from SEQ ID NO: 38 by 2 or 1 amino acids; and iii. An amino acid sequence having the same as SEQ ID NO: 15 or a CDR3 differing from SEQ ID NO: 15 by 2 or 1 amino acids; In some embodiments, CDR1 has the amino acid sequence of SEQ ID NO: 34, CDR2 has the amino acid sequence of SEQ ID NO: 38, and CDR3 has the amino acid sequence of SEQ ID NO: 15.

[0237] Non-limiting examples of such ISVDs that bind to human serum albumin have one or more or all of the architectural regions (and CDRs as defined in item D' above) as shown in Table A-2.1 for construct ALB23002, such as ISVDs having the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 3, see Tables A-1 and A-2.1).

[0238] Furthermore, in one embodiment, the amino acid sequence of the ISVD binding to human serum albumin may have greater than 90%, such as greater than 95% or greater than 99%, sequence identity with SEQ ID NO: 3, wherein the CDR is optionally as defined in item D above. In some embodiments, the ISVD binding to human serum albumin has the amino acid sequence of SEQ ID NO: 3.

[0239] When such an ISVD that binds to human serum albumin has a difference of 2 or 1 amino acids in at least one CDR relative to the corresponding reference CDR sequence (item D above), the ISVD has at least half, at least the same, or even higher binding affinity to human serum albumin compared to the construct ALB23002, wherein the binding affinity is measured using the same method (e.g., SPR).

[0240] When such an ISVD that binds to human serum albumin has a C-terminal position, it exhibits a C-terminal alanine (A) or glycine (G) extension and can be selected from SEQ ID NO: 52, 53, 55, 57, 58, 59, 60, 61, 62, and 63 (see Table A-4 below). In one embodiment, the ISVD that binds to human serum albumin has a position other than the C-terminal position (i.e., a C-terminal ISVD that is not the peptide described herein) and is selected from SEQ ID NO: 3, 50, 51, 54, and 56 (see Table A-4 below). [5.4] [Nucleic acid molecules]

[0241] A nucleic acid molecule encoding the polypeptide described herein is also provided.

[0242] A "nucleic acid molecule" (which may be used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked together by a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect host cells or host organisms, for example, for the expression and / or production of polypeptides. Suitable hosts or host cells for production purposes will be clear to those skilled in the art and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Hosts or host cells containing nucleic acids encoding the polypeptides described herein are also included herein.

[0243] Nucleic acids can be, for example, DNA, RNA, or hybrids thereof, and may also contain (e.g., chemically modified) nucleotides, such as PNA. They can be single-stranded or double-stranded DNA. For example, the nucleotide sequences described herein can be genomic DNA or cDNA.

[0244] The nucleic acids described herein can be prepared or obtained in a manner known per se, and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence variations. Similarly, as will be apparent to those skilled in the art, for the preparation of nucleic acids, several nucleotide sequences, such as at least one nucleotide sequence encoding a target moiety, can be linked together in a suitable manner with nucleic acids, for example, encoding one or more linkers.

[0245] The techniques used to generate nucleic acids will be clear to those familiar with the techniques and may include, for example, but 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) to introduce mutations that result in the expression of truncated products; introducing one or more restriction sites (e.g., to produce boxes and / or regions that may be easily digested and / or linked by suitable restriction enzymes); and / or introducing mutations by means of PCR reactions using one or more “mismatched” primers. [5.5] [Carrier]

[0246] A vector comprising a nucleic acid molecule encoding the polypeptide described herein is also provided. As used herein, the vector is suitable for carrying genetic material into cells. Vectors include naked nucleic acids (such as plasmids or mRNA) or nucleic acids embedded in larger structures (such as liposomes or viral vectors).

[0247] Vectors typically contain at least one nucleic acid, optionally linked to one or more regulatory elements (e.g., suitable promoters, enhancers, terminators, etc.). The vector is a performance vector, i.e., a vector suitable for expressing a encoded polypeptide or construct under suitable conditions (e.g., when the vector is introduced into cells, e.g., human cells). For DNA-based vectors, they typically include elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences).

[0248] In some embodiments, within the vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they have a functional relationship with each other. For example, a promoter is considered "operably linked" to a coding sequence if it is capable of initiating or otherwise controlling / regulating transcription and / or expression of the coding sequence (wherein the coding sequence should be understood as "under the control of the promoter"). Typically, when two nucleotide sequences are operably linked, they will be in the same orientation and usually in the same reading frame. They are also usually substantially continuous, although this may not be necessary.

[0249] In some embodiments, any regulatory elements of the vector enable it to provide its intended biological function in the intended host cell or host organism.

[0250] For example, a promoter, enhancer, or terminator should be "operable" in the intended host cell or host organism, meaning, for example, that the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence (e.g., a coding sequence) to which it is operably linked. [5.6] [Composition]

[0251] This document also provides a composition comprising at least one polypeptide of this document, at least one nucleic acid molecule encoding the polypeptide of this document, or at least one carrier comprising such a nucleic acid molecule. The composition may be a pharmaceutical composition. The composition may also comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally comprise one or more other pharmaceutically active polypeptides and / or compounds. [5.7] [Host organism]

[0252] This article also relates to host cells or host organisms that contain the polypeptides of this article, nucleic acids encoding the polypeptides of this article, and / or carriers containing nucleic acid molecules encoding the polypeptides of this article.

[0253] Suitable host cells or host organisms will be clear to those skilled in the art, and are, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, *Escherichia coli*, or *Pichia pastoris*. In some embodiments, the host is *Pichia pastoris*. [5.8] [Methods and Applications of Peptides]

[0254] This document also provides a method for generating the polypeptide of this document. The method may include transforming / transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, optionally followed by one or more isolation and / or purification steps. Specifically, the method may include: a) Expressing the nucleic acid sequence encoding the polypeptide in a suitable expression system (e.g., a suitable host cell or host organism or another expression system); optionally, then proceeding to: b) Isolate and / or purify the polypeptide.

[0255] The suitable host or host organism for the purpose of production will be clear to those skilled in the art, and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, *Escherichia coli*, or *Pichia pastoris*. In some embodiments, the host is *Pichia pastoris*.

[0256] The polypeptides described herein, nucleic acid molecules or carriers as described, or compositions comprising the polypeptides, nucleic acid molecules or carriers described herein—such as the polypeptides or compositions comprising the polypeptides—may be used as pharmaceuticals.

[0257] Therefore, this article provides the polypeptides, nucleic acid molecules or carriers as described herein, or compositions comprising the polypeptides, nucleic acid molecules or carriers described herein, for use as pharmaceuticals.

[0258] Also provided are polypeptides, nucleic acid molecules or carriers as described herein, or compositions comprising polypeptides, nucleic acid molecules or carriers described herein, for the (preventive or therapeutic) treatment of inflammatory diseases and / or autoimmune diseases.

[0259] A method for treating inflammatory diseases and / or autoimmune diseases (preventive and / or therapeutic) is also provided, wherein the method comprises administering to a subject in need a pharmaceutically active amount of the polypeptide, nucleic acid molecule or carrier described herein, or a composition comprising the polypeptide, nucleic acid molecule or carrier described herein.

[0260] The use of the peptides, nucleic acid molecules or carriers described herein, or compositions comprising the peptides, nucleic acid molecules or carriers described herein, in the preparation of pharmaceutical compositions (such as pharmaceutical compositions for the treatment of inflammatory diseases and / or autoimmune diseases) is also provided.

[0261] The inflammatory disease and / or autoimmune disease may be, for example, rheumatoid arthritis, hidradenitis suppurativa, and sarcoidosis. Preferably, the inflammatory disease and / or autoimmune disease is rheumatoid arthritis.

[0262] As used in the context of this article, "subject" can refer to any animal, such as a mammal. Among mammals, a distinction can be made between humans and non-human animals. Non-human animals can be, for example, companion animals (e.g., dogs, cats), livestock (e.g., cattle, horses, sheep, goats, or pigs), or animals commonly used for research purposes and / or for antibody production (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (e.g., cynomolgus monkeys), or camels (e.g., llamas or alpacas)).

[0263] In the context of preventive and / or therapeutic purposes, the subject can be any animal, and more specifically any mammal, such as a human subject.

[0264] Substances, including peptides, nucleic acid molecules and carriers, or components, can be administered to the subject via any suitable route of administration, such as enteral (e.g., oral or rectal) or parenteral (e.g., epidermal, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, percutaneous, or transmucosal). Parenteral administration, such as intramuscular, subcutaneous, or intradermal administration, can be used. In some embodiments, subcutaneous administration is used.

[0265] An effective amount of a polypeptide, a nucleic acid molecule or carrier as described, or a composition containing the polypeptide, nucleic acid molecule or carrier may be administered to a subject in order to provide the desired therapeutic outcome.

[0266] One or more doses may be administered. If more than one dose is administered, the doses may be administered at appropriate intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule, or carrier.

[0267] [surface] [A-0] [:] [F027201062] [Configuration] [name] [Building Blocks] [1] [Connector] [Building Blocks] [2] [Connector] [Building Blocks] [3] [Connector] [Building Blocks] [4] F027201062 17C04 9GS ALB23002 9GS 6B12 9GS 6C11+A* * C-terminal extension of a single alanine acid

[0268] [surface] [A-1] [In tetravalent polypeptides] [F027201062] Different unit prices for internal testing [V,HH , ] [Amino acid sequence of the building block (「) [ID] [ " refers to [as used in this article] [SEQ ID NO] [)] [name] [ID] [Amino acid sequence] 17C04 (anti-hIL-6) 2 DVQLVESGGGVVQPGGSLRLSCAASGRTFSNYAMAWFRQAPGKEREFVAVISYAGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAAVDSPLIATHPRGYDYWGQGTLVTVSS ALB23002 (Anti-HSA) 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS 6B12 (anti-hIL-6) 4 EVQLVESGGGVVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISGSVGTTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCVRSSWFDCGVQGRDLGNEYDYRGQGTLVTVSS 6C11 (anti-hTNF-α) 5 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVKVSS

[0269] [surface] [A-2] [:according to] [AbM] [Numbered] [CDR] [and the sequence of architectures()] [「] [ID] [」] [Refers to what is given] [SEQ ID NO] [)] [Building Blocks] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] [ID] 17C04 2 DVQLVESGGGVVQPGGSLRLSCAAS 18 GRTFSNYAMA 6 WFRQAPGKEREFVA twenty one VISYAGGRTY 10 YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAA 25 VDSPLIATHPRGYDY 14 WGQGTLVTVSS 29 ALB23002 3 EVQLVESGGGVVQPGGSLRLSCAAS 19 GFTFRSFGMS 7 WVRQAPGKGPEWVS 22 SISGSGSDTL 11 YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 26 GGSLSR 15 SSQGTLVTVSS 30 6B12 4 EVQLVESGGGVVQPGGSLRLSCAAS 19 GFTLAYYAIG 8 WFRQAPGKEREGVS 23 CISGSVGTTY 12 YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCVR 27 SSWFDCGVQGRDLGNEYDY 16 RGQGTLVTVSS 31 6C11 5 EVQLVESGGGVVQPGGSLRLSCTAS 20 GFTFSTADMG 9 WFRQAPGKGREFVA twenty four RISGIDGTTY 13 YDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRS 28 PRYADQWSAYDY 17 WGQGTLVKVSS 32

[0270] [surface] [A-2.1] [:according to] [Kabat] [Numbered] [CDR] [and the sequence of architectures()] [「] [ID] [」] [Refers to what is given] [SEQ ID NO] [)] [Building Blocks] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] [ID] 17C04 2 DVQLVESGGGVVQPGGSLRLSCAASGRTFS 41 NYAMA 33 WFRQAPGKEREFVA 21 VISYAGGRTYYADSVKG 37 RFTISRDNAKNTVYLQMNSLRPEDTALYYCAA 45 VDSPLIATHPRGYDY 14 WGQGTLVTVSS 29 ALB23002 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFR 42 SFGMS 34 WVRQAPGKGPEWVS 22 SISGSGSDTLYADSVKG 38 RFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 46 GGSLSR 15 SSQGTLVTVSS 30 6B12 4 EVQLVESGGGVVQPGGSLRLSCAASGFTLA 43 YYAIG 35 WFRQAPGKEREGVS twenty three CISGSVGTTYYADSVKG 39 RFTISRDNAKNTVYLQMNSLRPEDTALYYCVR 47 SSWFDCGVQGRDLGNEYDY 16 RGQGTLVTVSS 31 6C11 5 EVQLVESGGGVVQPGGSLRLSCTASGFTFS 44 TADMG 36 WFRQAPGKGREFVA twenty four RISGIDGTTYYDEPVKG 40 RFTISRDNSKNTVYLQMNSLRPEDTALYYCRS 48 PRYADQWSAYDY 17 WGQGTLVKVSS 32

[0271] [surface] [A-3] [:Selected amino acid sequence of multivalent polypeptide() [「] [ID] [」] [Refers to what is given] [SEQ ID NO] [)] [Name] [ID] [Amino acid sequence] F027201062 1 DVQLVESGGGVVQPGGSLRLSCAASGRTFSNYAMAWFRQAPGKEREFVAVISYAGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAAVDSPLIATHPRGYDYWGQGTLVTVSSGGGGS GGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGGSE VQLVESGGGVVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISGSVGTTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCVRSSWFDCGVQGRDLGNEYDYRGQGTLVTVSSGG GGSGGGSEVQLVESGGGVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVKVSSA

[0272] [surface] [A-4] [Bound to serum albumin] [ISVD] [sequence(] [「] [ID] [」] [Refers to what is used in this article] [SEQ ID NO] [)] [Name] [ID] [Amino acid sequence] Alb8 50 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 51 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 52 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 53 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 54 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11 (S112K)-A 55 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 56 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 57 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 58 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 59 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 60 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSG Alb82-GG 61 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 62 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG Alb23002 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 63 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA

[0273] [Table] [A-5] [: Linker sequence (] [「] [ID] [」] [refers to as used herein] [SEQ ID NO] [)] [Name] [ID] [Amino acid sequence] 3A connector 64 AAA 5GS connector 65 GGGGS 7GS connector 66 SGGSGGS 8GS connector 67 GGGGSGGS 9GS connector 68 GGGGSGGGS 10GS connector 69 GGGGSGGGGS 15GS connector 70 GGGGSGGGGSGGGGS 18GS connector 71 GGGGSGGGGSGGGGSGGS 20GS connector 72 GGGGSGGGGSGGGGSGGGGS 25GS connector 73 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS connector 74 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS connector 75 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS connector 76 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge 77 EPKSCDKTHTCPPCP 9GS-G1 hinge 78 GGGGSGGGSEPKSCDKTHTCPPCP Upper hinge area of ​​the llama 79 EPKTPKPQPAAA G3 hinge 80 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP [6] [Example] [6.1] [Example] [1] [:wild type resistance] [IL-6] [Bivalent or dual complementary positions] [ISVD] [Construction generation and in vitro characterization.]

[0274] Compared to the anti-IL-6 reference mAb 1 (the baseline monoclonal antibody against IL-6), the monovalent anti-IL-6 ISVDs IL6006B06, IL006B12, IL6007G04, IL6007G05, IL6007G09, IL6010A06, IL6013F12, and IL6017C04 (described in WO 2007104529) did not show sufficient IL-6 blocking ability when examined in TF-1 proliferation assays. To improve potency, the anti-IL-6 ISVDs were formatted as a dual-complementary-site ISVD construct. The building blocks in the construct were genetically linked via flexible 35GS or 9GS (GlySer) linkers. The ISVDs were expressed as FLAG3-HIS6-tagged proteins in *E. coli*. Expression was achieved via autoinduction and allowed to remain ON at 30ºC. Periplasmic extracts were prepared by rotating cell cultures, followed by freeze-thaw precipitate resuspending in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using a Nickel IDA / NTA column (Genscript-Atoll). ISVD was eluted from the column with 200 mM sodium acetate (pH 4), neutralized with TrisHCl (pH 8), and then desalted in dPBS.

[0275] The inhibitory efficacy of anti-IL-6 ISVD was determined in a cell-based assay for monitoring IL-6-mediated TF-1 cell proliferation. For this purpose, TF-1 cells were cultured in RPMI 1640, glutamax, and HEPES (Gibco) medium supplemented with 10% FBS and 1% sodium pyruvate. TF-1 cells were seeded at 12,500 cells per well in growth medium. A serial dilution of the purified anti-IL-6 ISVD or reference compound was added. After incubation at 37ºC for 30 min, 75 pM human IL-6 (R&D systems catalog number 200-IL-200|206-IL) was added. After 72 hours, TF-1 cell proliferation was measured using a CellTiter-Glo (Promega #G7571) reader on an EnVision Multilabel reader (Perkin Elmer).

[0276] Some dual complementary site constructs showed improved anti-human IL-6 potency and achieved potency similar to that of the anti-hIL-6 reference mAb 1 (Table 1).

[0277] In addition, bivalent anti-IL-6 ISVDs genetically linked via flexible 35GS (GlySer) linkers were generated, and their potency was evaluated in a second cell-based assay, monitoring IL-6-induced pSTAT3 production in THP-1 cells. For this purpose, THP-1 cells were cultured in RPMI 1640 medium supplemented with glutamic acid+ and 10% heat-inactivated FBS. The medium was replaced with HBSS before seeding into white 96-well plates at a density of 100,000 cells / well. A series of dilutions of purified anti-IL-6 ISVD or reference anti-IL-6 mAb1 were added along with 300 pM hIL-6 (R&D systems catalog number 200-IL-200|206-IL) and incubated at 37ºC for 20 min. Subsequently, the cells were centrifuged and lysed. 16 µl of lysed cell supernatant was mixed with an HTRF assay antibody mixture (PHOSPHO-STAT3(TYR705) KIT, Cisbio #62AT3PE) for pSTAT3 and total STAT3. Using this kit, pSTAT3 (Tyr705) was detected in a sandwich assay using two different specific antibodies (one labeled with an Eu3+-catechol compound (donor) and the other with d2 (recipient)). When the dyes were very close, excitation of the donor with a light source triggered a fluorescence resonance energy transfer (FRET) toward the recipient, which then emitted fluorescence at a specific wavelength (665 nm). This specific signal was positively modulated proportionally to pSTAT3. pSTAT was quantified by measuring the absorbance at 665 nm on an EnVision Multilabel reader (Perkin Elmer), and total STAT3 was quantified by measuring the absorbance at 620 nm.

[0278] Although the efficacy of the bivalent construct was not significantly increased compared to the monovalent anti-IL-6 ISDV, the bi-complementary site anti-IL-6 ISVD showed a significant increase in efficacy compared to either the monovalent or bivalent construct (Table 2).

[0279] From this experiment, 11 bicomplementary anti-IL-6 ISVDs were selected to conjugate to anti-TNF-α ISVDs: IL6013F12-IL6006B06, IL6006B06-IL6017C04, IL6013F12-IL6007G09, IL6006B06-IL6006B12, IL6006B06-IL6010A06, IL6017C04-IL6007G09, IL6006B12-IL6013F12, IL6010A06-IL6007G09, IL6006B12-IL007G09, IL6007G09-IL6006B12, or IL6017C04-IL6006B12.

[0280] [surface] [1] [:exist] [TF1] [In proliferation assays, compared with reference antibody] [IL-6 mAb 1] [Compared to single-unit price and dual complementary site resistance] [IL-6 ISVD] [of] [IC50] [Multiple Difference.] BB = Building Block [ISVD ID] [BB1] [Connector] [BB2] [IC50] [anti] [IL-6 ISVD / IC50] [Reference Antibody] [IL-6 mAb1] [Ratio] A007100001 10A06 2909 A007100002 13F12 70 A007100003 17C04 107 A007100005 6B12 77 A007100008 7G09 1489 A007100009 6B06 7 F027200055 7G04 35GS 13F12 5 – 7 F027200062 6B12 35GS 6B06 5 – 9 F027200064 17C04 35GS 7G09 1 – 3 F027200066 10A06 35GS 6B12 2 F027200067 13F12 35GS 6B06 1 – 3 F027200069 10A06 35GS 6B06 2 – 4 F027200070 13F12 35GS 6B12 1 – 2 F027200071 17C04 35GS 6B12 2 F027200072 6B12 35GS 17C04 2 F027200073 6B12 35GS 13F12 2 F027200075 10A06 35GS 7G09 2 F027200076 13F12 35GS 7G09 5 F027200077 13F12 35GS 7G05 6 – 7 F027200078 17C04 35GS 6B06 1 – 2 F027200079 17C04 35GS 7G05 4 – 9 F027200080 6B06 35GS 13F12 2 – 3 F027200081 6B06 35GS 6B12 4 – 6 F027200082 7G05 35GS 17C04 4 – 7 F027200084 7G09 35GS 6B12 2 F027200085 6B06 35GS 17C04 3 – 8 F027200113 6B06 9GS 10A06 twenty three

[0281] [surface] [2] [:exist] [THP1 pSTAT3] [During testing, compared with reference antibody] [IL-6 mAb 1] [Compared to monovalent, divalent, and bicomplementary isotropic agents] [IL-6 ISVD] [of] [IC50] [Multiple Difference.] BB = Building Block [ISVD ID] [BB1] [Connector] [BB2] [IC50] [anti] [IL-6 ISVD / IC50] [Reference Antibody] [IL-6 mAb1] [Ratio] A007100001 10A06 27 A007100002 13F12 86 A007100003 17C04 14 A007100005 6B12 14 A007100008 7G09 8 A007100009 6B06 22 F027200029 13F12 35GS 13F12 49 F027200030 17C04 35GS 17C04 14 F027200031 6B06 35GS 6B06 25 F027200032 6B12 35GS 6B12 46 F027200035 7G09 35GS 7G09 6 F027200036 10A06 35GS 10A06 13 F027200062 6B12 35GS 6B06 4 F027200064 17C04 35GS 7G09 2 F027200064 17C04 35GS 7G09 2 F027200067 13F12 35GS 6B06 7 F027200069 10A06 35GS 6B06 3 F027200073 6B12 35GS 13F12 6 F027200082 7G05 35GS 17C04 4 F027200084 7G09 35GS 6B12 1 F027200089 7G09 35GS 13F12 6 [6.2] [Example] [2] [:anti] [IL-6] [Unit Price] [ISVD] [Sequence optimization]

[0282] The sequences of anti-IL-6 ISVD IL6006B12 and IL6017C04 were further optimized.

[0283] Sequence optimization involves replacing one or more specific amino acid residues in the sequence to improve one or more (desired) properties of the ISVD.

[0284] Some examples of this sequence optimization are mentioned in further description in this paper, and are, for example, but not limited to:

[0285] Substitution in the parent wild-type Nanobody® sequence produces a Nanobody® sequence that is more identical to the human VH3-JH germline common sequence; this process is called humanization. To achieve this, specific amino acids (excluding so-called marker residues) that differ between the Nanobody® and human VH3-JH germline common sequences in the FR are modified to their human counterparts in a manner that preserves protein structure, activity, and stability.

[0286] The replacement with llama lineage to increase the stability of ISVD is defined as camelification. To this end, the amino acid sequences of the parent wild-type Nanobody® were compared with the amino acid sequences of the Nanobody® llama IGHV lineage (identified as the highest hit from the Nanobody® BlastP analysis for the llama IGHV lineage).

[0287] Substitutions that improve long-term stability or properties during storage, substitutions that increase performance levels in desired host cells or host organisms, and / or substitutions that eliminate or reduce one or more (unwanted) post-translational modifications (such as glycosylation or phosphorylation), depending on the desired host cell or host organism. To avoid N-terminal pyroglutamic acid formation, E1D mutations are typically introduced into the N-terminal building blocks of multivalent nanoantibodies without affecting potency or stability. Therefore, E1D mutations are not always introduced during the sequence optimization of the building blocks.

[0288] The mutation at position 11 is changed to Val and the mutation at position 89 is changed to Leu, in order to minimize the activity of binding any naturally occurring pre-existing antibodies.

[0289] Sequence optimization of the anti-IL-6 ISVD IL6006B12 yielded the final sequence-optimized variant F027201040, which contains five amino acid substitutions (i.e., L11V, S52aG, S60A, K83R, V89L) compared to the parental ISVD IL6006B12. Sequence optimization of the anti-IL-6 ISVD IL6017C04 yielded the final sequence-optimized variant F027200921, which contains six amino acid substitutions (i.e., E1D, L11V, A14P, D16G, K83R, V89L) compared to the parental ISVD IL6017C04.

[0290] Sequence-optimized variants were assembled from oligonucleotides using PCR overlap extension. These variants were expressed in *E. coli* and purified by IMAC and desalting. The hIL-6 binding capacity of F027201040 and the neutralizing activity of F027200921 in TF1 proliferation assays were evaluated by surface plasma resonance. The monomeric behavior of both variants was monitored by size exclusion HPLC (SE-HPLC). The thermal stability of the variants was tested using a Lightcycler (Roche) thermal displacement assay (TSA). In this assay, parental ISVDs and their variants were incubated at different pH values ​​in the presence of sypro orange with a temperature gradient applied. Sypro orange bound when ISVDs began to denature, and the measured fluorescence suddenly increased, allowing determination of the melting temperature at a specific pH. The results are summarized in Tables 3 and 4.

[0291] [surface] [3] [:anti] [IL-6 ISVD IL6006B12] [Sequence-optimized variants] [F027201040] [Analysis results] [ISVD ID] [One or more mutations] [k, off , ] [hIL-6 (1 / s)] [pH 7] [Down] [TSA] [,] [Tm] [(] [ºC] [)] [SE-HPLC] [, Main Peak] [%] [IL6006B12] [-] 6.5E-05 72 100 [F027201040] L11V, S52aG, S60A, K83R, V89L 5.9E-05 79 100

[0292] Compared to its parent ISVD IL006B12, F027201040 exhibits a similar dissociation rate with IL-6 binding in SPR. The Tm of F027201040 is 7ºC higher than that of its parent ISVD IL006B12. Based on the AbM definition (see Kontermann and Dübel (ed.) Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, 2010), the architectural identity % in the architectural region of F027201040 is 88%, and 86% based on the Kabat definition.

[0293] [surface] [4] [:anti] [IL-6 ISVD IL6017C04] [Analysis results of sequence-optimized variants] [ISVD ID] [One or more mutations] [TF-1] [Proliferation Assay] [hIL-6] [IC50 (M)] [, , ] [pH 7] [Down] [TSA] [,] [Tm] [(] [ºC] [)] [SE-HPLC] [, Main Peak] [%] [IL6017C04] - 3.8E-08 85 99 [F027200921] E1D, L11V, A14P, D16G, K83R, V89L 5.5E-08 84 95

[0294] The efficacy of F027200921 in TF1 proliferation assays is similar to that of the WT sequence. The Tm of F027200921 is 1°C lower than that of the parental ISVD F027200921. Based on the AbM definition, the structural identity % in the structural regions of F027200921 is 88%, and based on the Kabat definition, it is 86%.

[0295] [surface] [5] [:] [ISVD] [of] [IL6006B12] [and] [IL017C04] [Amino acid sequence in its optimized form] [ISVD ID] [ISVD] [describe] [sequence] F027200921 IL6017C04(E1D, L11V, A14P, D16G, K83R, V89L) DVQLVESGGGVVQPGGSLRLSCAASGRTFSNYAMAWFRQAPGKEREFVAVISYAGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAAVDSPLIATHPRGYDYWGQGTLVTVSS F027201040 IL6006B12(L11V, S52aG, S60A, K83R, V89L) EVQLVESGGGVVQPGGSLRLSCAASGFTLAYYAIGWFRQAPGKEREGVSCISGSVGTTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCVRSSWFDCGVQGRDLGNEYDYRGQGTLVTVSS [6.3] [Example] [3] [Multispecificity] [ISVD] [Construction Generation]

[0296] The identification of the ISVD-containing peptide F027201062 (SEQ ID NO: 1) that binds to TNFα and IL-6 stemmed from data-driven multispecific engineering and formatting activities that included three anti-TNFα VHH constructs (TNF06C11 (WO 2017081320), TNF01C02 (WO 2015173325, SEQ ID NO: 327), and VHH#3 (WO 2004041862)) and six anti-IL-6 VHH constructs (IL6006B06, IL006B12, IL6007G04, IL6007G05, IL6007G09, IL6010A06, IL6013F12, and IL6017C04, WO 2007104529) and an anti-HSA VHH construct ALB23002 (see WO 2007104529). 2017134234, SEQ ID NO:10 / WO 2018131234). Different positions / orientations of the building blocks were applied, and these were demonstrated to be crucial for various parameters (potency, cross-reactivity, performance, etc.). For all builds, the linkers between the building blocks were kept at 9GS to minimize the binding of pre-existing antibodies as much as possible.

[0297] A group containing 87 constructs (Table 6) was transformed into *Pichia pastoris* for small-scale production. ISVD expression was induced by stepwise addition of methanol. Clarified medium containing secreted ISVDs was used as the starting material for purification via protein A affinity chromatography, followed by desalting. The purified samples were used for performance evaluation and functional characterization. For the latter, potency was determined by measuring inhibition of TNFα-induced NFκB initiation and IL-6-induced TF-1 cell proliferation in vitro (as described in Examples 8 and 9).

[0298] In addition, ISVD performance levels were monitored in clarified media. Constructs were classified according to the following performance level criteria: low = <50 µg / ml, medium = 51-100 µg / ml, and high = >101 µg / ml (Table 6).

[0299] [surface] [6] [Evaluation]

[87] [Different species, multiple specificities] [ISVD] [Form relates to their respective performance levels] [Nfkb] [In the determination of reporter substances] [IC50] [、] [TF1] [In proliferation assays] [IC50] [And in the relationship between humans and crab-eating macaques] [TNF-] [α] [and] [IL-6] [List of effectiveness differences between them.] BB = building block, ALB = ALB23002. nd = undetermined. Performance level standards: Low = <50 µg / ml, Medium = 51-100 µg / ml, High = >101 µg / ml [ISVD] [Constructor] [ID] [Anti-resistance in constructs] [IL-6] [Double complementary positions] [ISDV] [BB1] [Connector] [1] [BB2] [Connector] [2] [BB3] [Connector] [3] [BB4] [Connector] [4] [BB5] [Performance Level] [hTNF-α Nfkb] [Measurement] [(IC50, M)] [IC50] Crab-eating macaques [TNFα / hTNFα] [ratio] [hIL-6 TF1] [Proliferation Assay] [(IC50, M)] [IC50] Crab-eating macaques [IL-6 / hIL-6] [ratio] F027200926 10A06-7G09 1C02 9GS 10A06 9GS ALB 9GS 7G09 high 4.53E-10 5.9 5.86E-11 1.2 F027200927 10A06-7G09 6C11 9GS 10A06 9GS ALB 9GS 7G09 high 5.93E-11 4.3 7.12E-11 1.0 F027200928 10A06-7G09 10A06 9GS ALB 9GS 7G09 9GS 1C02 high 7.88E-10 4.2 6.89E-11 1.0 F027200929 10A06-7G09 10A06 9GS ALB 9GS 7G09 9GS 6C11 high 8.58E-11 3.2 7.38E-11 1.0 F027200930 10A06-7G09 10A06 9GS 1C02 9GS 7G09 9GS 1C02 9GS ALB medium 2.98E-11 3.4 7.23E-11 1.0 F027200158 13F12-6B06 1C02 9GS ALB 9GS 13F12 9GS 6B06 high 1.02E-09 3.3 F027200159 13F12-6B06 6C11 9GS ALB 9GS 13F12 9GS 6B06 medium 1.30E-10 3.9 3.73E-10 2.4 F027200162 13F12-6B06 13F12 9GS 6B06 9GS ALB 9GS 1C02 medium 1.04E-09 4.0 2.87E-10 2.5 F027200163 13F12-6B06 13F12 9GS 6B06 9GS ALB 9GS 6C11 Low 1.31E-10 3.6 3.02E-10 2.4 F027200178 13F12-6B06 13F12 9GS 6B06 9GS 1C02 9GS ALB 9GS 1C02 Medium 7.8E-11 2.1 4.37E-10 2.2 F027200181 13F12-6B06 13F12 9GS 6B06 9GS VHH#3E 9GS ALB 9GS VHH#3E Low 7.01E-11 3.6 3.14E-10 1.9 F027200202 13F12-6B06 1C02 9GS ALB 9GS 1C02 9GS 13F12 9GS 6B06 Medium 8.55E-11 3.2 5.52E-10 2.7 F027200205 13F12-6B06 VHH#3E 9GS ALB 9GS VHH#3E 9GS 13F12 9GS 6B06 Medium 8.75E-11 4.6 9.43E-10 2.9 F027200160 13F12-7G09 6C11 9GS ALB 9GS 13F12 9GS 7G09 High 1.21E-10 3.2 3.41E-10 0.9 F027200161 13F12-7G09 1C02 9GS ALB 9GS 13F12 9GS 7G09 High 6.34E-10 3.7 2.68E-10 0.8 F027200191 13F12-7G09 1C02 9GS ALB 9GS 1C02 9GS 13F12 9GS 7G09 Medium 5.47E-11 3.4 3.05E-10 1.0 F027200195 13F12-7G09 VHH#3E 9GS ALB 9GS VHH#3E 9GS 13F12 9GS 7G09 Low 6.46E-11 4.1 3.30E-10 1.0 F027200208 13F12-7G09 13F12 9GS 7G09 9GS 1C02 9GS ALB 9GS 1C02 Medium 8.02E-11 3.4 3.80E-10 1 F027200209 13F12-7G09 13F12 9GS 7G09 9GS VHH#3E 9GS ALB 9GS VHH#3E Low 7.24E-11 5.5 3.19E-10 1 F027200214 13F12-7G09 13F12 9GS 7G09 9GS ALB 9GS 6C11 Medium 2.02E-10 2.6 3.68E-10 1 F027200771 13F12-7G09 13F12 9GS 7G09 9GS ALB 9GS 1C02 High 7.88E-10 7.2 3.57E-10 0.7 F027201024 17C04-6B06 17C04 9GS 6B06 9GS 1C02 9GS ALB 9GS 1C02 High 1.71E-11 3.8 2.86E-10 1.3 F027201025 17C04-6B06 1C02 9GS ALB 9GS 1C02 9GS 17C04 9GS 6B06 high 1.64E-11 4.5 2.85E-10 1.3 F027201026 17C04-6B06 6C11 9GS ALB 9GS 17C04 9GS 6B06 medium 8.52E-11 2.2 3.03E-10 1.2 F027201027 17C04-6B06 17C04 9GS ALB 9GS 6B06 9GS 6C11 medium 6.03E-11 2.5 3.17E-10 0.6 F027201028 17C04-6B06 17C04 9GS 6B06 9GS ALB 9GS 6C11 Low 6.10E-11 3.1 2.93E-10 1.1 F027201029 17C04-6B06 6C11 9GS 17C04 9GS ALB 9GS 6B06 medium 6.85E-11 2.0 3.47E-10 0.6 F027201030 17C04-6B06 17C04 9GS 1C02 9GS 6B06 9GS 1C02 9GS ALB medium 1.91E-11 4.2 3.08E-10 0.6 F027201031 17C04-6B06 1C02 9GS 17C04 9GS 1C02 9GS 6B06 9GS ALB medium 2.08E-11 3.3 3.08E-10 0.9 F027201058 17C04-6B12 17C04 9GS 1C02 9GS 6B12 9GS 1C02 9GS ALB High 4.029E-11 2.3 1.55E-10 0.6 F027201059 17C04-6B12 1C02 9GS 17C04 9GS 1C02 9GS 6B12 9GS ALB Medium 4.155E-11 2.2 1.35E-10 0.7 F027201062 17C04-6B12 17C04 9GS ALB 9GS 6B12 9GS 6C11 Medium 9.784E-11 2.0 1.34E-10 0.5 F027200204 17C04-7G09 VHH#3E 9GS 17C04 9GS VHH#3E 9GS 7G09 9GS ALB Medium 6.37E-11 5.9 7.71E-11 0.7 F027200212 17C04-7G09 17C04 9GS VHH#3E 9GS 7G09 9GS VHH#3E 9GS ALB Low 8.10E-11 5.5 8.21E-11 1 F027200216 17C04-7G09 17C04 9GS ALB 9GS 7G09 9GS 1C02 Medium 1.44E-09 6.9 8.90E-11 1 F027200809 17C04-7G09 6C11 9GS 17C04 9GS ALB 9GS 7G09 High 1.41E-10 2.3 6.82E-11 0.9 F027200812 17C04-7G09 17C04 9GS ALB 9GS 7G09 9GS 6C11 Medium 2.06E-10 1.7 7.2E-11 0.7 F027200817 17C04 - 7G09 1C02 9GS 17C04 9GS 1C02 9GS 7G09 9GS ALB High 6.80E-11 1.6 6.44E-11 1.1 F027200818 17C04 - 7G09 17C04 9GS 1C02 9GS 7G09 9GS 1C02 9GS ALB High 8.91E-11 2.1 8.29E-11 0.9 F027200823 17C04 - 7G09 1C02 9GS 17C04 9GS ALB 9GS 7G09 high 1.55E-09 4.5 7.81E-11 0.8 F027200153 6B06-10A06 6C11 9GS ALB 9GS 6B06 9GS 10A06 high 1.39E-10 2.7 1.03E-09 2.5 F027200165 6B06-10A06 6B06 9GS 10A06 9GS ALB 9GS 1C02 medium 9.94E-10 5.2 5.98E-10 3.9 F027200167 6B06-10A06 6B06 9GS 10A06 9GS ALB 9GS 6C11 medium 1.71E-10 3.1 1.27E-09 2.5 F027200168 6B06-10A06 6B06 9GS ALB 9GS 10A06 9GS 6C11 medium 1.40E-10 3.3 9.66E-10 1.6 F027200169 6B06-10A06 6B06 9GS ALB 9GS 10A06 9GS 1C02 medium 1.18E-09 3.7 3.82E-10 2.1 F027200179 6B06-10A06 6B06 9GS 10A06 9GS 1C02 9GS ALB 9GS 1C02 medium 7.59E-11 2.0 5.83E-10 2.5 F027200183 6B06-10A06 6B06 9GS 1C02 9GS 10A06 9GS 1C02 9GS ALB Medium 1.03E-10 2.4 4.61E-10 1.6 F027200185 6B06-10A06 6B06 9GS VHH#3E 9GS 10A06 9GS VHH#3E 9GS ALB Low 7.43E-11 4.1 5.98E-10 1.8 F027200192 6B06-10A06 VHH#3E 9GS ALB 9GS VHH#3E 9GS 6B06 9GS 10A06 Low 6.85E-11 4.3 8.16E-10 1.9 F027200196 6B06-10A06 VHH#3E 9GS 6B06 9GS VHH#3E 9GS 10A06 9GS ALB Low 7.31E-11 5.8 5.50E-10 1.5 F027200201 6B06-10A06 1C02 9GS ALB 9GS 1C02 9GS 6B06 9GS 10A06 Medium 6.80E-11 3.5 6.98E-10 3.0 F027200218 6B06-10A06 1C02 9GS 6B06 9GS 1C02 9GS 10A06 9GS ALB Low 1.02E-10 3.4 7.41E-10 2 F027200219 6B06-10A06 6B06 9GS 10A06 9GS VHH#3E 9GS ALB 9GS VHH#3E Low 7.30E-11 9.6 5.85E-10 2.5 F027200782 6B06-10A06 1C02 9GS 6B06 9GS ALB 9GS 10A06 medium 9.53E-10 3.32 3.27E-10 3.0 F027200925 6B06-10A06 6C11 9GS 6B06 9GS ALB 9GS 10A06 high 6.43E-11 4.1 6.1E-10 2.8 F027200154 6B06-17C04 6C11 9GS ALB 9GS 6B06 9GS 17C04 Low 1.64E-10 2.8 4.15E-10 2.1 F027200155 6B06-17C04 1C02 9GS ALB 9GS 6B06 9GS 17C04 High 1.15E-09 3.1 4.05E-10 2.2 F027200187 6B06-17C04 6B06 9GS 17C04 9GS 1C02 9GS ALB 9GS 1C02 Medium 5.64E-11 2.7 3.43E-10 2.6 F027200189 6B06-17C04 1C02 9GS ALB 9GS 1C02 9GS 6B06 9GS 17C04 Medium 5.31E-11 3.1 4.91E-10 2.2 F027200193 6B06-17C04 VHH#3E 9GS ALB 9GS VHH#3E 9GS 6B06 9GS 17C04 Low 5.95E-11 4.7 4.23E-10 1.8 F027200210 6B06-17C04 6B06 9GS 17C04 9GS VHH#3E 9GS ALB 9GS VHH#3E Low 7.20E-11 4.8 3.23E-10 2 F027200213 6B06-17C04 6B06 9GS 17C04 9GS ALB 9GS 1C02 Medium 1.53E-09 9.5 2.94E-10 2 F027200215 6B06-17C04 6B06 9GS 17C04 9GS ALB 9GS 6C11 Medium 2.39E-10 2.7 3.02E-10 2 F027200156 6B06-6B12 1C02 9GS ALB 9GS 6B06 9GS 6B12 medium 1.10E-09 3.5 3.05E-09 9.9 F027200157 6B06-6B12 6C11 9GS ALB 9GS 6B06 9GS 6B12 medium 1.24E-10 4.0 3.33E-09 7.1 F027200164 6B06-6B12 6B06 9GS 6B12 9GS ALB 9GS 1C02 medium 1.12E-09 5.4 2.40E-09 10.3 F027200166 6B06-6B12 6B06 9GS 6B12 9GS ALB 9GS 6C11 Medium 1.60E-10 3.3 2.25E-09 10.0 F027200180 6B06-6B12 6B06 9GS 6B12 9GS 1C02 9GS ALB 9GS 1C02 Medium 7.13E-11 2.9 3.55E-09 2.4 F027200188 6B06-6B12 6B06 9GS 6B12 9GS VHH#3E 9GS ALB 9GS e VHH#3E<� Low <� 5.58E-11 4.4 2.68E-09 2.7 F027200190 6B06-6B12 1C02 9GS ALB 9GS 1C02 9GS 6B06 9GS 6B12 Low 7.93E-11 3.2 2.77E-09 2.9 F027200194 6B06-6B12 VHH#3E 9GS ALB 9GS VHH#3E 9GS 6B06 9GS 6B12 Low 6.47E-11 4.3 2.36E-09 4.4 F027200170 6B12-13F12 6B12 9GS ALB 9GS 13F12 9GS 6C11 Medium 1.09E-10 4.1 9.04E-11 1.2 F027200171 6B12-13F12 6B12 9GS ALB 9GS 13F12 9GS 1C02 Medium 8.42E-10 4.5 1.02E-10 1.0 F027200172 6B12-13F12 6C11 9GS 6B12 9GS ALB 9GS 13F12 Medium 8.64E-11 4.5 1.12E-10 1.7 F027200173 6B12-13F12 1C02 9GS 6B12 9GS ALB 9GS 13F12 Medium 6.27E-10 4.4 1.15E-10 1.7 F027200184 6B12-13F12 6B12 9GS 1C02 9GS 13F12 9GS 1C02 9GS ALB Medium <000斯288> 1.04E-10 2.1 1.06E-10 2.6 F027200186 6B12-13F12 6B12 9GS VHH#3E 9GS 13F12 9GS VHH#3E 9GS ALB Low 6.51E-11 4.8 2.05E-1×10 3.0 F027200197 6B12-13F12 VHH#3E 9GS 6B12 9GS VHH#3E 9GS 13F12 9GS ALB Low 7.72E-11 5.9 2.56E-1×10 2.8 F027200198 6B12-13F12 1C02 9GS 6B12 9GS 1C02 9GS 13F12 9GS ALB Low 7.77E-11 2.6 2.51E-1×10 3.2 F027201056 6B12-7G09 1C02 9GS 6B12 9GS 1C02 9GS 7G09 9GS It should be noted that in the translation of scientific and technical terms, it is necessary to ensure accuracy and consistency. If there are specific professional standards or common translations for certain terms, they should be followed. In this text, some numbers and scientific notations are directly retained, and only the Chinese words are translated into English.ALB Low 3.426E-11 2.2 1.44E-10 0.5 F027201057 6B12-7G09 6B12 9GS 1C02 9GS 7G09 9GS 1C02 9GS ALB Medium 1.9E-10 2.0 5.77E-10 0.4 F027201060 6B12-7G9 6B12 9GS ALB 9GS 7G09 9GS 6C11 Medium 9.995E-11 1.8 1.27E-10 0.4 F027201061 6B12-7G09 6C11 9GS 6B12 9GS ALB 9GS 7G09 High 7.125E-11 2.5 1.47E-10 0.7 F027200981 7G09-6B12 1C02 9GS 7G09 9GS 1C02 9GS 6B12 9GS ALB nd nd 1.86E-10 59.7 F027200983 7G09-6B12 7G09 9GS 1C02 9GS 6B12 9GS 1C02 9GS ALB nd nd 1.38E-10 42.7 F027200987 7G09-6B12 7G09 9GS ALB 9GS 6B12 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​medium nd 1.27E-10 38.3

[0300] Depending on the valence, the ISVD building blocks used, and the relative positions of the ISVD building blocks, some constructs exhibited impaired potency and performance. Bispecific ISVDs containing anti-TNFa ISVDs TNF006C11, bivalent TNF001C02, and bivalent VHH#3E showed similar potency to the reference anti-TNFa mAb (the benchmark monoclonal antibody against TNF-α), while ISVDs containing monovalent TNF001C02 were 5 to 25 times less potent than the reference anti-TNFa mAb. All ISVDs containing bivalent VHH#3E showed low performance levels and were therefore deselected.

[0301] All bispecific ISVDs containing the anti-IL-6 ISVD IL6006B06 showed impaired potency, with the 6B06-6B12 combination performing the worst. All bispecific ISVDs containing the anti-IL-6 ISVD IL6013F12 showed degradation after expression in *Pichia pastoris*, making them unsuitable for production. The remaining bispecific ISVDs containing the dual complementary anti-IL-6 ISVDs 10A06-7G09, 17C04-7G09, 17C04-6B12, 6B12-7G09, and 7G09-6B12 generally showed similar potency to the anti-IL-6 reference mAb 1; however, 7G09-6B12 showed significantly impaired cross-reactivity against cynomolgus monkey IL-6.

[0302] Subsequently, the large group was reduced to a smaller group of multispecific constructs consisting of seven ISVD constructs: F027200809, F027200812, F027200817, F027200927, F027201060, F027201061, and F027201062. Preliminary yield estimates demonstrated that these ISVD constructs were effective against both targets (humans and cynomolgus monkeys) and possessed high performance potential. Three additional ISVD constructs, F027200925, F027200926, and F027201029, were selected primarily based on their high performance potential. However, the latter three constructs showed high potency against one target but only moderate potency against the other (Table 7a).

[0303] The configurations for ISVD constructs F027200927 and F027200925 are given in Table 7b.

[0304] Tables 7c and 7d provide the sequence of each individual building block for ISVD constructs F027200927 and F027200925, respectively.

[0305] Tables 7e, 7f, 7g, and 7h provide the sequences of the three CDR regions and four architecture regions present in each individual building block of ISVD constructs F027200927 and F027200925 (both numbered according to AbM and Kabat). Finally, Table 7i provides the complete amino acid sequences of ISVD constructs F027200927 and F027200925.

[0306] [surface] [7a] [:for]

[10] [Seed Resistance] [TNFa / ] [anti] [IL-6] [Bispecific] [ISVD] [The selected group compared to the reference compound] [Nfkb] [Humans and cynomolgus monkeys being measured] [TNFa] [and in] [TF1] [Humans and cynomolgus monkeys in proliferation assays] [IL-6] [neutralizing] [IC50] [Value.] ALB = ALB23002, BB = Building block [ISVD] [Constructor] [ID] [BB1] [Connector] [1] [BB2] [Connector] [2] [BB3] [Connector] [3] [BB4] [Connector] [4] [BB5] [hTNF-α Nfkb] [Measurement] [(IC50, pM)] [IC50] [Reference Antibody] [TNF-α mAb / Nb] [ratio] Crab-eating macaques [TNF-α NFkb] [Measurement] [(IC50, pM)] [hIL-6 TF1] [Proliferation Assay] [(IC50, pM)] [IC50] [Reference Antibody] [IL-6 mAb1 / Nb] [ratio] Crab-eating macaques [IL-6 TF1] [Proliferation Assay] [(IC50, pM)] [F027200809] 6C11 9GS 17C04 9GS ALB 9GS 7G09 141 0.91 329 68 1.7 61 [F027200812] 17C04 9GS ALB 9GS 7G09 9GS 6C11 206 0.62 360 72 1.7 54 [F027200817] 1C02 9GS 17C04 9GS 1C02 9GS 7G09 9GS ALB 68 0.50 108 64 1.6 69 [F027200925] 6C11 9GS 6B06 9GS ALB 9GS 10A06 64 1.43 265 610 14.8 1710 [F027200926] 1C02 9GS 10A06 9GS ALB 9GS 7G09 453 5.20 2670 59 1.5 72 [F027200927] 6C11 9GS 10A06 9GS ALB 9GS 7G09 59 4.20 254 71 1.8 72 [F027201029] 6C11 9GS 17C04 9GS ALB 9GS 6B06 69 0.83 139 347 5.3 209 [F027201060] 06B12 9GS ALB 9GS 7G09 9GS 6C11 100 0.67 176 127 2.3 49 [F027201061] 6C11 9GS 06B12 9GS ALB 9GS 7G09 71 0.91 176 147 2.7 101 [F027201062] 17C04 9GS ALB 9GS 06B12 9GS 6C11 98 0.67 193 134 2.4 65

[0307] [surface] [7b] [:] [F027200927] [and] [F027200925] [Configuration] [Name] [Constructor Block] [1] [Connector] [Constructor Block] [2] [Connector] [Building Blocks] [3] [Connector] [Building Blocks] [4] F027200927 6C11 9GS 10A06 9GS ALB23002 9GS 7G09+A* F027200925 6C11 9GS 6B06 9GS ALB23002 9GS 10A06+A* * C-terminal extension of a single alanine acid

[0308] [surface] [7c] [In tetravalent polypeptides] [F027200927] Different unit prices for internal testing [V, HH , ] [Amino acid sequence of the building block (「) [ID] [ " refers to [as used in this article] [SEQ ID NO] [)] [name] [ID] [Amino acid sequence] 6C11 (anti-hTNF-α) 5 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVKVSS 10A06 (Anti-hIL-6) 148 EVQLVESGGGVVQPGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVSTINWAGSRGYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAASAGGFLVPRVGQGYDYWGQGTLVTVSS ALB23002 (Anti-HSA) 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS 7G09 (anti-hIL-6) 149 EVQLVESGGGVVQPGGSLRLSCAASGFSLDYYGVGWFRQAPGKEREGVSCISSSEGDTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCATDLSDYGVCSRWPPSYDYWGQGTLVKVSS

[0309] [surface] [7d] [In tetravalent polypeptides] [F027200925] [Different unit prices for internal appraisal] [V, HH , ] [Constructing the amino acid sequence of the block ("") [ID] [ " refers to [as used in this article] [SEQ ID NO] [)] [Name] [ID] [Amino acid sequence] 6C11 (anti-hTNF-α) 5 EVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVKVSS 6B06 (anti-hIL-6) 159 EVQLVESGGGVVQPGGSLRLSCAASGIIFSINAMGWYRQAPGKQRELVADIFPFGSTEYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCHSYDPRGDDYWGQGTLVTVSS ALB23002 (anti-HSA) 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS 10A06 (anti-hIL-6) 148 EVQLVESGGGVVQPGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVSTINWAGSRGYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAASAGGFLVPRVGQGYDYWGQGTLVTVSS

[0310] [Table] [7e] [: According to] [AbM] [Numbered tetravalent polypeptide] [F027200927] [alone] [V, HH , ] [Building blocks] [CDR] [and architecture sequence (「) [ID] [ " refers to the given information. [SEQ ID NO] [)] [Building Blocks] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] [ID] 6C11 5 EVQLVESGGGVVQPGGSLRLSCTAS 20 GFTFSTADMG 9 WFRQAPGKGREFVA twenty four RISGIDGTTY 13 YDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRS 28 PRYADQWSAYDY 17 WGQGTLVKVSS 32 10A06 148 EVQLVESGGGVVQPGGSLRLSCAAS 19 GRTFSSYVMG 150 WFRQAPGKEREFVS 156 TINWAGSRGY 151 YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAA 25 SAGGFLVPRVGQGYDY 152 WGQGTLVTVSS 29 ALB23002 3 EVQLVESGGGVVQPGGSLRLSCAAS 19 GFTFRSFGMS 7 WVRQAPGKGPEWVS 22 SISGSGSDTL 11 YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 26 GGSLSR 15 SSQGTLVTVSS 30 7G09 149 EVQLVESGGGVVQPGGSLRLSCAAS 19 GFSLDYYGVG 153 WFRQAPGKEREGVS twenty three CISSSEGDTY 154 YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAT 157 DLSDYGVCSRWPSPYDY 155 WGQGTLVKVSS 32

[0311] [surface] [7f] [:according to] [AbM] [Numbered tetravalent polypeptide] [F027200925] [Alone] [V, HH , ] [Building blocks] [CDR] [and architecture sequence (「) [ID] [ " refers to the given information. [SEQ ID NO] [)] [Building Blocks] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] [ID] 6C11 5 EVQLVESGGGVVQPGGSLRLSCTAS 20 GFTFSTADMG 9 WFRQAPGKGREFVA 24 RISGIDGTTY 13 YDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRS 28 PRYADQWSAYDY 17 WGQGTLVKVSS 32 6B06 159 EVQLVESGGGVVQPGGSLRLSCAAS 19 GIIFSINAMG 160 WYRQAPGKQRELVA 163 DIFPFGSTE 161 YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCHS 164 YDPRGDDY 162 WGQGTLVTVSS 29 ALB23002 3 EVQLVESGGGVVQPGGSLRLSCAAS 19 GFTFRSFGMS 7 WVRQAPGKGPEWVS twenty two SISGSGSDTL 11 YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 26 GGSLSR 15 SSQGTLVTVSS 30 10A06 148 EVQLVESGGGVVQPGGSLRLSCAAS 19 GRTFSSYVMG 150 WFRQAPGKEREFVS 156 TINWAGSRGY 151 YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAA 25 SAGGFLVPRVGQGYDY 152 WGQGTLVTVSS 29

[0312] [surface] [7g] [:according to] [Kabat] [Numbered tetravalent polypeptide] [F027200927] [The only one] [V, HH , ] [Constructing blocks] [CDR] [and architectural sequence (" [ID] [ " refers to the given information. [SEQ ID NO] [)] [Building Blocks] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] [ID] 6C11 5 EVQLVESGGGVVQPGGSLRLSCTASGFTFS 44 TADMG 36 WFRQAPGKGREFVA twenty four RISGIDGTTYYDEPVKG 40 RFTISRDNSKNTVYLQMNSLRPEDTALYYCRS 48 PRYADQWSAYDY 17 WGQGTLVKVSS 32 10A06 148 EVQLVESGGGVVQPGGSLRLSCAASGRTFS 169 SYVMG 165 WFRQAPGKEREFVS 156 TINWAGSRGYYADSVKG 166 RFTISRDNAKNTVYLQMNSLRPEDTALYYCAA 45 SAGGFLVPRVGQGYDY 152 WGQGTLVTVSS 29 ALB23002 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFR 42 SFGMS 34 WVRQAPGKGPEWVS 22 SISGSGSDTLYADSVKG 38 RFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 46 GGSLSR 15 SSQGTLVTVSS 30 7G09 149 EVQLVESGGGVVQPGGSLRLSCAASGFSLD 175 YYGVG 167 WFRQAPGKEREGVS 23 CISSSEGDTYYADSVKG 168 RFTISRDNAKNTVYLQMNSLRPEDTALYYCAT 170 DLSDYGVCSRWPSPYDY 155 WGQGTLVKVSS 32

[0313] [surface] [7h] [:according to] [Kabat] [Numbered tetravalent polypeptide] [F027200925] [Alone] [V, HH , ] [Building blocks] [CDR] [and architecture sequence (「) [ID] [ " refers to the given information. [SEQ ID NO] [)] [Building Blocks] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] [ID] 6C11 5 EVQLVESGGGVVQPGGSLRLSCTASGFTFS 44 TADMG 36 WFRQAPGKGREFVA 24 RISGIDGTTYYDEPVKG 40 RFTISRDNSKNTVYLQMNSLRPEDTALYYCRS 48 PRYADQWSAYDY 17 WGQGTLVKVSS 32 6B06 159 EVQLVESGGGVVQPGGSLRLSCAASGIIFS 173 INAMG 171 WYRQAPGKQRELVA 163 DIFPFGSTEYADSVKG 172 RFTISRDNAKNTVYLQMNSLRPEDTALYYCHS 174 YDPRGDDY 162 WGQGTLVTVSS 29 ALB23002 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFR 42 SFGMS 34 WVRQAPGKGPEWVS 22 SISGSGSDTLYADSVKG 38 RFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 46 GGSLSR 15 SSQGTLVTVSS 30 10A06 148 EVQLVESGGGVVQPGGSLRLSCAASGRTFS 169 SYVMG 165 WFRQAPGKEREFVS 156 TINWAGSRGYYADSVKG 166 RFTISRDNAKNTVYLQMNSLRPEDTALYYCAA 45 SAGGFLVPRVGQGYDY 152 WGQGTLVTVSS 29

[0314] [surface] [7i] [Selected multivalent peptides] [F027200927] [and] [F027200925] [Amino acid sequence () [「] [ID] [」] [Refers to a given] [SEQ ID NO] [)] [Name] [ID] [Amino acid sequence] F027200927 147 DVQLVESGGGVVQPGGSLRLSCTASGFTFSTADMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVSTINWAGSRGYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAASAGGFLVPRVGQGYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFSLDYYGVGWFRQAPGKEREGVSCISSSEGDTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCATDLSDYGVCSRWPSPYDYWGQGTLVKVSSA F027200925 158 DVQLVESGGGVVQPGGSLRLSCTASGFTFSTADDMGWFRQAPGKGREFVARISGIDGTTYYDEPVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCRSPRYADQWSAYDYWGQGTLVTVSSGGGGS GGGSEVQLVESGGGVVQPGGSLRLSCAASGIIFSINAMGWYRQAPGKQRELVADIFPFGSTEYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCHSYDPRGDDYWGQGTLVTVSSGGGGSG GGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGGGSGGG SEVQLVESGGGVVQPGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVSTINWAGSRGYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAASAGGFLVPRVGQGYDYWGQGTLVKVSSA

[0315] Large-scale 2L and 5L production of groups containing 11 ISVD constructs was conducted in *Pichia pastoris* to determine performance yields and evaluate biophysical properties. It was demonstrated that specific combinations of the anti-IL-6 construct with the anti-TNF-α construct are required to obtain high performance yields as well as adequate solubility and biophysical stability. This is illustrated in Table 8. For example, constructs F027201062 and F027200812, which are very similar in composition except for one construct at the third position, showed significantly different performance and solubility profiles.

[0316] [surface] [8] [:]

[10] [Seed Resistance] [TNFa / ] [anti] [IL-6] [Dual heterosexuality] [ISVD] [The selected group exhibited yield, solubility, and biophysical properties at the highest concentration.] ALB = ALB23002, BB = building block, SVP = particles visible under a microscope, HMW = high molecular weight, nd = not determined. [ISVD] [Constructor] [ID] [BB1] [Connector] [1] [BB2] [Connector] [2] [BB3] [Connector] [3] [BB4] [Connector] [4] [BB5] [2 L] [Fermentation tank performance yield ()] [g / L] [)] [Solubility()] [mg / ml] [)] [F027200809] 6C11 9GS 17C04 9GS ALB 9GS 7G09 3.6 <50 [F027200812] 17C04 9GS ALB 9GS 7G09 9GS 6C11 2.5 <50 [F027200817] 1C02 9GS 17C04 9GS 1C02 9GS 7G09 9GS ALB 3.5 132 [F027200925] 6C11 9GS 6B06 9GS ALB 9GS 10A06 6.7 124 [F027200926] 1C02 9GS 10A06 9GS ALB 9GS 7G09 5.4 141 HMW formation at 5ºC [F027200927] 6C11 9GS 10A06 9GS ALB 9GS 7G09 5.1 112 Phase separation and gel formation [F027201029] 6C11 9GS 17C04 9GS ALB 9GS 6B06 6.4 152 Gel formation [F027201060] 06B12 9GS ALB 9GS 7G09 9GS 6C11 6.5 153 Gel formation [F027201061] 6C11 9GS 06B12 9GS ALB 9GS 7G09 4.9 152 Gel formation [F027201062] 17C04 9GS ALB 9GS 06B12 9GS 6C11 5.1 149

[0317] Finally, ISVD construct F027201062 was selected for further characterization because it exhibits good overall performance in terms of potency and CMC characteristics (e.g., solubility and performance). [Example] [4] [Multispecific] [ISVD] [Constructor: AND] [TNF-] [α] [、] [IL-6] [Binding affinity with serum albumin]

[0318] The affinity (expressed as equilibrium dissociation constant (KD)) of F027201062 for human and cynomolgus monkey TNF-α and IL-6, as well as human, cynomolgus monkey and mouse serum albumin (SA), was quantified on a Gyrolab xP workstation (Gyros) using in-solution affinity measurements.

[0319] Under KD-controlled measurements, serial dilutions of TNF-α or IL-6 (range 1.3 µM - 0.1 pM) or human or cynomolgus monkey SA (range 13 µM - 1 pM) or mouse SA (range 133 µM - 30 pM) and fixed amounts of F027201062 (80 pM for TNF-α, 20 pM for IL-6, 300 pM for human and cynomolgus monkey SA, and 600 pM for mouse SA) were mixed to allow interaction and incubated for 48 or 72 hours (for IL-6 and TNF-α) or 2 hours (for SA) to reach equilibrium.

[0320] Under receptor-controlled measurements, serial dilutions of TNF-α or IL-6 (range 1.3 µM - 0.1 pM) or human cynomolgus monkey SA (range 13 µM - 1 pM) or mouse SA (range 133 µM - 30 pM) and fixed amounts of F027201062 (30 nM for TNFα, 5 nM for IL-6, 1 µM for human cynomolgus monkey SA, and 2 µM for mouse SA) were mixed to allow interaction and incubated for 48 or 72 hours (for IL-6 and TNFα) or 2 hours (for SA) to reach equilibrium.

[0321] Biotinylated human TNF-α / IL-6 / serum albumin was captured in a microstructure of a Gyrolab Bioaffy 1000 CD containing a beaded column that served as a molecular probe to capture free F027201062 from an equilibrium solution. A mixture of TNF-α / IL-6 / serum albumin and F027201062 (containing free TNF-α / IL-6 / serum albumin, free F027201062, and the TNF-α / IL-6 / serum albumin-F027201062 complex) was passed through the beads, capturing a small percentage of free F027201062 proportional to the concentration of the free ISVD construct. A fluorescently labeled anti-VHH antibody ABH0086-Alexa647 was then injected to label any captured F027201062, and changes in fluorescence were measured after washing away excess fluorescent probe. The dilution series were fitted using Gyrolab Analysis software, where KD-controlled and receptor-controlled curves were analyzed to determine KD values. The results (Table 9) confirmed that the multispecific ISVD construct binds to human / cynomolgus monkey IL-6 and human / cynomolgus monkey TNF-α with high affinity.

[0322] [surface] [9] [:] [F027201062] [With humans and cynomolgus monkeys] [TNF-] [α] [and] [IL-6] [And human, cynomolgus monkey and mouse serum albumin () [SA] [) binding affinity. [antigen] [Human] [K, D , (pM) ] Crab-eating macaques [K, D , (pM) ] [Mouse] [K, D , (pM) ] [TNF-α] [n=1] 5.7 53.8 / [n=2] 5.2 23.7 [n=3] 9.9 25.9 [IL-6] [n=1] 7.3 13.7 / [n=2] 5.3 3.0 [n=3] 3.0 6.2 [SA] [n=1] 7600 6700 58500 [n=2] 6400 4900 62200 [n=3] 6200 6100 56000 [6.4] [Example] [5] [Multispecific] [ISVD] [Construction and Membrane Integration] [TNF] [α] [The combination of]

[0323] Flow cytometry was used to demonstrate the binding of F027201062 to membrane-bound TNFα in HEK293H cells expressing human membrane TNFα. Briefly, cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (to enhance the detection of membrane-bound TNFα). Cells were then seeded at a density of 1 × 10⁴ cells / well and incubated for 24 hours at room temperature with a series of dilutions of F027201062 or an anti-TNFα reference mAb, ranging from 100 nM to 0.5 pM, in the absence or presence of 30 µM HSA. Cells were washed three times, then incubated with anti-VHH mAb (ABH00119) at 4ºC for 30 min, washed again, and incubated with goat anti-mouse or anti-human PE-labeled antibodies at 4ºC for 30 min. Samples were washed and resuspended in FACS buffer (D-PBS containing 10% FBS and 0.05% sodium azide, supplemented with 5 nM TOPRO3). The cell suspension was then analyzed on an iQuescreener. EC50 values ​​were calculated using GraphPad Prism. The EC50 values ​​of F027201062 and the anti-TNFα reference mAb were comparable (Table 10).

[0324] [surface]

[10] [:and resistance] [hTNF] [α] [refer to] [mAb] [Compared to, in] [twenty four] [After hours of cultivation,] [F027201062] [Regarding membrane performance] [TNF] [α] [The affinity between them.] [condition] [+HSA] [-HSA] [n =] [1] [2] [3] [1] [2] [3] [sample] [ID] [F027201062] [EC50 (M)] 3.10E-10 2.90E-10 3.60E-10 1.20E-10 1.80E-10 1.70E-10 [sample] [ID] [Reference Antibody] [TNFa mAb] [EC50 (M)] 3.3E-11 2.93E-11 1.95E-11 3.8E-11 nd 9.80E-12 [6.5] [Example] [6] [Multispecific] [ISVD] [Constructors and] [TNF-] [α] [and] [IL-6] [Selective combination]

[0325] The absence of binding to TNF-α and IL-6-related human targets was assessed using SPR (Proteon XPR36). Human IL23, IL27, CNTF, OSM (OSM), and IL11 were evaluated as IL-6-related cytokines or cytokines sharing the gp130 receptor. Human FASL, TNFβ, LIGHT, TL-1A, and RANKL, members of the TNF superfamily and TNFα-related cytokines, were tested.

[0326] For this purpose, the target was immobilized on the Proteon GLC sensor chip at 25 μg / mL using amine coupling for 200 s, with 80 s of EDC / NHS injection for inactivation and 1 M ethanolamine HCl injection for 150 s of inactivation (ProteOn Amine Coupling Kit, catalog number 176-2410). The flow rate during inactivation, inactivation, and ligand injection was set to 30 µl / min. The pH of the 10 mM acetate immobilization buffer was selected by subtracting approximately 1.5 from the pI of each ligand. Next, 300 nM F027201062 was injected for 2 min, followed by dissociation at a flow rate of 45 µL / min for 600 s. PBS (pH 7.4) + 0.005% Tween 20 was used as the run buffer. As a positive control, 0.3 μM α-IL11 Ab, α-OSM Ab, α-CNTF Ab, α-IL27 Ab, α-IL27A Ab, α-IL23 p19 Ab, α-hFASL Ab, 0.3 μM α-hTNFβ Ab, 0.5 μM α-hLIGHT Ab, 0.3 μM α-hTL-1A Ab, and 0.5 μM α-hRANKL VHH were injected.

[0327] The interaction between F027201062 and the positive control with the immobilized target was measured by detecting an increase in refractive index, which was due to a change in quality on the wafer after bonding.

[0328] All positive controls were indeed bound to their respective targets. No binding was detected between the ISVD construct F027201062 and human TRAIL, CD30L, CD40L, FASL, TNF, LIGHT, TL-1A, RANKL, IL23, IL27, CNTF, tumor suppressor M, and IL11. [6.6] [Example] [7] [Multispecific] [ISVD] [Constructors and] [hIL-6] [、] [hTNFa] [and] [HSA] [Simultaneous combination]

[0329] The ability of the ISVD construct F027201062 to simultaneously bind to recombinant soluble hTNF-α and hIL-6 was determined using a Biacore 8K+ instrument. To this end, HSA was immobilized on a CM5 sensor chip at approximately 1600 RU via amine coupling. 100 nM of F027201062 was injected onto the HSA surface at 10 μL / min for 2 min to capture the ISVD construct via an ALB23002 building block. Subsequently, 100 nM hIL-6, hTNF-α, or hOX40L, or a mixture of 100 nM IL-6 + 100 nM TNFα, 100 nM IL-6 + 100 nM OX40L, or 100 nM TNF-α + 100 nM OX40L, were injected at a flow rate of 45 µL / min for 2 min, followed by a subsequent 600-second dissociation step. The HSA surface was regenerated by injecting HCl (100 mM) at 45 μL / min for 2 minutes. The sensor plot (Fig. 1) shows that the ISVD construct F027201062 can bind both hIL-6 and hTNF-α simultaneously, as indicated by the increase in response units after capture on the HSA: approximately 150 RU from hTNF-α alone, approximately 120 RU from hIL-6 alone, and approximately 340 RU for a mixture of IL-6 and TNF-α. [6.7] [Example] [8] [Multispecific] [ISVD] [Construction in vitro] [TNF-] [α] [Induced] [NFkB] [Inhibition of initiation]

[0330] HEK293_NFκB-NLucP cells are cells that express the TNF receptor and are stably transfected with a reporter construct encoding Nanoluciferase under the control of an NFκB-dependent promoter. Culture of these cells with soluble human and cynomolgus monkey TNF-α resulted in NFκB-mediated Nanoluciferase gene expression. Nanoluciferase luminescence was measured using Nano-Glo luciferase substrate added to the cells at a 1:50 ratio with lysis buffer. Samples were mixed on a shaker for 5 min to achieve complete lysis. Glo response™ HEK293_NFκB-NLucP cells were seeded at 20,000 cells / well in normal growth medium in white tissue culture (TC) treated 96-well plates with clear bottoms. Serial dilutions of F027201062 or an anti-TNF-α reference mAb were added to 25 pM human or 70 pM cynomolgus monkey TNF-α and incubated with the cells at 37ºC for 5 h in the presence of 30 µM HSA.

[0331] F027201062 inhibited TNF-α-induced NFκB initiation in humans and cynomolgus monkeys in a concentration-dependent manner, with mean IC50 values ​​of 53 pM (for human TNF-α) and 158 pM (for cynomolgus monkey TNF-α), comparable to the reference compound anti-hTNF-α mAb (Table 11, Figure 2). The negative control ISVD, i.e., IRR00096, showed no inhibition.

[0332] [surface]

[11] Compared to resistance [TNFα] [refer to] [mAb] [,exist] [Glo response™ HEK293_NFκB-NLucP] [Report analyte determination in progress,] [F027201062] [Mediation of human-cynomolgus monkey interaction] [TNFα] [neutralized] [IC50] [and] [IC90] [Value.] [F027201062] [Human] [TNF-α] Crab-eating macaques [TNF-α] [n=1] [n=2] [n=3] [n=1] [n=2] [n=3] [IC50 (M)] 7.57E-11 2.57E-11 5.86E-11 1.60E-10 8.37E-11 2.30E-10 [IC90 (M)] 3.61E-10 2.70E-10 4.60E-10 6.98E-10 7.50E-10 1.45E-09 [anti] [TNF-α] [refer to] [mAb] [Human] [TNF-α] Crab-eating macaques [TNF-α] [n=1] [n=2] [n=3] [n=1] [n=2] [n=3] [IC50 (M)] 5.54E-11 5.92E-11 5.90E-11 8.45E-11 1.05E-10 1.28E-10 [IC90 (M)] 2.97E-10 4.09E-10 3.42E-10 4.38E-10 8.85E-10 8.40E-10 [6.8] [Example] [9] [Multispecific] [ISVD] [Construction Pairs] [IL-6] [Induced] [TF-1] [Inhibition of cell proliferation]

[0333] The inhibitory efficacy of F027201062 was determined in a cell-based assay for monitoring IL-6-mediated TF-1 cell proliferation. For this purpose, TF-1 cells were cultured in RPMI 1640, glutamax, and HEPES (Gibco) medium supplemented with 10% FBS and 1% sodium pyruvate. TF-1 cells were seeded at 12,500 cells per well in growth medium. A serial dilution of purified anti-IL-6 ISVD or a reference compound was added. After incubation at 37ºC for 30 min, 75 pM of human IL-6 (R&D systems catalog 200-IL-200|206-IL) or cynomolgus monkey IL-6 (Evotek, catalog APP-7634) was added. After 72 hours, TF-1 cell proliferation was measured using a CellTiter-Glo (Promega #G7571) reader on an EnVision Multilabel reader (Perkin Elmer).

[0334] F027201062 inhibited IL-6-induced TF-1 cell proliferation in humans and cynomolgus monkeys in a concentration-dependent manner, with mean IC50 values ​​of 34 pM (for human IL-6) and 56 pM (for cynomolgus monkey IL-6), which were comparable to and superior to anti-IL-6 reference mAb 1 and anti-IL-6 reference mAb 2 (Table 12, Figure 3).

[0335] [surface]

[12] [:and resistance] [IL-6] [Compared to the reference compound,] [F027201062] [Mediated interaction between humans and cynomolgus monkeys] [IL-6] [Induced] [TF-1] [Inhibitory to cell proliferation] [IC50] [and] [IC90] [value] [F027201062] [people] [IL-6] Crab-eating macaques [IL-6] [n=1] [n=2] [n=3] [n=1] [n=2] [n=3] [IC50 (M)] 1.36E-10 4.62E-11 4.23E-11 6.46E-11 5.92E-11 4.34E-11 [IC90 (M)] 3.75E-10 9.63E-11 8.10E-11 2.15E-10 1.97E-10 1.68E-10 [Reference Antibody] [IL-6 mAb1] [people] [IL-6] Crab-eating macaques [IL-6] [n=1] [n=2] [n=3] [n=1] [n=2] [n=3] [IC50 (M)] uncertain 2.78E-11 3.30E-11 3.19E-11 3.06E-11 3.18E-11 [IC90 (M)] uncertain 4.48E-11 5.70E-11 7.50E-11 7.45E-11 7.08E-11 [Reference Antibody] [IL-6 mAb2] [people] [IL-6] Crab-eating macaques [IL-6] [n=1] [n=2] [n=3] [n=1] [n=2] [n=3] [IC50 (M)] 1.55E-10 5.51E-11 5.73E-11 7.86E-11 5.85E-11 5.47E-11 [IC90 (M)] 1.54E-09 7.35E-10 3.80E-10 5.06E-10 4.58E-10 3.48E-10 [6.9] [Example]

[10] [Multispecificity with pre-existing antibodies] [ISVD] [Constructor]

[0336] The reactivity of the ISVD construct F027201062 to a pre-existing antibody was evaluated in normal human serum (n = 96) using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) was used as the run buffer, and experiments were performed at 25ºC.

[0337] ISVD was captured on the wafer by binding to HSA immobilized on the wafer via an ALB23002 building block. To immobilize the HSA, the ligand channels of the ProteOn GLC sensor wafer were activated using an EDC / NHS (flow rate 30 μl / min), and the HSA was injected at 100 μl / ml into ProteOn acetate buffer at pH 4.5 to achieve an immobilization level of approximately 2500 RU. After immobilization, the surface was deactivated using ethanolamine hydrochloride (flow rate 30 μl / min).

[0338] Subsequently, the ISVD construct was injected onto the HSA surface at 45 μl / min for 2 min to achieve an ISVD capture level of approximately 800 RU. Samples containing the pre-existing antibody were centrifuged at 14,000 rpm for 2 min, and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%) and injected at 45 μl / min for 2 min, followed by a subsequent 400-second dissociation step. After each loop (i.e., before the new ISVD capture and blood sample injection steps), the HSA surface was regenerated by injecting HCl (100 mM) at 45 μl / min for 2 min. A sensor map showing the binding of the pre-existing antibody was obtained after subtracting two references: 1) ISVD-HSA dissociation and 2) non-specific binding with the reference ligand channel. The binding level of the pre-existing antibody was determined by setting the reporter point to 125 seconds (5 seconds after the end of association). The percentage reduction in pre-existing antibody binding was calculated relative to the binding level of the reference ISVD at 125 seconds.

[0339] Compared to the unoptimized pentavalent ISVD construct F027301186, the quadrivalent ISVD construct F027201062, optimized by introducing mutants L11V and V89L and C-terminal alanine in each building block to reduce binding to pre-existing antibodies, showed a significant reduction in binding to pre-existing antibodies (Figure 4). [6.10] [Example]

[11] [Use rats to resist] [TNF-] [α] [and resistance] [IL-6] [A combination of alternative antibodies provides sustained long-term relief from collagen-induced arthritis in mice]

[0340] Rheumatoid arthritis is a destructive autoimmune disease that attacks peripheral joints. A mouse model of collagen-induced arthritis (CIA) is reproducing this erosive disease. To elicit an immune response against joint components, susceptible DBA / 1 mice were immunized twice with 100 µg of adjuvant chicken collagen II. The immune response against collagen II spread to endogenous articular cartilage and resulted in clinically apparent arthritis. After the second immunization on day 21, progressive arthritis became apparent, manifested as swelling and erythema in the ankles and feet, and sometimes joint stiffness. The severity of arthritis in each of the four feet was clinically assessed using a scoring system detailed in Table 13 below.

[0341] [surface]

[13] Arthritis scoring systems used to determine the severity of arthritis phenotypes [Arthritis Score] [, 1 , ] [feature] 0 No clinical symptoms were detected. 1. Mild Swelling of the ankle or sole 2. Moderate Swelling and mild erythema in the ankle and sole. 3. Severe Severe swelling of the ankle and sole, severe erythema, and stiff joints. Arthritis score on the y-axis in Figure 5: sum of individual claw scores. Maximum total score: 4 x 3 = 12.

[0342] To assess the impact of combined TNF and IL-6 blockade on disease severity and progression, N=13 immunized male DBA / 1 mice were treated with blocking antibodies against mouse TNF, mouse IL-6, or a combination of both. Mice were treated twice weekly via intraperitoneal injection, starting on day 22 post-immunization and continuing until day 55. As shown in Figure 5, clinical arthritis gradually developed from day 21. Mice treated with anti-muTNF or a combination of anti-muTNF and anti-muIL-6 showed delayed and less severe arthritis progression compared to mice treated with isotype control antibodies or anti-muIL-6 alone. Upon discontinuation of treatment on day 55, disease rebound was observed in mice treated with anti-muTNF alone, rapidly reaching severity comparable to isotype control or anti-muIL-6. However, mice treated with the combination of anti-muTNF and anti-muIL-6 did not rebound to arthritis progression and maintained a response despite discontinuation of treatment. Figure 6 shows the analysis of the area under the curve (AUC) of arthritis scores over time throughout the study duration, during the treatment period, and at the treatment-off period. The latter was significantly suppressed by the combination therapy, indicating a sustained effect on disease progression.

[0343] Arthritis in this model was induced by an antibody response to the collagen II vaccine. On day 91, plasma levels of anti-collagen II antibodies were measured by ELISA. As shown in Figure 7, all treatments reduced anti-collagen II antibody titers, with the largest reductions observed in mice treated with both anti-muTNF and anti-muIL-6.

[0344] After euthanizing mice on day 91, hind paws were collected, and metatarsal joints were processed for histological evaluation of arthritis. Hematoxylin and eosin, as well as safranin-O stained sections were scored in a blinded manner across four aspects in a range of 0-5 (Table 14).

[0345] [surface]

[14] [Arthritis Histological Scoring System] [Histological score] [, * , ] [feature] inflammation 1. Immune cells infiltrate the synovium and / or adjacent areas minimally. 2. Mild infiltration, fewer than 3 affected joints 3. Moderate infiltration, with swelling of 3-4 affected joints and connective tissue. 4. Severe infiltration in most joints 5. Very severe infiltration in all joints Pannus formation 1. Minimal pannus formation in the surrounding area, with only one affected joint. 2. Mild pannus formation, 1-3 joints 3. Moderate pannus formation, 1-3 joints 4. Severe pannus formation and joint deformities in most joints. 5. Very severe pannus formation and joint deformities in all joints. Cartilage damage 1. Minimal proteoglycan loss (Safranin-O staining), only 1 affected joint. 2. Proteoglycan loss, affecting up to 3 joints. 3. Most of the proteoglycans are lost in many joints. 4. Severe loss of proteoglycans, rough surface, and necrotic cartilage cells. 5. No cartilage remaining. Bone destruction 1. Minimal bone resorption visible only at high magnification 2. Mild bone resorption visible at low magnification 3. Moderate bone resorption on certain cortical surfaces 4. Severe bone resorption on all cortical surfaces 5. Bone deformities *Arthritis scores are plotted on the y-axis in Figure 8.

[0346] As shown in Figure 8, in mice treated with a combination of anti-muTNF and anti-muIL-6, the histological scores for pannus formation and bone destruction were statistically significantly improved relative to the isotype control antibody.

[0347] In summary, these data indicate that combination therapy, which blocks both the TNF and IL-6 inflammatory pathways, has the highest therapeutic efficacy. Importantly, combination therapy can produce a sustained response even without aggressive treatment. [6.11] [Example]

[12] [From using anti] [TNF-] [α] [,anti] [IL6] [and combined resistance] [TNF-] [α] [IL6] [Therapeutic] [CIA] [A model of collagen-induced arthritis] [RNA-seq] [Data Analysis]

[0348] Total RNA from forefoot tissue samples of CIA mice was purified using the RNAeasy kit (Qiagen), and paired-end sequencing of 2x51-66 million reads was performed on the NovaSeq platform (Illumina) at ATLAS Biolabs GmbH in Berlin. Bioinformatics analysis of the raw RNA-seq data was performed using OmicsSoft studio software version 10.01.118 (Qiagen). Using OmicSoftGenCode.V19 as the gene model, mouse B.38 as the reference genome, and OSA4 as the alignment, RNA-seq reads (fastq files) were mapped against the mouse genome.

[0349] [Genes with differential expression ()] [DEG] [Wayne's analysis]

[0350] Figure 9(A) shows the overlap of DEGs identified from anti-TNF-α, anti-IL6, and combined anti-TNF-α / IL6 treatments in the CIA model. DEGs were determined by comparing RNA-seq samples treated with standard conventional anti-TNF-α antibody (n = 13 samples), standard conventional anti-IL6 antibody (MP5-20F3; n = 13), and combined anti-TNF-α / anti-IL-6 antibody (n = 13) with samples from allotype treatment (IgG control; n = 13) using the Benjamini-Hochberg (BH-FDR) method. DEGs with log2 fold changes > 1 or 2 and adjusted p-values ​​< 0.05 were considered significant. Based on the number of DEGs, the Wayne analysis demonstrated the additive effect of combination anti-TNF-α / IL-6 therapy relative to monotherapy with either anti-TNF-α or anti-IL-6.

[0351] [Genes with differential expression ()] [DEG] [) Plotting the path []

[0352] The pathway diagram in Figure 9(B) shows the top 20 classic pathways obtained from genomic enrichment analysis of DEGs using a combined biological knowledge base planned from Ingenuity (Qiagen) and MetaCore (Clarivate). DEGs from collagen-induced arthritis (CIA) were determined using DESeq2 by comparing samples from allotype-treated (IgG control group, n = 13) with untreated samples from collagen-free arthritis (untreated group, n = 4). DEGs from combined anti-TNF-α / IL-6 treatment were determined using DESeq2 by comparing samples from anti-TNF-α / IL-6 (XT.3 + MP5-20F3; n = 13) with samples from allotype-treated (IgG control group, n = 13). The metabolic and immune signaling pathways in both diagrams show opposite scores for collagen-induced arthritis and combined anti-TNF-α / IL-6 treatment at different false discovery rates (FDRs). [6.12] [Example]

[13] Quantitative Systems Pharmacology of Rheumatoid Arthritis [QSP] [Model Prediction] [F027201062] [Increased remission at lower doses (compared to anti-)] [hTNF-] [α] [and resistance] [hIL-6] [refer to] [mAb] [compared to)]

[0353] The applicant has developed a proprietary quantitative systems pharmacology model for rheumatoid arthritis (RA) that considers relevant tissues, cells, and mediators in the blood and synovium. Mechanistic details of the included biological interactions were parameterized using extensive in vitro data from both internal and publicly available external sources. The model was subsequently tested and validated using clinical data from various studies of methotrexate, JAK inhibitors, anti-IL-6R, anti-IL-6, and anti-TNF treatments.

[0354] Based on this model, a 52-week treatment period simulated the reduction in DAS28-CRP in ordinary patients with moderate to severe rheumatoid arthritis due to decreased disease activity in the synovium. The nanoantibody F027201062 simultaneously binds to TNF-α and IL-6 and achieved a greater reduction in DAS28-CRP compared to monotherapy with a 20 mg dose every 2 weeks, predicting DAS28-CRP remission in reference patients after 24 weeks. The nanoantibody simulation considered pharmacokinetic predictions from animal and in vitro data. For target binding, in vitro IC50 data from cell assays were used together with the target binding parameters of published anti-hTNF and anti-hIL-6 comparative mAbs to calculate the target binding parameters of the nanoantibody (Figure 10). [6.13] [Example]

[14] [:anti] [TNF-α] [and resistance] [IL-6] [In humanity] [RA] [-FLS / T] [In the cell co-culture model] [MMP-1] [and] [G-CSF] [The cumulative effect]

[0355] An in vitro model of rheumatoid arthritis was developed, analogous to the concentrations of TNF-α and IL-6 in patient joints. In short, fibroblast-like synovial cells (RA-FLS) from rheumatoid arthritis patients were co-cultured with CD4+ T cells from healthy human donors. Additional stimulation induced endogenous secretion of TNF-α and IL-6. Treatment with anti-hTNF-α and anti-hIL-6 reference mAbs partially reduced the secretion of MMP-1 and G-CSF, while the combination of these and F027201062 achieved stronger maximal inhibition.

[0356] The detailed protocol for measuring IL-6 transduction is described below:

[0357] RA-FLS cells were seeded at a density of 10,000 cells / well in 96-well synovial cell basal medium supplemented with growth supplement (Pelobiotech). The following day, PBMCs were isolated from blood from healthy human donors using Ficoll gradient centrifugation. CD4+ T cells were isolated from the PBMCs using magnetic separation (negative selection).

[0358] The RA-FLS medium was replaced with medium containing appropriate concentrations of isotype control, comparative antibody, and ISVD (diluted 1:10 from 200 nM, 6 concentrations, triplicate). The IgG1 isotype control served as a negative control for the comparative antibody, while VHH IRR00119 served as a negative control for the ISVD isotype. Anti-human TNF-α and anti-human IL-6 reference antibodies were used as comparatives. Additionally, a combination of full doses of two comparatives was used as an additional positive control, demonstrating additive power. The following ISVD constructs, F027200926 and F027201062, were evaluated in this model. The intraplate position of all constructs was varied between three replicates to avoid plate effects.

[0359] 100,000 CD4+ T cells were then added to the FLS (in synovial cell basal medium with growth supplements). Finally, the co-culture was stimulated for 48 h with 100 ng / ml recombinant human IL-17A, 100 ng / ml sIL-6R, and 100 ng / ml soluble anti-CD3. After 48 h, the cells were centrifuged, the supernatant was collected, and stored at -20ºC. MMP-1 and G-CSF levels were measured using Luminex technology. Donors that did not respond to the anti-hTNF-α comparison mAb (which could not demonstrate the additive efficacy and dual targeting of the ISVD construct) were excluded.

[0360] Endogenous secretion of TNF-α and IL-6 was measured 48 h after stimulation with IL-17A, sIL-6R, and anti-CD3. Stimulated cocultures induced increased secretion of TNF-α (without stimulation below the detection limit) and IL-6. Stimulated cocultures secreted 4.4 pg / ml IL-6 and 7977 pg / ml TNF-α (mean from 8 donors, Figure 11). These values ​​are comparable to the median values ​​collected from human rheumatoid arthritis joints from various publications: 24 ± 21 pg / ml TNF-α and 13400 ± 12700 pg / ml IL-6.

[0361] To evaluate the efficacy of ISVDs against MMP-1 and G-CSF, RA-FLS cells from one donor were co-cultured with eight different human donor T cells. We did not observe a dose-dependent effect on MMP-1 secretion from the two isotype controls. Anti-hTNF-α reference mAbs partially reduced MMP-1 secretion. Anti-hIL-6 reference mAbs were more effective than the anti-hTNF-α comparative mAbs, while the combination of both antibodies showed the highest efficacy. The combination of the two ISVD constructs with the comparative antibody (administered at 200 nM Ab1 + 200 nM Ab2) showed similar efficacy and a dose-dependent effect (Fig. 12, 8 donors). Similar results were obtained for G-CSF (Fig. 13, 8 donors). [6.14] [Example]

[15] [Anti-] in human adenoid model [TNF-] [α] [and resistance] [IL-6] [right] [CXCL13] [The cumulative effect]

[0362] We evaluated the additive efficacy of F027201062 in a human pharyngeal tonsil (adenoid) model composed of follicular helper T cells (Tfh) and germinal center B cells. Briefly, we performed high-density lymphocyte aggregate cultures using cryopreserved lymphocytes. These cultures were stimulated with mutant pertussis toxin to induce an AIM (Initiation Inducible Marker) response (Schmidt, A. et al. 2020 Complex human adenoid tissue-based ex vivo culture systems reveal anti-inflammatory drug effects on germinal center T and B cells. EBioMedicine 53, 102684, doi:10.1016 / j.ebiom.2020.102684). Treatment with anti-hTNF-α and anti-hIL-6 reference mAbs partially reduced CXCL13 secretion, while the combination of these and F027201062 achieved a stronger maximal inhibition.

[0363] The detailed plan is described below:

[0364] Adenoid tissue from surgery was collected in RPMI medium (without supplement) at 4ºC. Postoperatively, the tissue was further processed as follows: Adenoid-derived tissues and cells were cultured in RPMI medium containing 15% (v / v) FBS (qualified heat-inactivated Gibco fetal bovine serum) and supplements (0.1 mM MEM non-essential amino acids, 1 mM MEM sodium pyruvate, 50 μg / ml gentamicin, 2.5 μg / ml amphotericin B, 0.3 μg / ml tekcillin, 0.01 μg / ml clavulanate). Tissue was washed twice with PBS and dissected in culture dishes containing CMT medium (containing 15% (v / v) FBS (qualified heat-inactivated Gibco fetal bovine serum) and supplements (0.1 mM MEM non-essential amino acids, 1 mM MEM sodium pyruvate, 50 μg / ml gentamicin, 2.5 μg / ml amphotericin B, 0.3 μg / ml tekcillin, 0.01 μg / ml clavulanate) and RPMI medium (containing L-glutamic acid). Bloody cauterized tissue was discarded. Remaining tissue and dissection medium were routinely filtered, while mechanically destroyed using a syringe plunger via a 40 µm cell screening procedure immersed in CMT medium. The suspended cells were then washed in CMT medium (500 x g, 5 min), counted, and aspirated at 12.5–100 Mio cells / ml in 10% DMSO / 90% FCS for cryopreservation.

[0365] On the day of the experiment, cryopreserved adenoid suspension cells were thawed and cultured in 96U wells at 1 × 10⁶ cells / well. Cells were either unstimulated or stimulated with a pertussis toxin mutant (PT; an enzymatically ineffective point mutant, tested as highly purified and low in endotoxin, List Biological Laboratories via Biotrend) at a final concentration of 1 µg / ml. Cultures were treated with the following compounds, as indicated: media containing appropriate concentrations of isotype control, comparative antibody, and ISVD (diluted 1:10 from 200 nM, 4–5 concentrations, in duplicate). The IgG1 isotype control served as a negative control for the comparative antibody, while VHH IRR00119 served as an isotype negative control for ISVD. Anti-human TNF-α and anti-human IL-6 reference antibodies were used as comparatives. Additionally, a combination of full doses of two comparatives was used as an additional positive control to demonstrate additive efficacy. The following ISVD construct, F027201062, was evaluated in this model. Eighteen h post-stimulation, cells were centrifuged, the supernatant was collected, and stored at -20ºC. CXCL13 levels were determined by ELISA.

[0366] To evaluate the efficacy of ISVD against MMP-1 and G-CSF, up to seven adenoid donors were evaluated (depending on the test concentration). Anti-hTNF-α and anti-hIL-6 reference mAbs partially reduced CXCL13 secretion. The combination of anti-hTNF-α and anti-hIL-6 completely inhibited pertussis toxin-induced CXCL13 increase in a dose-dependent manner. F027201062, in combination with two comparative antibody antibodies, showed similar efficacy (Figure 14, 4–7 donors).

[0367] To determine the maximum efficacy at high concentrations (data for all seven donors were available at 200 nM), we then evaluated the additive effects of the comparator antibody combination and F027201062 compared to the monospecific anti-hTNF-α and anti-hIL-6 comparator antibodies using a one-way variance analysis with Tukey correction. At the same molar dose (200 nM), F027201062 was significantly more effective than both the anti-hTNF-α comparator antibody (p: 0.0002) and the anti-hIL-6 comparator antibody (p: 0.0077). There were no significant or nominal differences between F027201062 and the combination of 200 nM anti-hTNF-α and 200 nM anti-hIL-6 reference antibodies (Figure 15, seven donors). [6.15] [Example]

[16] [:different] [ISVD] [Antibody in human whole blood assay] [TNF-] [α] [effect]

[0368] We analyzed the efficacy of blocking TNF-α in human whole blood (a more physiological state). Human whole blood was stimulated with SEB to secrete endogenous TNF-α, and treated with different concentrations of anti-hTNF-α reference antibody, different ISVDs, and corresponding isotype controls.

[0369] Details: Blood from a healthy donor was aspirated into a vacuum blood collection tube (BD #368480) containing sodium heparin [17 IU / ml] as an anticoagulant. SEB was reconstituted into a stock solution [1 mg / ml] in sterile water, and working solutions containing SEB were prepared. Working solutions of negative IgG1 control antibody, anti-hTNF-α comparator antibody (positive control), negative control VHH IRR00119, and multispecific anti-TNF-α / anti-IL-6 ISVD constructs F027200926, F027201029, F027201060, F027201061, and F027201062 were prepared.

[0370] A serial dilution of antibody and ISVD construct at final concentrations between 13 pM and 200 nM in medium [RPMI-1640 (from Gibco) + 10% human AB serum (from Sigma; order number H3667) + 1% PenStrep] was added in 10 µL to a 96-well V-bottom microplate. 10 μL of SEB in medium was added to each well of the pre-cultured mixture of human blood and antibody or ISVD construct in the 96-well plate. Finally, 80 µL of human blood was added to each well. The samples were gently mixed, the plate was sealed with a sterile cap, and the plate was incubated at 37ºC, 5% CO2, and 95% rH for 6 h. After incubation, 200 μL of PBS was added, and the blood sample was centrifuged at 2000 xg for 15 min. The plasma supernatant was harvested and stored in a new 96-well microplate at -80ºC for further analysis by ELISA. MCP-1 levels were determined using an ELISA (Invitrogen) according to the manufacturer's protocol. CCL4 levels were determined using Luminex technology (R&D). For each donor, dose-response curves were fitted using the XLfit program in Speed ​​and IC50 values ​​were calculated in Figures 16 and 18. Geometric means for all seven donors are reported. The data presented are based on seven human blood donors.

[0371] Cultured human whole blood with a negative control IgG1 isotype antibody or negative VHH IRR00119 did not result in any inhibition of SEB-induced MCP-1 release (data not shown). In contrast, cultured human whole blood with a monospecific anti-TNF-α monoclonal reference antibody induced strong inhibition of SEB-induced MCP-1 release, with an IC50 of 2.8 nM (Figure 15). Cultured human whole blood with the multispecific anti-TNF-α / anti-IL-6 ISVD construct F027201062 showed a similar degree of inhibition of MCP-1 secretion, with an IC50 of 3.2 nM. Multispecific anti-TNF-α / anti-IL-6 ISVD constructs F027200926, F027201029, F027201060, and F027201061 inhibited MCP-1 release, with IC50 values ​​of 8.9 nM, 1 nM, 3.8 nM, 4.1 nM, and 3.5 nM, respectively (Figure 16). The IC50 values ​​reported here are based on geometric mean, while Figure 16 shows the mean for all seven donors.

[0372] The anti-hIL-6 comparative antibody did not induce any inhibition of MCP-1, demonstrating that the assay was dependent solely on TNF-α. This is further reinforced by the lack of additive efficacy of the anti-hTNF-α and anti-hIL-6 comparative antibodies compared to anti-hTNF-α alone (Figure 16).

[0373] The efficacy against TNF-α was further evaluated using a second chemokine to read out CCL4 (Figures 17 and 18). The analysis focused on the comparative antibody and F027201062 as an ISVD construct. Culture of human whole blood with a monospecific anti-TNF-α monoclonal reference antibody induced a strong dose-dependent inhibition of SEB-induced CCL4 release, with an IC50 of 0.96 nM (Figures 17 and 18). Culture of human whole blood with the multispecific anti-TNF-α / anti-IL-6 ISVD construct F027201062 showed a similar degree of inhibition of CCL4 secretion, with an IC50 of 0.92 nM (Figures 17 and 18). The IC50 values ​​reported here are based on the geometric mean, while Figure 18 shows the mean for all seven donors.

[0374] The anti-hIL-6 comparative antibody did not induce any inhibition of CCL4, demonstrating that the assay was dependent solely on TNF-α (Figure 17). This is further reinforced by the lack of additive efficacy of the anti-hTNF-α and anti-hIL-6 comparative antibodies compared to anti-hTNF-α alone (Figures 17 and 18). [6.16] [Example]

[17] [:different] [ISVD] [The construct in human fibroblast-like synovial cells from rheumatoid arthritis patients] [IL-6] [effect]

[0375] We analyzed the efficacy of blocking IL-6 in primary fibroblast-like synovial cells (RA-FLS) from patients with rheumatoid arthritis. RA-FLS cells were stimulated with IL-17A and soluble IL-6R, as they lack membrane-bound IL-6R. This FLS assay thus reflects IL-6 transtransduction compared to a TF-1 proliferation assay. RA-FLS cells do not secrete human TNF-α, therefore the system is IL-6-dependent. Stimulated RA-FLS cells were then treated with different concentrations of anti-hIL-6 reference antibody, different ISVDs, and corresponding isotype controls.

[0376] More detailed plan:

[0377] RA-FLS cells were seeded at a density of 10,000 cells / well in 96-well synovial cell basal medium supplemented with growth supplement (Pelobiotech). The next day, the RA-FLS medium was replaced with medium containing appropriate concentrations of isotype control, comparative antibody, and ISVD (diluted 1:10 from 200 nM, six concentrations, in duplicate). The IgG1 isotype control served as a negative control for the comparative antibody, while VHH IRR00119 served as a negative control for the ISVD isotype. Anti-human IL-6 reference antibody was used as a comparative. The following ISVD constructs were evaluated in this model: F027200926, F027201029, F027201060, F027201061, and F027201062.

[0378] The intradisc position of all constructs was varied between two replicate experiments to avoid disk effects. Finally, RA-FLS cells were stimulated for 24 h with 100 ng / ml human recombinant IL-17A and 100 ng / ml sIL-6R. After 24 h, cells were centrifuged, and the supernatant was collected and stored at -80ºC. VEGF-A levels were measured using Luminex technology. Measurements were performed in three different rheumatoid arthritis donors, and for each donor, measurements were taken at two distinct stages.

[0379] Culture of RA-FLS with negative control IgG1 isotype antibody or negative control VHH IRR00119 did not block IL-17A / sIL-6R-induced VEGF-A secretion (data not shown). In contrast, culture of RA-FLS with a monospecific anti-IL-6 reference antibody induced a strong dose-dependent inhibition of IL-17A / sIL-6R-induced VEGF-A release, with an IC50 of 0.67 nM (Figures 19 and 20). Culture of RA-FLS with the multispecific anti-TNF-α / anti-IL-6 ISVD construct F027201062 inhibited VEGF-A secretion to a slightly lesser extent, with an IC50 of 2 nM. Multispecific anti-TNF-α / anti-IL-6 ISVD constructs F027200926, F027201029, F027201060, and F027201061 inhibited VEGF-A release, with IC50 values ​​of 2 nM, 1 nM, 1.4 nM, 2.9 nM, and 2.7 nM, respectively (Figure 20). The IC50 values ​​reported here are based on geometric mean, while Figure 20 shows the mean for all donors and stages.

[0380] The anti-hTNF-α comparative antibody did not induce any inhibition of VEGF-A, demonstrating that the assay was IL-6 dependent only (Figure 19). This is further reinforced by the lack of additive efficacy of the anti-hTNF-α and anti-hIL-6 comparative antibodies compared to anti-IL-6 alone (Figure 19). [6.17] [Example]

[18] [In humanity] [TNF-] [α] [genetically modified] [Tg197] [In a model of multiple arthritis] [F027201062] [Evaluation.]

[0381] F027201062 was profiled in the Tg197 mouse model of TNF-driven progressive polyarthritis (Keffer, J. et al. Transgenic mice expressing human tumor necrosis factor: a predictive genetic model of arthritis. EMBO J (1991)).

[10] , 4025-4031). In these mice, a modified human TNF-α gene was inserted as a transgene. The human gene was modified in a way that made the transcribed mRNA more stable, resulting in overexpression of TNF-α and spontaneous progressive arthritis with 100% penetrance in all four paws. Without treatment, signs and symptoms became apparent at about 6 weeks of age and continued to increase until, from about 10 weeks of age, the signs and symptoms led to obvious impending death. The severity of the arthritis was clinically assessed using a scoring system, as detailed in Table 15 below.

[0382] [surface]

[15] Arthritis scoring systems used to determine the severity of arthritis phenotypes [Arthritis Score] [, 1 , ] [feature] 0 / No disease No arthritis (normal appearance; mice can support their weight and lie flat against an inverted or tilted surface (such as a wire fence or cage cover) for a period of time; normal overall flexibility / escape ability; maximum grip strength). 0.5 / Mild illness Arthritis flare-up (mild joint swelling, all other parameters as above) 1 / Mild to moderate illness Mild to moderate (joint deformity due to swelling, inflammation of the soles of the feet, all other parameters as above) 1.5 / Moderate illness Moderate arthritis (joint-foot swelling and deformity + tail-pointing inward deformity, brief support to inverted or tilted surfaces (such as wire mesh or cage covers), decreased overall flexibility, and reduced grip strength) 2 / Moderate to severe illness Moderate to severe arthritis (severe swelling of joints, soles and fingers, joint-leg deformity, inability to support when pressed against an inverted or tilted surface (such as wire mesh or a cage), lack of overall flexibility, lack of grip, difficulty climbing / eating, wobbling when attempting to move but able to move forward) 2.5 / Severe Illness Severe arthritis (such as the finger deformities in the forefoot mentioned above, impaired movement in mice, wobbling, and reluctance to move) 3 / Extremely serious illness Severe arthritis (rigidity was detected when bending forward, and movement was severely impaired; the mouse was near death, no longer swaying, and could not easily turn / roll over when tilted to one side). 1. Arthritis scores are shown on the y-axis in Figure 21.

[0383] Arthritis is sensitive to treatments targeting inhibitors of human TNFα (Shealy, DJ et al.). Anti-TNF-alpha antibody allows healing of joint damage in polyarthritic transgenic mice. Arthritis Res [4](2002), R7, doi:10.1186 / ar430).

[0384] To establish dose-dependent efficacy, 6-week-old animals with obvious signs and symptoms of arthritis were treated with different doses of F027201062 via intraperitoneal injection twice weekly (n=8 animals per group). Human IgG1 purified from human myeloma serum (BioXcell #BE0297) was used as a negative control, and an anti-human TNF reference mAb was used as a positive control for arthritis inhibition. Additionally, an anti-hTNF monospecific nanoantibody RA15627569 was used as a second positive control. F027201062 was administered at four different dose intensities: 3 mg / kg body weight, 10 mg / kg, 30 mg / kg, and 100 mg / kg. Treatment continued until 11 weeks of age. Clinical arthritis scores were determined weekly. Figure 21 shows the dose-dependent inhibition of clinical arthritis scores over time resulting from treatment with F027201062. By week 11, the mean arthritis score in animals treated with the human IgG1 negative control antibody reached 1.571 ± 0.1086. Anti-hTNFα reference mAb and anti-hTNF nanoantibody RA15627569 inhibited arthritis progression, with mean scores of 0.5156 ± 0.0898 and 0.2344 ± 0.0156, respectively, at week 11. Incremental doses of F027201062 reduced arthritis progression, with mean scores of 1.203 ± 0.0943 (3 mg / kg), 0.8214 ± 0.161 (10 mg / kg), 0.3393 ± 0.0592 (30 mg / kg), and 0.25 ± 0.0579 (100 mg / kg) at week 11. Statistical analysis was performed using two-factor variance analysis over time and treatment, with Bonferroni-corrected group comparisons (Figure 21).

[0385] The overall inhibition of arthritis in the Tg197 arthritis model was analyzed using the area under the curve (AUC, Figure 22). When analyzed by one-way ANOVA followed by Bonferroni-corrected group comparisons, doses greater than 3 mg / kg of F027201062 significantly inhibited arthritis progression, and the degree of inhibition was comparable to that of the anti-hTNF reference mAb and the anti-hTNF nanoantibody RA15627569.

[0386] After treatment, the hind limb ankle joints were processed for histological analysis, and the arthritic structural signs of the sections were evaluated using the scoring system outlined in Table 16.

[0387] [surface]

[16] [Cumulative histopathological criteria used to score the arthritis phenotype in the ankle joint] [Score] [, 1 , ] [disease] [standard] 0 normal No detectable symptoms 1 Mild Synovial hyperplasia with polymorphonuclear infiltrates is present. Mild tendinitis may be present. 2 moderate Pannus and fibrous tissue formation, as well as focal subchondral bone erosion 3 Moderate to severe Cartilage destruction and bone erosion 4 Severe Extensive cartilage destruction and bone erosion. Loss of bone contour structure. 1. Arthritis scores are shown on the y-axis of Figure 23.

[0388] The results of the histological scoring are depicted in Figure 23. At higher doses, F027201062 significantly inhibited structural arthritis and joint destruction.

[0389] In summary, the results indicate that F027201062 exerts a dose-dependent inhibition of arthritis signs and symptoms, as well as an inhibition of structural progression, to a degree comparable to that of the anti-hTNF reference mAb and the anti-hTNF nanoantibody RA15627569. [6.18] [Example]

[19] [:exist] [hIL-6] [Evaluation in an in vivo model of induced haptoglobin] [F027201062]

[0390] In vivo IL-6 inhibition was investigated in a mechanophagocytic mouse model. Female BALB / c mice were injected intraperitoneally with F027201062 (the reference anti-hIL-6 mAb) or a mediator. Eight hours later, the mice were injected with PBS or 25 µg of recombinant human IL-6. Sixteen hours later, the mice were bled and plasma was prepared. The IL-6-induced acute-phase reactant haptoglobin in the plasma samples was measured by fluorescent bead conjugation assay. As shown in Figure 24, F027201062 at doses of 1 mg / kg and 3 mg / kg completely inhibited IL-6-induced plasma haptoglobin, similar to the reference anti-hIL-6 mAb. [6.19] [Example]

[20] [:exist] [hIL-6] [Evaluation in a transgenic splenomegaly model] [F027201062]

[0391] In vivo inhibition of IL-6 was further tested in a transgenic mouse model overexpressing hIL-6. The C.B6-Tg(H2-L-IL6)1 Kish / J strain (Suematsu S et al., 1992: Generation of plasmacytomas with the chromosomal translocation t (12;15) in interleukin 6 transgenic mice. Proc Natl Acad Sci USA 89(1):232-5) overexpresses human IL-6 under the control of the H-2Ld major histocompatibility promoter. At approximately 7 to 10 weeks of age, lymphoproliferative changes such as plasmacytosis and subsequent hyperglobulinemia became apparent (Suematsu S et al., 1989: IgG1 plasmacytosis in interleukin 6 transgenic mice. Proc Natl Acad Sci USA 86(19):7547-51).

[0392] Male and female hemizygous C.B6-Tg(H2-L-IL6)1 Kish / J mice aged approximately 2-2.5 months were treated three times weekly with intraperitoneal injection of F027201062 (reference anti-hIL-6 mAb) or a non-specific control nanoantibody. Two weeks later, the mice were sacrificed, and splenomegaly and hypergammaglobulinemia were measured. Non-transgenic wild-type littermates were used as controls.

[0393] In this model, both F027201062 and the anti-hIL-6 reference mAb significantly reduced splenomegaly (Figure 25). Consistent with the suppression of plasmacytosis, treatment with F027201062 and the anti-hIL-6 mAb also suppressed plasma levels of IgG1 and IgG2a (Figure 26). [6.20] [Example] [twenty one] [:] [F027201062] Single-dose pharmacokinetics in non-human primates

[0394] The aim of this study was to investigate the pharmacokinetics of a single dose of F027201062 in non-human primates. For this non-GLP study, a total of nine male untreated cynomolgus monkeys (Macaca fascicularis) were used. Animals were administered the drug according to the protocol in Table 17.

[0395] [surface]

[17] [:] [NHP PK] [The drug administration regimen studied.] [stage] [animal] [ID] [Administration route] [Dose Level] [(mg / kg)] [Dose Concentration] [(mg / mL)] [Dose-volume] [(mL / kg)] 1 001M-003M IV 3 3 1 2 004M-006M SC 3 3 1 3 007M-009M SC 30 30 1

[0396] Before centrifugation (approximately 1500 g, at +4ºC for 10 minutes), keep the blood at room temperature to allow clotting (up to 90 minutes). Pour the resulting serum into labeled polypropylene tubes and store in a freezer set to ≤-65ºC. Measure the samples using a developed, unvalidated, universal ELISA method.

[0397] Pharmacokinetic curves are shown in Figure 27. Following IV administration, the clearance was 0.273 L / h / kg and the volume of distribution (Vss) was 0.0464 L / kg. Pharmacokinetics were affected by ADA. All previously untreated, ADA-negative animals tested positive at 360 h and 672 h. [7] [Industrial Applicability]

[0398] The polypeptide, the nucleic acid molecule encoding it, and the carrier containing the nucleic acid and composition described herein can be used, for example, to treat subjects suffering from inflammatory diseases and / or autoimmune diseases.

[0399] The publications discussed herein are provided only because their publications predate the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to pre-existing inventions prior to such publications.

[0400] Although the invention has been described in conjunction with specific embodiments thereof, it should be understood that further modifications can be made to the invention, and this application is intended to cover any changes, uses or adaptations of the invention that generally follow the principles of the invention and include any known or customary practices within the field to which the invention pertains and that can be applied to the essential features described above and as follows within the scope of the appended claims that deviate from such description herein.

[0401] none

[0402] none.

[0403]

Claims

1. A polypeptide, a composition comprising said polypeptide, or a composition comprising a nucleic acid containing a nucleotide sequence encoding said polypeptide, wherein said polypeptide comprises or is composed of at least three immunoglobulin single variable domains (ISVDs), wherein each said ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein: a) a first ISVD binds to IL-6 and comprises i. an amino acid sequence having SEQ ID NO: 6 or a CDR1 differing from SEQ ID NO: 6 by 2 or 1 amino acids; ii. an amino acid sequence having SEQ ID NO: 10 or a CDR2 differing from SEQ ID NO: 10 by 2 or 1 amino acids; and iii. an amino acid sequence having SEQ ID NO: 14 or a CDR3 differing from SEQ ID NO: 14 by 2 or 1 amino acids; b) a second ISVD binds to IL-6 and comprises iv. an amino acid sequence having SEQ ID NO: 8 or a CDR3 differing from SEQ ID NO: 9 by 1 or 1 amino acids; v. CDR1 with SEQ ID NO: 8 having 2 or 1 different amino acids; v. CDR2 having the amino acid sequence of SEQ ID NO: 12 or having 2 or 1 different amino acids than SEQ ID NO: 12; and vi. CDR3 having the amino acid sequence of SEQ ID NO: 16 or having 2 or 1 different amino acids than SEQ ID NO: 16; and c) a third ISVD binding to TNF-α and comprising vii. CDR1 having the amino acid sequence of SEQ ID NO: 9 or having 2 or 1 different amino acids than SEQ ID NO: 9; viii. CDR2 having the amino acid sequence of SEQ ID NO: 13 or having 2 or 1 different amino acids than SEQ ID NO: 13; and ix. CDR3 having the amino acid sequence of SEQ ID NO: 17 or having 2 or 1 different amino acids than SEQ ID NO:

17.

2. The polypeptide or composition as claimed in claim 1, which is a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more other pharmaceutically active polypeptides and / or compounds.

3. The polypeptide or composition as claimed in claim 1 or 2, wherein: a) the first ISVD comprises CDR1 having an amino acid sequence of SEQ ID NO: 6, CDR2 having an amino acid sequence of SEQ ID NO: 10, and CDR3 having an amino acid sequence of SEQ ID NO: 14; b) the second ISVD comprises CDR1 having an amino acid sequence of SEQ ID NO: 8, CDR2 having an amino acid sequence of SEQ ID NO: 12, and CDR3 having an amino acid sequence of SEQ ID NO: 16; and c) the third ISVD comprises CDR1 having an amino acid sequence of SEQ ID NO: 9, CDR2 having an amino acid sequence of SEQ ID NO: 13, and CDR3 having an amino acid sequence of SEQ ID NO:

17.

4. The polypeptide or composition of any one of claims 1 to 3, wherein: a) the amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO: 2; b) the amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO: 4; and c) the amino acid sequence of the third ISVD has greater than 90% sequence identity with SEQ ID NO:

5.

5. The polypeptide or composition of any one of claims 1 to 4, wherein: a) the first ISVD has an amino acid sequence of SEQ ID NO: 2; b) the second ISVD has an amino acid sequence of SEQ ID NO: 4; and c) the third ISVD has an amino acid sequence of SEQ ID NO:

5.

6. A polypeptide or composition as claimed in any one of claims 1 to 5, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without said one or more other groups, residues, portions or binding units.

7. The polypeptide or composition as claimed in claim 6, wherein the one or more other groups, residues, portions or binding units that provide an increased half-life for the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

8. The polypeptide or composition as claimed in claim 7, wherein the binding unit providing the increased half-life of the polypeptide is an ISVD that can bind to human serum albumin.

9. The polypeptide or composition as claimed in claim 8, wherein the ISVD binding to human serum albumin comprises i. an amino acid sequence having the amino acid sequence of SEQ ID NO: 7 or a CDR1 having two or one amino acid difference from SEQ ID NO: 7; ii. an amino acid sequence having the amino acid sequence of SEQ ID NO: 11 or a CDR2 having two or one amino acid difference from SEQ ID NO: 11; and iii. an amino acid sequence having the amino acid sequence of SEQ ID NO: 15 or a CDR3 having two or one amino acid difference from SEQ ID NO:

15.

10. The polypeptide or composition as claimed in claim 8 or 9, wherein the ISVD that binds to human serum albumin comprises CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO:

15.

11. The polypeptide or composition of any one of claims 8 to 10, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity with SEQ ID NO:

3.

12. A polypeptide or composition as claimed in any one of claims 1 to 11, wherein the polypeptide comprises or is composed of an amino acid sequence having a sequence identity greater than 90% with SEQ ID NO:

1.

13. The polypeptide or composition as claimed in any one of claims 1 to 12, wherein the polypeptide or composition is used as a medicine.

14. The polypeptide or composition as claimed in any one of claims 1 to 12, said polypeptide or composition for treating inflammatory diseases and / or autoimmune diseases.

15. The polypeptide or composition for the stated use as described in claim 14, wherein the inflammatory disease and / or autoimmune disease is rheumatoid arthritis.

16. A nucleic acid comprising a nucleotide sequence encoding a polypeptide as described in any one of claims 1 to 15.

17. A host or host cell comprising nucleic acid as described in claim 16.

18. A method for generating a polypeptide as described in any one of claims 1 to 15, the method comprising at least the following steps: a) expressing a nucleic acid as described in claim 16; optionally followed by: b) isolating and / or purifying the polypeptide.

19. A composition comprising at least one polypeptide as described in any one of claims 1 to 15 or a nucleic acid as described in claim 16.

20. A composition as claimed in claim 19, which is a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more other pharmaceutically active peptides and / or compounds.

21. A method for treating inflammatory diseases and / or autoimmune diseases, wherein the method comprises administering to a subject in need a pharmaceutically active amount of any one of claims 1 to 15, or a composition as described in claim 19 or 20.

22. The method as described in claim 21, wherein the inflammatory disease and / or autoimmune disease is rheumatoid arthritis.

23. Use of the polypeptide as claimed in any one of claims 1 to 15 or the composition as claimed in any one of claims 19 to 20 in the preparation of pharmaceutical compositions for the treatment of inflammatory diseases and / or autoimmune diseases.

24. Use of the polypeptide or composition as claimed in claim 23, wherein the inflammatory disease and / or autoimmune disease is rheumatoid arthritis.