A peptide containing a single variable domain of an immunoglobulin targeting TNFα and IL-23

Abstract

This technology aims to provide novel drugs. Specifically, this technology provides polypeptides comprising at least three immunoglobulin single variable domains (ISVDs), characterized in that at least one ISVD binds to TNFα and at least two ISVDs bind to IL-23. This technology also provides nucleic acids, vectors, and compositions.

Description

Technical Field

[0001] This technology relates to polypeptides targeting the p19 subunits of TNFα and IL-23. It also relates to nucleic acid molecules encoding said polypeptides and carriers comprising said nucleic acids, as well as compositions comprising said polypeptides, nucleic acids, or carriers. This technology further relates to these products in methods for treating subjects suffering from inflammatory bowel disease, psoriasis, psoriatic arthritis, or hidradenitis suppurativa. Furthermore, this technology relates to methods for manufacturing these products. Background Technology

[0002] Autoimmune or inflammatory diseases are the result of an immune response produced by the body against its own tissues. Autoimmune or inflammatory diseases are often chronic and can even be life-threatening. Notable examples of autoimmune or inflammatory diseases include inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, and hidradenitis suppurativa. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are chronic inflammatory diseases involving inflammation of the intestines and associated epithelial damage. Other chronic autoimmune diseases, such as psoriasis, psoriatic arthritis, and hidradenitis suppurativa, are characterized by red, dry, itchy, or scaly patches of skin, painful inflammation of the joints, or inflammatory and red, swollen patches on the skin. It has been found that patients with psoriasis are more likely to have certain comorbidities, including diabetes and inflammatory bowel diseases (such as Crohn's disease or ulcerative colitis), as well as cancer.

[0003] Interleukin-23 (IL-23) is a cytokine that plays a crucial role in the activation of various immune cells during chronic inflammation. IL-23 is an upstream regulator of the cytokines IL-6, IL-17, GM-CSF, and IL-22, and is a heterodimer composed of the p19 subunit (IL-23α subunit, also referred to herein as IL-23p19) covalently linked to the p40 subunit (which is shared with the cytokine IL-12 and is also called the IL-12β subunit). Furthermore, IL-23 plays an important role in regulating T-cell inflammatory immune responses and the inflammatory activity of innate lymphoid cells. IL-23 is associated with inflammatory diseases, including inflammatory bowel disease and other autoimmune diseases.

[0004] Tumor necrosis factor-α (TNFα) is a homotrimeric cytokine primarily produced by monocytes and macrophages, but it is also known to be produced by CD4+. + and CD8 + TNFα is secreted by peripheral blood T lymphocytes. It can exist in a soluble form or as a transmembrane protein. The main function of TNFα is to regulate immune cells. TNFα 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 autoimmune diseases such as psoriasis.

[0005] Currently approved FDA treatments for inflammatory bowel disease include anti-TNFα biologics (such as...) [golimumab], [Inerasipu], [Infliximab] and [adalimumab]). However, current anti-TNFα treatment for inflammatory bowel disease faces a large number of patients who do not respond to currently available treatments, and most patients experience loss of response to anti-TNFα treatment after 12 months of treatment.

[0006] For psoriasis and psoriatic arthritis, only a small number of patients have received biologic therapy (including...). [Infliximab] and [Adalimumab], and the shared p40 subunit targeting the cytokines IL-12 and IL-23. [Utecumab]). Further antibody therapies targeting IL-23 are underway, including guselkumab (Tremfya; approved in a Phase 3 clinical trial for psoriasis, as well as inflammatory bowel disease and psoriatic arthritis) and risankizumab (Skyrizi; approved in a Phase 3 clinical trial for psoriasis, as well as psoriatic arthritis and Crohn's disease). While anti-IL-23 antibody classes targeting p19 may confer some disease-suppressive efficacy in psoriasis, patients with different and / or other autoimmune diseases do not necessarily benefit from these treatments to the same extent. For example, in psoriatic arthritis, anti-IL23 treatment does not improve the response to inflamed joints. In inflammatory bowel disease, more than half of patients do not respond to or lose response to TNF inhibitors. The same is true for hidradenitis suppurativa, where, to date, the only approved treatment is... [Adalimumab], but only about 50% of patients respond.

[0007] Targeting multiple disease factors can be achieved, for example, through the co-administration or combination of two separate biologics (e.g., antibodies that bind to different therapeutic targets). However, from both a practical and commercial perspective, co-administering or combining separate biologics can be challenging. For example, two injections of a single product make the treatment regimen more inconvenient and painful for patients, which can negatively impact adherence. For a single injection of two separate products, providing a formulation that allows for acceptable viscosity and suitable stability at the desired concentrations of both products may be difficult or impossible. Furthermore, co-administration and co-formulation require the production of two separate drugs, which can increase the overall cost.

[0008] Bispecific antibodies capable of binding to two different antigens have been suggested as a strategy to address such limitations associated with the co-administration or combination of individual biologics, such as antibodies.

[0009] Various forms of bispecific antibody constructs have been proposed. For example, bispecific antibody forms may 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). In some forms, a single-chain Fv (scFv) fragment that binds to a specific antigen is linked to an IgG antibody that binds to a single antigen (e.g., WO 2016 / 073406, which describes an anti-TNFα / anti-IL-23 IgG-scFv bispecific antibody). WO 2019 / 027780 describes a heterodimeric IgG antibody in which one pair of heavy and light chain variable regions targets TNFα, and another pair of heavy and light chain variable regions targets IL-23.

[0010] However, 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 and high-affinity binding per binding unit, which can impact peptide stability and production efficiency. This type of bispecific antibody may also potentially lead to chemistry, manufacturing, and control (CMC) problems related to light chain or heavy chain mismatches. Summary of the Invention

[0011] In some embodiments, this technology involves peptides or constructs that specifically target TNFα and IL-23. Simultaneous targeting of TNFα and IL-23 results in increased efficiency in modulating the inflammatory response compared to single-specific anti-TNFα or anti-IL-23 peptides.

[0012] The peptides exhibit high potency against TNFα and IL-23 (e.g., TNFα and IL-23 in humans and cynomolgus monkeys), can be efficiently produced (e.g., in microbial hosts such as Pichia, e.g., Pichia pastoris), and exhibit low viscosity at high concentrations, which is advantageous and convenient for subcutaneous administration. Furthermore, limited reactivity of such peptides with pre-existing antibodies in the subjects to be treated (i.e., antibodies present in the subjects prior to the first treatment with the antibody construct) can be demonstrated. In other embodiments, such peptides exhibit a sufficiently long half-life in the subjects to be treated, allowing for convenient spacing of successive treatments.

[0013] The polypeptide of this technology comprises or is composed of at least three immunoglobulin single variable domains (ISVDs), wherein at least one ISVD specifically binds TNFα, and at least two ISVDs specifically bind the p19 subunit of IL-23. In one embodiment, at least one ISVD binding to TNFα specifically binds human TNFα, and at least two ISVDs binding to IL-23 specifically bind the p19 subunit of human IL-23.

[0014] In one embodiment, 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 a polypeptide with an increased half-life 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 (human) serum protein (such as human serum albumin).

[0015] Also provided are nucleic acid molecules capable of expressing the polypeptide of the present technology, vectors containing said nucleic acid, and compositions containing said polypeptide, nucleic acid, or vector.

[0016] The polypeptides of this technology, compositions comprising said polypeptides, and compositions comprising nucleic acids containing a nucleotide sequence encoding said polypeptide can be used as pharmaceuticals. The polypeptide used as a pharmaceutical 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:

[0017] a) The first ISVD contains

[0018] i.CDR1, which is the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 by 2 or 1 amino acids;

[0019] ii. CDR2, which is the amino acid sequence of SEQ ID NO:10 or an amino acid sequence differing from SEQ ID NO:10 by 2 or 1 amino acids; and

[0020] iii. CDR3, which is the amino acid sequence of SEQ ID NO:14 or an amino acid sequence that differs from SEQ ID NO:14 by 2 or 1 amino acids;

[0021] b) The second ISVD contains

[0022] iv.CDR1, which is the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that differs from SEQ ID NO:7 by 2 or 1 amino acids;

[0023] v.CDR2, which is the amino acid sequence of SEQ ID NO:11 or an amino acid sequence differing from SEQ ID NO:11 by 2 or 1 amino acids; and

[0024] vi. CDR3, which is the amino acid sequence of SEQ ID NO:15 or an amino acid sequence that differs from SEQ ID NO:15 by 2 or 1 amino acids; and

[0025] c) The third ISVD contains

[0026] vii.CDR1, which is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that differs from SEQ ID NO:9 by 2 or 1 amino acids;

[0027] viii.CDR2, which is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence that differs from SEQ ID NO:13 by 2 or 1 amino acids; and

[0028] ix.CDR3, which is the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that differs from SEQ ID NO:17 by 2 or 1 amino acids.

[0029] The order of ISVDs starts from the end of N.

[0030] The compositions of this technology are intended for use as pharmaceuticals. The compositions may be pharmaceutical compositions 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.

[0031] The polypeptide (which may be present in the composition or may be encoded by nucleic acids) therefore comprises or is composed 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:

[0032] a) The first ISVD contains

[0033] i.CDR1, which is the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 by 2 or 1 amino acids;

[0034] ii. CDR2, which is the amino acid sequence of SEQ ID NO:10 or an amino acid sequence differing from SEQ ID NO:10 by 2 or 1 amino acids; and

[0035] iii. CDR3, which is the amino acid sequence of SEQ ID NO:14 or an amino acid sequence that differs from SEQ ID NO:14 by 2 or 1 amino acids;

[0036] b) The second ISVD contains

[0037] iv.CDR1, which is the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that differs from SEQ ID NO:7 by 2 or 1 amino acids;

[0038] v.CDR2, which is the amino acid sequence of SEQ ID NO:11 or an amino acid sequence differing from SEQ ID NO:11 by 2 or 1 amino acids; and

[0039] vi. CDR3, which is the amino acid sequence of SEQ ID NO:15 or an amino acid sequence that differs from SEQ ID NO:15 by 2 or 1 amino acids; and

[0040] c) The third ISVD contains

[0041] vii.CDR1, which is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that differs from SEQ ID NO:9 by 2 or 1 amino acids;

[0042] viii.CDR2, which is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence that differs from SEQ ID NO:13 by 2 or 1 amino acids; and

[0043] ix.CDR3, which is the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that differs from SEQ ID NO:17 by 2 or 1 amino acids.

[0044] The order of ISVDs starts from the end of N.

[0045] In one embodiment, the polypeptide specifically binds to the p19 subunits of TNFα and IL-23. In one embodiment, the polypeptide specifically binds to the p19 subunits of human TNFα and human IL-23. In one embodiment, a first ISVD present in the polypeptide specifically binds to TNFα, and a second and third ISVD present in the polypeptide specifically bind to the p19 subunits of IL-23. In one embodiment, a first ISVD present in the polypeptide specifically binds to human TNFα, and a second and third ISVD present in the polypeptide specifically bind to the p19 subunits of human IL-23.

[0046] The polypeptides in this technology may contain:

[0047] a) A first ISVD comprising CDR1 of the amino acid sequence of SEQ ID NO:6, CDR2 of the amino acid sequence of SEQ ID NO:10 and CDR3 of the amino acid sequence of SEQ ID NO:14;

[0048] b) A second ISVD comprising CDR1 of the amino acid sequence of SEQ ID NO:7, CDR2 of the amino acid sequence of SEQ ID NO:11, and CDR3 of the amino acid sequence of SEQ ID NO:15; and

[0049] c) A third ISVD comprising CDR1 of the amino acid sequence of SEQ ID NO:9; CDR2 of the amino acid sequence of SEQ ID NO:13; and CDR3 of the amino acid sequence of SEQ ID NO:17.

[0050] The polypeptides in this technology may contain:

[0051] a) A first ISVD having an amino acid sequence containing more than 90% (such as 95%) of the sequence identity with SEQ ID NO:2;

[0052] b) A second ISVD having an amino acid sequence containing greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:3; and

[0053] c) The third amino acid sequence, which contains greater than 90% (such as 95%) sequence identity with SEQ ID NO:5.

[0054] The polypeptides in this technology may contain:

[0055] a) First ISVD, which contains the amino acid sequence of SEQ ID NO:2;

[0056] b) A second ISVD containing the amino acid sequence of SEQ ID NO:3; and

[0057] c) A third ISVD, which contains the amino acid sequence of SEQ ID NO:5.

[0058] The polypeptide of this technology may further comprise 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 a polypeptide with an increased half-life compared to a corresponding polypeptide without said one or more other groups, residues, portions, or binding units. The one or more other groups, residues, portions, or binding units providing the polypeptide with an increased half-life may be selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding serum proteins, Fc moieties, and small proteins or peptides capable of binding serum proteins. The binding units providing the polypeptide with an increased half-life may be selected from binding units capable of binding serum albumin (such as human serum albumin) or serum immunoglobulins (such as IgG), for example, binding units of human serum albumin.

[0059] The polypeptide of this technology may contain an ISVD that binds to human serum albumin, which includes:

[0060] i.CDR1, which is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence that differs from SEQ ID NO:8 by 2 or 1 amino acids;

[0061] ii. CDR2, which is the amino acid sequence of SEQ ID NO:12 or an amino acid sequence differing from SEQ ID NO:12 by 2 or 1 amino acids; and

[0062] iii. CDR3, which is the amino acid sequence of SEQ ID NO:16 or an amino acid sequence that differs from SEQ ID NO:16 by 2 or 1 amino acids.

[0063] In one embodiment, the ISVD binding to human serum albumin comprises CDR1 of the amino acid sequence of SEQ ID NO:8, CDR2 of the amino acid sequence of SEQ ID NO:12, and CDR3 of the amino acid sequence of SEQ ID NO:16. In one embodiment, the ISVD binding to human serum albumin comprises a sequence identity greater than 90% (e.g., 95%) with SEQ ID NO:4. In one embodiment, the ISVD binding to human serum albumin comprises or consists of the amino acid sequence of SEQ ID NO:4.

[0064] The ISVD that binds to human serum albumin can be located at any location on the polypeptide of this technology (i.e., the N-terminus, between two building blocks, or the C-terminus). In one embodiment, the ISVD that binds to human serum albumin is located between the second and third ISVDs that specifically bind to the p19 subunit of IL-23.

[0065] The polypeptide of this technology may comprise an amino acid sequence that is more than 90% (e.g., 95%) identical to the sequence of SEQ ID NO:1. In one embodiment, the polypeptide of this technology comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0066] It also provides nucleic acids containing nucleotide sequences encoding the polypeptides of this technology.

[0067] It also provides a host or host cell containing this nucleic acid.

[0068] A method for generating the polypeptide of this technology is also provided, the method comprising at least the following steps:

[0069] a) Expressing the nucleic acid encoding the polypeptide of this technology in a suitable host cell or host organism or another suitable expression system; optionally, this is followed by:

[0070] b) Isolate and / or purify the polypeptide.

[0071] Compositions comprising at least one polypeptide of the present technology or a nucleic acid encoding a polypeptide of the present technology are also provided. The composition may be a 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.

[0072] The peptides of this technology can be used for treatment. More specifically, the peptides of this technology can be used to treat autoimmune or inflammatory diseases, such as those selected from inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, and hidradenitis suppurativa.

[0073] Therefore, this technology also covers methods for treating autoimmune or inflammatory diseases. In some embodiments, this technology covers methods for treating diseases selected from inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, and hidradenitis suppurativa, wherein said methods include administering to a subject in need a pharmaceutically active amount of the present technology polypeptide, a nucleic acid encoding the present technology polypeptide, or a composition comprising the present technology polypeptide.

[0074] Therefore, this technology also covers the use of the polypeptides of this technology in the preparation of pharmaceutical compositions for treating autoimmune or inflammatory diseases. In some embodiments, this technology also covers the use of the polypeptides of this technology in the preparation of pharmaceutical compositions for treating diseases selected from inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, and hidradenitis suppurativa.

[0075] Specifically, this technology provides the following implementation scheme:

[0076] Implementation Scheme 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 pharmaceutical, 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:

[0077] a) The first ISVD contains

[0078] x.CDR1, which is the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 by 2 or 1 amino acids;

[0079] xi.CDR2, which is the amino acid sequence of SEQ ID NO:10 or an amino acid sequence that differs from SEQ ID NO:10 by 2 or 1 amino acids; and

[0080] xii.CDR3, which is the amino acid sequence of SEQ ID NO:14 or an amino acid sequence that differs from SEQ ID NO:14 by 2 or 1 amino acids;

[0081] b) The second ISVD contains

[0082] xiii.CDR1, which is the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that differs from SEQ ID NO:7 by 2 or 1 amino acids;

[0083] xiv.CDR2, which is the amino acid sequence of SEQ ID NO:11 or an amino acid sequence that differs from SEQ ID NO:11 by 2 or 1 amino acids; and

[0084] xv.CDR3, which is the amino acid sequence of SEQ ID NO:15 or an amino acid sequence that differs from SEQ ID NO:15 by 2 or 1 amino acids; and

[0085] c) The third ISVD contains

[0086] xvi.CDR1, which is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that differs from SEQ ID NO:9 by 2 or 1 amino acids;

[0087] xvii.CDR2, which is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence that differs from SEQ ID NO:13 by 2 or 1 amino acids; and

[0088] xviii.CDR3, which is the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that differs from SEQ ID NO:17 by 2 or 1 amino acids.

[0089] The order of ISVDs starts from the end of N.

[0090] Implementation Scheme 2. The composition for the stated use according to Implementation Scheme 1 is a 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.

[0091] Implementation Scheme 3. A polypeptide or composition for the purpose according to Implementation Scheme 1 or 2, wherein the polypeptide specifically binds to the p19 subunit of TNFα and IL-23.

[0092] Implementation Scheme 4. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 3, wherein the polypeptide specifically binds to the p19 subunit of human TNFα and human IL-23.

[0093] Implementation Scheme 5. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 4, wherein the first ISVD specifically binds to TNFα, and the second and third ISVDs specifically bind to the p19 subunit of IL-23.

[0094] Implementation Scheme 6. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 5, wherein the first ISVD specifically binds to human TNFα, and the second and third ISVDs specifically bind to the p19 subunit of human IL-23.

[0095] Implementation Scheme 7. A polypeptide or composition for the said use according to any one of Implementation Schemes 1 to 6, wherein:

[0096] a) The first ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:6, CDR2 of the amino acid sequence of SEQ ID NO:10 and CDR3 of the amino acid sequence of SEQ ID NO:14;

[0097] b) The second ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:7, CDR2 of the amino acid sequence of SEQ ID NO:11, and CDR3 of the amino acid sequence of SEQ ID NO:15; and

[0098] c) The third ISVD comprises CDR1 with the amino acid sequence of SEQ ID NO:9, CDR2 with the amino acid sequence of SEQ ID NO:13, and CDR3 with the amino acid sequence of SEQ ID NO:17.

[0099] Implementation Scheme 8. A polypeptide or composition for the said use according to any one of Implementation Schemes 1 to 7, wherein:

[0100] a) The amino acid sequence of the first ISVD contains more than 90% (such as 95%) sequence identity with SEQ ID NO:2;

[0101] b) The amino acid sequence of the second ISVD contains greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:3; and

[0102] c) The amino acid sequence of the third ISVD contains greater than 90% (such as 95%) sequence identity with SEQ ID NO:5.

[0103] Implementation Scheme 9. A polypeptide or composition for the said use according to any one of Implementation Schemes 1 to 8, wherein:

[0104] a) The first ISVD contains the amino acid sequence of SEQ ID NO:2;

[0105] b) The second ISVD contains the amino acid sequence of SEQ ID NO:3; and

[0106] c) The third ISVD contains the amino acid sequence of SEQ ID NO:5.

[0107] Implementation Scheme 10. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 9, 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 a polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0108] Implementation Scheme 11. A polypeptide or composition for the said use according to Implementation Scheme 10, wherein the one or more other groups, residues, portions or binding units of the polypeptide providing an increased half-life are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind serum proteins, Fc portions, and small proteins or peptides that can bind serum proteins.

[0109] Implementation Scheme 12. A polypeptide or composition for the purpose according to any one of Implementation Schemes 10 to 11, wherein the binding unit of the polypeptide providing an increased half-life is selected from binding units that can bind serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0110] Implementation Scheme 13. The polypeptide or composition for the said use according to Implementation Scheme 12, wherein the binding unit of the polypeptide providing an increased half-life is an ISVD capable of binding human serum albumin.

[0111] Implementation Scheme 14. The polypeptide or composition for the said use according to Implementation Scheme 13, wherein the ISVD bound to human serum albumin comprises

[0112] i.CDR1, which is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence that differs from SEQ ID NO:8 by 2 or 1 amino acids;

[0113] ii. CDR2, which is the amino acid sequence of SEQ ID NO:12 or an amino acid sequence differing from SEQ ID NO:12 by 2 or 1 amino acids; and

[0114] iii. CDR3, which is the amino acid sequence of SEQ ID NO:16 or an amino acid sequence that differs from SEQ ID NO:16 by 2 or 1 amino acids.

[0115] Implementation Scheme 15. A polypeptide or composition for the purpose according to any one of Implementation Schemes 13 to 14, wherein the ISVD that binds to human serum albumin comprises CDR1 of the amino acid sequence of SEQ ID NO: 8, CDR2 of the amino acid sequence of SEQ ID NO: 12, and CDR3 of the amino acid sequence of SEQ ID NO: 16.

[0116] Implementation Scheme 16. A polypeptide or composition for the purpose according to any one of Implementation Schemes 13 to 15, wherein the amino acid sequence of the ISVD bound to human serum albumin contains greater than 90% (such as 95%) sequence identity with SEQ ID NO:4.

[0117] Implementation Scheme 17. A polypeptide or composition for the purpose according to any one of Implementation Schemes 13 to 16, wherein the ISVD bound to human serum albumin comprises or is composed of the amino acid sequence of SEQ ID NO:4.

[0118] Implementation Scheme 18. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 17, wherein the amino acid sequence of the polypeptide contains greater than 90% (such as 95%) sequence identity with SEQ ID NO:1.

[0119] Implementation Scheme 19. A polypeptide or composition for the purpose according to any one of Implementation Schemes 1 to 18, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO:1.

[0120] Implementation Scheme 20. A polypeptide or composition according to any one of Implementation Schemes 1 to 19 for the purpose of said use, for the treatment of autoimmune or inflammatory diseases, such as diseases selected from inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis and hidradenitis suppurativa.

[0121] Implementation Scheme 21. 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:

[0122] a) The first ISVD contains

[0123] x.CDR1, which is the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 by 2 or 1 amino acids;

[0124] xi.CDR2, which is the amino acid sequence of SEQ ID NO:10 or an amino acid sequence that differs from SEQ ID NO:10 by 2 or 1 amino acids; and

[0125] xii.CDR3, which is the amino acid sequence of SEQ ID NO:14 or an amino acid sequence that differs from SEQ ID NO:14 by 2 or 1 amino acids;

[0126] b) The second ISVD contains

[0127] xiii.CDR1, which is the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that differs from SEQ ID NO:7 by 2 or 1 amino acids;

[0128] xiv.CDR2, which is the amino acid sequence of SEQ ID NO:11 or an amino acid sequence that differs from SEQ ID NO:11 by 2 or 1 amino acids; and

[0129] xv.CDR3, which is the amino acid sequence of SEQ ID NO:15 or an amino acid sequence that differs from SEQ ID NO:15 by 2 or 1 amino acids; and

[0130] c) The third ISVD contains

[0131] xvi.CDR1, which is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that differs from SEQ ID NO:9 by 2 or 1 amino acids;

[0132] xvii.CDR2, which is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence that differs from SEQ ID NO:13 by 2 or 1 amino acids; and

[0133] xviii.CDR3, which is the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that differs from SEQ ID NO:17 by 2 or 1 amino acids.

[0134] The order of ISVDs starts from the end of N.

[0135] Implementation Scheme 22. The polypeptide according to Implementation Scheme 21, wherein the polypeptide specifically binds to the p19 subunit of TNFα and IL-23.

[0136] Implementation Scheme 23. The polypeptide according to Implementation Scheme 21 or 22, wherein the polypeptide specifically binds to the p19 subunit of human TNFα and human IL-23.

[0137] Implementation Scheme 24. The polypeptide according to any one of Implementation Schemes 21 to 23, wherein the first ISVD specifically binds to TNFα, and the second and third ISVDs specifically bind to the p19 subunit of IL-23.

[0138] Implementation Scheme 25. The polypeptide according to any one of Implementation Schemes 21 to 24, wherein the first ISVD specifically binds to human TNFα, and the second and third ISVDs specifically bind to the p19 subunit of human IL-23.

[0139] Implementation Scheme 26. The polypeptide according to any one of Implementation Schemes 21 to 25, wherein:

[0140] a) The first ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:6, CDR2 of the amino acid sequence of SEQ ID NO:10 and CDR3 of the amino acid sequence of SEQ ID NO:14;

[0141] b) The second ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:7, CDR2 of the amino acid sequence of SEQ ID NO:11, and CDR3 of the amino acid sequence of SEQ ID NO:15; and

[0142] c) The third ISVD comprises CDR1 with the amino acid sequence of SEQ ID NO:9, CDR2 with the amino acid sequence of SEQ ID NO:13, and CDR3 with the amino acid sequence of SEQ ID NO:17.

[0143] Implementation Scheme 27. The polypeptide according to any one of Implementation Schemes 21 to 26, wherein:

[0144] a) The amino acid sequence of the first ISVD contains more than 90% (such as 95%) sequence identity with SEQ ID NO:2;

[0145] b) The amino acid sequence of the second ISVD contains greater than 90% (e.g., 95%) sequence identity with SEQ ID NO:3; and

[0146] c) The amino acid sequence of the third ISVD contains greater than 90% (such as 95%) sequence identity with SEQ ID NO:5.

[0147] Implementation Scheme 28. The polypeptide according to any one of Implementation Schemes 21 to 27, wherein:

[0148] a) The first ISVD contains the amino acid sequence of SEQ ID NO:2;

[0149] b) The second ISVD contains the amino acid sequence of SEQ ID NO:3; and

[0150] c) The third ISVD contains the amino acid sequence of SEQ ID NO:5.

[0151] Implementation Scheme 29. A polypeptide according to any one of Implementation Schemes 21 to 28, 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 a polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0152] Implementation Scheme 30. The polypeptide according to Implementation Scheme 29, wherein the one or more other groups, residues, portions or binding units of the polypeptide providing an increased half-life are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind serum proteins, Fc portions, and small proteins or peptides that can bind serum proteins.

[0153] Implementation Scheme 31. The polypeptide according to any one of Implementation Schemes 29 to 30, wherein the one or more other groups, residues, portions or binding units of the polypeptide providing an increased half-life are selected from binding units that can bind serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0154] Implementation Scheme 32. The polypeptide according to Implementation Scheme 31, wherein the binding unit of the polypeptide having an increased half-life is an ISVD capable of binding human serum albumin.

[0155] Implementation Scheme 33. The polypeptide according to Implementation Scheme 32, wherein the ISVD bound to human serum albumin comprises:

[0156] i.CDR1, which is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence that differs from SEQ ID NO:8 by 2 or 1 amino acids;

[0157] ii. CDR2, which is the amino acid sequence of SEQ ID NO:12 or an amino acid sequence differing from SEQ ID NO:12 by 2 or 1 amino acids; and

[0158] iii. CDR3, which is the amino acid sequence of SEQ ID NO:16 or an amino acid sequence that differs from SEQ ID NO:16 by 2 or 1 amino acids.

[0159] Implementation Scheme 34. The polypeptide according to any one of Implementation Schemes 32 to 33, wherein the ISVD binding to human serum albumin comprises CDR1 of the amino acid sequence of SEQ ID NO:8, CDR2 of the amino acid sequence of SEQ ID NO:12, and CDR3 of the amino acid sequence of SEQ ID NO:16.

[0160] Implementation Scheme 35. The polypeptide according to any one of Implementation Schemes 32 to 34, wherein the amino acid sequence of the ISVD that binds to human serum albumin contains greater than 90% (such as 95%) sequence identity with SEQ ID NO:4.

[0161] Implementation Scheme 36. The polypeptide according to any one of Implementation Schemes 32 to 35, wherein the ISVD bound to human serum albumin comprises or is composed of the amino acid sequence of SEQ ID NO:4.

[0162] Implementation Scheme 37. The polypeptide according to any one of Implementation Schemes 21 to 36, wherein the amino acid sequence of the polypeptide contains greater than 90% (such as 95%) sequence identity with SEQ ID NO:1.

[0163] Implementation Scheme 38. The polypeptide according to any one of Implementation Schemes 21 to 37, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO:1.

[0164] Implementation Scheme 39. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of Implementation Schemes 21 to 38.

[0165] Implementation Scheme 40. A host or host cell comprising the nucleic acid according to Implementation Scheme 39.

[0166] Implementation Scheme 41. A method for generating a polypeptide according to any one of Implementation Schemes 21 to 38, said method comprising at least the following steps:

[0167] a) Express the nucleic acid as described in embodiment 39 in a suitable host cell or host organism or another suitable expression system; optionally, this is performed subsequently:

[0168] b) Isolate and / or purify the polypeptide according to any one of embodiments 21 to 38.

[0169] Implementation Scheme 42. A composition comprising at least one polypeptide according to any one of Implementation Schemes 21 to 38, or a nucleic acid according to Implementation Scheme 39.

[0170] Implementation Scheme 43. The composition according to Implementation Scheme 42 is a 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.

[0171] Implementation Scheme 44. A method for treating an autoimmune disease or an inflammatory disease, wherein the method comprises administering to a subject in need a pharmaceutically active amount of a polypeptide according to any one of Implementation Schemes 21 to 38, or a composition according to any one of Implementation Schemes 42 to 43.

[0172] Implementation Scheme 45. The method according to Implementation Scheme 44, wherein the autoimmune disease or inflammatory disease is selected from inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, and hidradenitis suppurativa.

[0173] Implementation Scheme 45. Use of the polypeptide according to any one of Implementation Schemes 21 to 38 or the composition according to any one of Implementation Schemes 42 to 43 in the preparation of a pharmaceutical composition for treating autoimmune diseases or inflammatory diseases.

[0174] Implementation Scheme 46. Use of the polypeptide or composition according to Implementation Scheme 45, wherein the autoimmune disease or inflammatory disease is selected from inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, and hidradenitis suppurativa. Attached Figure Description

[0175] Figure 1 : A sensor map showing the simultaneous binding of TNFα and IL-23 to F027500069 captured via human serum albumin (HSA).

[0176] Figure 2: F027500069 and anti-hTNFα reference mAb in Glo response TM Inhibition of soluble human (A) and cynomolgus monkey (B) TNFα in the HEK293_NFκB-NLucP reporter assay. IRR00096 is a negative control. HH The data points are the global average (n=2), and the error bars represent + / -SD.

[0177] Figure 3: F027500069, anti-hIL-23 reference mAb1, and anti-hIL-23 reference mAb2 in Glo response TM Inhibition of human (A) and cynomolgus monkey (B) IL-23 in the HEK293_human IL-23R / IL-12Rb1-Luc2P reporter assay. IRR00096 is a negative control. HH The data points are the global average (n=2), and the error bars represent + / -SD.

[0178] Figure 4 The block diagram shows the binding of the pre-existing antibody present in 96 human serum samples to F027500069, compared to the control F027301099.

[0179] Figure 5The block diagram shows the binding of pre-existing antibodies present in 96 human serum samples to F027500069, F027500093, F027500095, and F027500096, compared to control peptides F027301099 and F027301186.

[0180] Figure 6 Inhibition of polyarthritis in the Tg197 human TNF transgenic mouse model. Arthritis scores over time were plotted in different treatment groups (n = 8 mice / group). Animals received intraperitoneal injections of the specified compound twice weekly starting at 6 weeks of age. The mean weekly arthritis score is shown as mean ± SEM. Statistical analysis was performed using two-way ANOVA and Bonferroni multiple comparison test.

[0181] Figure 7 Bar graph showing the area under the curve of arthritis score over time in the Tg197 mouse model. Individual values ​​(symbols) and mean ± SEM (bars) are displayed. Statistical analysis was performed using one-way ANOVA and the Bonferroni multiple comparison test.

[0182] Figure 8 Bar graph of histological scores of the paws of the Tg197 mouse model. Individual values ​​(symbols) and mean ± SEM (bars) are shown. Statistical analysis was performed using one-way ANOVA and Bonferroni multiple comparison test.

[0183] Figure 9 Suppression of skin inflammation in a human IL-23 model. Bar graphs illustrate ear skin swelling in different treatment groups (n = 10 mice / group). Animals received intradermal injections of recombinant human IL-23 or PBS daily from day 1 to day 4. On days 1 and 3, animals received intraperitoneal injections of the designated compound. The mean change in ear skin thickness relative to baseline ± SEM is shown on day 5. Statistical analysis was performed using ANOVA and Bonferroni multiple comparison tests.

[0184] Figure 10 On day 5 of a human IL-23-induced skin inflammation model, bar graphs of tissue IL-22 concentrations from skin biopsies were generated. Individual values ​​(symbols) and mean ± SEM (bars) are shown. Statistical analysis was performed using ANOVA and Bonferroni multiple comparison tests.

[0185] Figure 11The inhibition of skin inflammation by F027500069 administered via both intraperitoneal and subcutaneous routes. Bar graphs illustrate ear skin swelling in different treatment groups (n = 8 mice / group). Animals received intradermal injections of recombinant human IL-23 or PBS daily from day 1 to day 4. Specified doses were administered via intraperitoneal (IP) or subcutaneous (SC) injections on days 1 and 3. The mean change in ear skin thickness relative to baseline ± SEM is shown on day 5. Statistical analysis was performed using ANOVA and Bonferroni multiple comparison tests.

[0186] Figure 12 In cases where F027500069 was delivered via both intraperitoneal and subcutaneous routes, a bar graph of tissue IL-22 concentration from skin biopsies was generated on day 5. Individual values ​​(symbols) and mean ± SEM (bars) are shown. Statistical analysis was performed using ANOVA and Bonferroni multiple comparison tests.

[0187] Figure 13 Inhibition of arthritis scores in a collagen antibody-induced arthritis (CAIA) model in human TNFα / TNFR1 knock-in mice. Arthritis scores over time are shown in different treatment groups (n = 8 mice / group). On day 1, animals received a single intraperitoneal injection of the designated compound 6 hours after LPS injection. Average daily arthritis scores ± SEM are shown. Statistical analysis was performed using two-way ANOVA and Bonferroni multiple comparison test.

[0188] Figure 14 Dual inhibition of IL-23 and TNFα-induced inflammation in a human TNFα knock-in mouse model induced by human IL-23 skin injection (n = 4–10 mice / group). Animals received intradermal injections of recombinant human IL-23 or PBS daily from day 1 to day 4. The designated compound was administered intraperitoneally on days 1 and 3. The mean change in ear skin thickness relative to baseline ± SEM is shown on day 5. Statistical analysis was performed using ANOVA and Bonferroni multiple comparison tests.

[0189] Figure 15 Differential skin tissue gene expression in inflamed skin tissue following single- or bispecific inhibition of cytokines. Venn diagram of differentially expressed genes (DEGs, fold change >2, p < 0.001). Of the 769 DEGs in F027500069, 199 were specific to this treatment.

[0190] Figure 16: Schematic diagram of ISVD build F027500069, showing the unitary building blocks / ISVD 6C11, 119A03 / 1, ALB23002 and 81A12 connected via 9GS connectors from N end to C end. Detailed Implementation

[0191] This technology aims to provide novel drugs for the treatment of inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, or hidradenitis suppurativa.

[0192] The inventors have surprisingly discovered that peptides containing at least three ISVDs (where at least one ISVD specifically binds to TNFα and at least two ISVDs specifically bind to the p19 subunit of IL-23) can be used to treat autoimmune or inflammatory diseases more effectively than monospecific anti-TNFα or anti-IL-23p19 peptides.

[0193] In some embodiments, the peptides of this technology exhibit high potency against TNFα and IL-23 (e.g., human or cynomolgus monkey TNFα and IL-23). ​​In some embodiments, the peptides of this technology are efficiently produced (e.g., in a microbial host such as Pichia pastoris). In some embodiments, the peptides of this technology have low viscosity at high concentrations, which is advantageous and convenient for subcutaneous administration. Furthermore, in some embodiments, the peptides of this technology have limited reactivity with pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with the antibody construct). In other embodiments, such peptides exhibit a sufficiently long half-life in the subject to be treated, allowing for convenient spacing of successive treatments.

[0194] 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 or pentavalent.

[0195] 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,” or “pentavalent” all fall under the term “multivalent,” and respectively indicate the presence of two, three, four, or five binding units (such as ISVDs). For example, the polypeptide could be trispecific and tetravalent, such as a polypeptide containing or composed of four ISVDs, one of which binds to human TNFα, two ISVDs to human IL-23, and one ISVD to human serum albumin. For example, if two ISVDs bind to two different epitopes on the p19 subunit of IL-23, then such a polypeptide can be simultaneously bicomplementary. The term “bicomplementary” refers to binding to two different parts (i.e., epitopes) of the same target molecule.

[0196] As used herein, the terms "first ISVD," "second ISVD," "third ISVD," etc., refer only to the relative positions of the ISVDs to each other, with the numbering starting from the N-terminus of the polypeptide of this technique. Thus, "first ISVD" is 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 only indicates the relative positions of the at least three ISVDs, the presence of other binding units / building blocks within the polypeptide, such as additional ISVDs binding to the p19 subunit of TNFα or IL-23, 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 further include another ISVD that binds to human serum albumin, which may even be located between, for example, a “second ISVD” and a “third ISVD”.

[0197] In view of the above, this technology provides a polypeptide comprising at least three ISVDs or thereof, wherein at least one ISVD specifically binds to TNFα and at least two ISVDs specifically bind to the p19 subunit of IL-23.

[0198] The at least two ISVDs that specifically bind to IL-23 bind to the p19 subunit of IL-23. The at least two ISVDs that specifically bind to IL-23 may bind to different epitopes on the p19 subunit of IL-23. At least one of the ISVDs that specifically bind to IL-23 may be able to block the function of IL-23, such as blocking the interaction between IL-23 and IL-23R and / or inhibiting IL-23-induced IL-22 release.

[0199] The components of the polypeptide, such as ISVD, can be linked together by one or more suitable adapters (such as peptide adapters).

[0200] The use of linkers to connect two or more (multi)peptides is well known in the art. Exemplary peptide linkers are shown in Tables A-5. One class of commonly used peptide linkers is referred to as “Gly-Ser” or “GS” linkers. These are linkers consisting 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:47) 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 common examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO:50), the 15GS linker (n = 3; SEQ ID NO:52), and the 35GS linker (n = 7; SEQ ID NO:57). For example, references are made to 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. In one embodiment of the polypeptide of this technique, 9GS connectors are used to link the components of the polypeptide to each other.

[0201] In one embodiment, the ISVD that specifically binds to TNFα is located at the N-terminus of the peptide. The inventors surprisingly discovered that this configuration can increase the product yield of the peptide.

[0202] In another embodiment, one of the ISVDs that specifically binds to IL-23 is located at the C-terminus of the polypeptide.

[0203] Therefore, the polypeptide, starting from its N-terminus, sequentially comprises or consists of the following: an ISVD that specifically binds TNFα, a first ISVD that specifically binds IL-23 and can block IL-23 function, an optional binding unit that provides a polypeptide with an increased half-life as defined herein, and a second ISVD that specifically binds IL-23. In one embodiment, the binding unit that provides a polypeptide with an increased half-life is an ISVD.

[0204] In one embodiment, the polypeptide, starting from its N-terminus, sequentially comprises or consists of the following: an ISVD that specifically binds to TNFα, a linker, a first ISVD that specifically binds to IL-23 and can block IL-23 function, a linker, an ISVD that binds to human serum albumin, a linker, and a second ISVD that specifically binds to IL-23. In one aspect, each linker is a 9GS linker.

[0205] Such a configuration of the polypeptide can provide increased product yield and favorable CMC characteristics, including expression yield, viscosity, and other biophysical properties.

[0206] In one embodiment, the polypeptide of this technology exhibits reduced binding to pre-existing antibodies in human serum. To this end, in one embodiment, the polypeptide contains valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat designation) in at least one ISVD. In another embodiment, the polypeptide contains valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat designation) in each ISVD. In another embodiment, the polypeptide contains 1 to 5 (naturally occurring) amino acid extensions, such as a single alanine (A) extension, at the C-terminus of the C-terminus of the ISVD. The C-terminus of the ISVD is typically VTVSS (SEQ ID NO: 112). In another embodiment, the polypeptide contains lysine (K) or glutamine (Q) at position 110 (according to Kabat designation) in at least one ISVD. In yet another embodiment, the ISVD contains lysine (K) or glutamine (Q) at position 112 (according to Kabat designation) in at least one ISVD. In these embodiments, the C-terminus of ISVD is VKVSS (SEQ ID NO:113), VQVSS (SEQ ID NO:114), VTVKS (SEQ ID NO:146), VTVQS (SEQ ID NO:147), VKVKS (SEQ ID NO:148), VKVQS (SEQ ID NO:149), VQVKS (SEQ ID NO:150), or VQVQS (SEQ ID NO:151), such that after the addition of a single alanine, the C-terminus of the polypeptide includes, for example, the sequences VTVSSA (SEQ ID NO:115), VKVSSA (SEQ ID NO:116), VQVSSA (SEQ ID NO:117), VTVKSA (SEQ ID NO:152), VTVQSA (SEQ ID NO:153), VKVKSA (SEQ ID NO:154), VKVQSA (SEQ ID NO:155), or VQVKSA (SEQ ID NO:151). No:156) or VQVQSA (SEQ ID NO:157). In one embodiment, the C-terminus contains VKVSSA (SEQ ID NO:116).In another embodiment, the polypeptide comprises 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 glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and includes a C-terminus of 1 to 5 (naturally occurring) amino acid extensions, such as a single alanine (A) extension, at the C-terminus of the polypeptide, such that the C-terminus of the polypeptide comprises, for example, the sequence VTVSSA (SEQ ID NO: 115), VKVSSA (SEQ ID NO: 116), or VQVSSA (SEQ ID NO: 117), such as VKVSSA (SEQ ID NO: 116). For more information in this regard, see, for example, WO 2012 / 175741 and WO 2015 / 173325.

[0207] In another embodiment, the polypeptide of this technology comprises or is composed of an amino acid sequence that is more than 90%, such as more than 95% or more than 99%, identical to the sequence of SEQ ID NO:1, wherein the CDRs of the four ISVDs are defined as items A to D (or A' to D', if Kabat's definition is used) as shown in the following sections “5.1 Immunoglobulin Single Variable Domain” and “5.3 (In vivo) Half-life Extension”, wherein in particular:

[0208] • The ISVD that specifically binds to TNFα contains CDR1 with the amino acid sequence of SEQ ID NO:6, CDR2 with the amino acid sequence of SEQ ID NO:10, and CDR3 with the amino acid sequence of SEQ ID NO:14;

[0209] The first ISVD that specifically binds to the p19 subunit of IL-23 comprises CDR1 of the amino acid sequence of SEQ ID NO:7, CDR2 of the amino acid sequence of SEQ ID NO:11, and CDR3 of the amino acid sequence of SEQ ID NO:15.

[0210] The second ISVD that specifically binds to the p19 subunit of IL-23 comprises CDR1 of the amino acid sequence SEQ ID NO:9, CDR2 of the amino acid sequence SEQ ID NO:13, and CDR3 of the amino acid sequence SEQ ID NO:17; and

[0211] The ISVD that binds to human serum albumin comprises CDR1 with the amino acid sequence of SEQ ID NO:8, CDR2 with the amino acid sequence of SEQ ID NO:12, and CDR3 with the amino acid sequence of SEQ ID NO:16.

[0212] Alternatively, if Kabat is used:

[0213] • The ISVD that specifically binds to TNFα contains CDR1 with the amino acid sequence of SEQ ID NO:122, CDR2 with the amino acid sequence of SEQ ID NO:130, and CDR3 with the amino acid sequence of SEQ ID NO:138.

[0214] The first ISVD that specifically binds to the p19 subunit of IL-23 comprises CDR1 of the amino acid sequence SEQ ID NO:123, CDR2 of the amino acid sequence SEQ ID NO:131, and CDR3 of the amino acid sequence SEQ ID NO:139.

[0215] The second ISVD that specifically binds to the p19 subunit of IL-23 comprises CDR1 of the amino acid sequence SEQ ID NO:125, CDR2 of the amino acid sequence SEQ ID NO:133, and CDR3 of the amino acid sequence SEQ ID NO:141; and

[0216] • The ISVD that binds to human serum albumin comprises CDR1 with the amino acid sequence of SEQ ID NO:124, CDR2 with the amino acid sequence of SEQ ID NO:132, and CDR3 with the amino acid sequence of SEQ ID NO:140.

[0217] In another embodiment, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:1. In yet another embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:1.

[0218] In one embodiment, the polypeptide of this technology has at least half or at least the same binding affinity for human TNFα and human IL-23 compared to the polypeptide composed of the amino acids of SEQ ID NO:1, wherein the binding affinity is measured using the same method, such as surface plasmon resonance (SPR).

[0219] 5.1 Single variable domain of immunoglobulin

[0220] The term "single immunoglobulin variable domain" (ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which an antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes ISVDs from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, discFv), where two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V... H ) and light chain variable structural domain (V L ) interact to form antigen-binding sites. In this case, V H and V L The complementary determinant regions (CDRs) of both will contribute to the formation of antigen binding sites; that is, a total of 6 CDRs will participate in the formation of antigen binding sites.

[0221] 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 art) or the antigen-binding domain of Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or biantibodies (all known in the art) derived from such conventional four-chain antibodies are generally not considered ISVDs because, in these cases, binding to the corresponding epitope of the antigen typically does not occur through a single immunoglobulin domain, but rather through a pair of associated immunoglobulin domains, such as light chain and heavy chain variable domains, i.e., through the V domains of the immunoglobulin domains that bind together to the epitope of the corresponding antigen. H -V L It happened.

[0222] In contrast, ISVDs can specifically bind to antigen epitopes without pairing with additional immunoglobulin variable domains. The binding site of an ISVD consists of a single V... H A single V HH Or a single V L Domain formation.

[0223] Thus, ISVD can be a sequence of light chain variable structural domains (e.g., V). L Sequences) or suitable fragments thereof; or heavy-chain variable domain sequences (e.g., V H Sequence or V HH (sequence) or a suitable fragment thereof; provided that it can form a single antigen-binding unit; that is, a functional antigen-binding unit consisting essentially of ISVDs, such that a single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit.

[0224] ISVDs can be, for example, heavy-chain ISVDs, such as V H V HH Including camel-derived V H Or humanized V HH In one implementation, it is V. HH Including camel-derived V H Or humanized V HH Heavy chain ISVDs can be derived from conventional four-chain antibodies or heavy chain antibodies.

[0225] For example, an ISVD can be a single-domain antibody (or an amino acid sequence suitable for use as a single-domain antibody), a "dAb", or a dAb (or an amino acid sequence suitable for use as a dAb). (as defined in this document, and including but not limited to V) HH ); other single variable domains, or any suitable fragment of any of them.

[0226] Specifically, ISVD can be (such as V) HH Including humanized V HH or camel-derived V H (or a suitable fragment thereof.) and It is a registered trademark.

[0227] “V HH "Structural domain", also known as V HH V HH Antibody fragments and V HH Antibodies, initially described as "heavy-chain antibodies" (i.e., "antibodies without light chains"), are antigen-binding immunoglobulin variable domains; see Hamers-Casterman et al., Nature 363:446-448, 1993. The term "V" is chosen as a separate point. HH "V" domains are used to combine these variable domains with the heavy chain variable domains present in conventional 4-chain antibodies (referred to as "V" in this paper). H The light chain variable domain (referred to as "V" in this paper) and the light chain variable domain present in conventional 4-chain antibodies. L Distinguish between "structural domain" and "structural domain". Regarding V... HH For further description, see Muyldermans' review article (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0228] 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, immunoglobulin-producing, or synthetic libraries, for example, via phage display.

[0229] Immunoglobulin sequences (such as) The generation of [a specific antigen] has been widely described in various publications, with WO 94 / 04678, Hamers-Casterman et al. 1993 (Nature 363:446-448, 1993) and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001) serving as examples. In these methods, camels are immunized with a target antigen to induce an immune response against the target antigen. The [response] obtained from the immunization... The library further screens for antibodies that bind to the target antigen.

[0230] In these cases, antibody production requires purified antigens for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production.

[0231] Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0232] This technique can utilize immunoglobulin sequences from various sources, including mouse, rat, rabbit, donkey, human, and cameloid immunoglobulin sequences. This technique also includes fully human sequences, humanized sequences, or chimeric sequences. For example, this technique includes cameloid immunoglobulin sequences and humanized cameloid immunoglobulin sequences, or camel-derived domain antibodies, such as camel-derived dAbs, as described by Ward et al. (Nature 341:544, 1989) (see, for example, WO 94 / 04678 and Davies and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531-537, 1996). Furthermore, this technique also utilizes fused immunoglobulin sequences, for example, to form multivalent and / or multispecific constructs (for those containing one or more V...). HHMultivalent and multispecific polypeptides with domains and their preparation are also described in Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, and, for example, WO 96 / 34103 and WO 99 / 23221, as well as immunoglobulin sequences containing tags or other functional parts (e.g., toxins, labels, radiochemicals, etc.) that may be derived from immunoglobulin sequences of this technique.

[0233] "Humanization V" HH "Contains V corresponding to natural occurrences" HH The amino acid sequence of the domain, but which has been "humanized," i.e., through use in conventional 4-chain antibodies derived from humans (e.g., as noted above) V H One or more amino acid residues appearing at one or more corresponding positions in the domain replace the naturally occurring V. HH This is performed on one or more amino acid residues in the amino acid sequence of the sequence (and particularly in the framework sequence). This can be done in ways known per se, as will be apparent to those skilled in the art, for example, based on further description herein and prior art (e.g., WO2008 / 020079). Similarly, it should be noted that such humanized V... HH It can be obtained in any suitable manner known in itself, and is therefore not strictly limited to those already in use that contain naturally occurring V. HH Peptides obtained from peptides whose structural domains are used as starting materials.

[0234] "Camel Source V" H "Contains V corresponding to natural occurrences" H The amino acid sequence of the domain, but which has been "camelized," i.e., through its use in heavy chain antibodies. HH One or more amino acid residues appearing at one or more corresponding positions in the domain replace the naturally occurring V from a conventional 4-chain antibody. H This is carried out by one or more amino acid residues in the amino acid sequence of the domain. This can be done in ways known per se, as will be apparent to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). As defined herein, such “camelization” substitution typically involves insertions that form and / or are present in V H -V L The amino acid sites at the interface and / or so-called cameloid marker residues (see, for example, WO 94 / 04678 and Davies and Riechmann, 1994 and 1996, ibid.). In one embodiment, used to generate or design camel-derived V H V of the starting material or starting point H The sequence is derived from the V sequence of mammals.H Sequence, or human V H Sequences, such as V H 3. Sequence. However, it should be noted that this camel-derived V can be obtained in any suitable manner known per se. H And therefore not strictly limited to the use of naturally occurring V H Peptides obtained from peptides whose structural domains are used as starting materials.

[0235] The structure of an ISVD sequence can be considered to consist of four frame regions (“FR”), referred to in the art and herein as “Frame Region 1” (“FR1”); “Frame Region 2” (“FR2”); “Frame Region 3” (“FR3”); and “Frame Region 4” (“FR4”); said frame regions are interrupted by three complementary decision regions (“CDR”), referred to in the art and herein as “Complementary Decision Region 1” (“CDR1”); “Complementary Decision Region 2” (“CDR2”); and “Complementary Decision Region 3” (“CDR3”).

[0236] As further described in paragraphs q) on pages 58 and 59 of WO 08 / 020079, the amino acid residues of ISVD can be based on the sequence of proteins of immunological interest given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, 91). H The domains are numbered using common designations, as in the article by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240(1-2):185-195; see, for example, Figure 2 of that publication) applied to V from camelids. HH Structural domain. It should be noted that, as in the art, V... H Domain and V HH As is well known in structural domains, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number. That is, 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 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. H Domain and V HHThe total number of amino acid residues in the domain is typically in the range of 110 to 120, and usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0237] In this application, unless otherwise stated, the CDR sequence is determined according to the AbM numbering as described in Kontermann and Dübel (ed. 2010, 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 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acid residues at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113.

[0238] The determination of the CDR region can also be performed using different methods. In the CDR determination according to Kabat, ISVD FR1 contains amino acid residues at positions 1-30, ISVD CDR1 contains amino acid residues at positions 31-35, ISVD FR2 contains amino acids at positions 36-49, ISVD CDR2 contains amino acid residues at positions 50-65, ISVD FR3 contains amino acid residues at positions 66-94, ISVD CDR3 contains amino acid residues at positions 95-102, and ISVD FR4 contains amino acid residues at positions 103-113.

[0239] In such immunoglobulin sequences, the frame sequence can be any suitable frame sequence, and examples of suitable frame sequences will be clear to those skilled in the art, for example based on standard manuals and further disclosures and prior art mentioned herein.

[0240] The framework sequence is a suitable combination of immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences, for example, through humanization or camelification. For example, the framework sequence could be derived from a light chain variable domain (e.g., V...). L Sequence) and / or heavy-chain variable structural domains (e.g., V) H Sequence or V HH A frame sequence (often a sequence of elements). On one hand, a frame sequence is derived from V. HH A frame sequence of sequences, wherein the frame sequence may optionally be partially or fully humanized; or a conventional V sequence that has already been camel-derived. H Sequences (as defined in this article).

[0241] In particular, the framework sequence present in the ISVD sequence used in this technique may contain one or more marker residues (as defined herein), such that the ISVD sequence is Such as V HH Including humanized V HH or camel-derived V H Some non-limiting examples of suitable combinations of such frame sequences will become clear from further disclosure herein.

[0242] 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 frame sequences and / or linked via one or more frame sequences; for example, in the full-size immunoglobulin sequence from which said fragment is derived, the fragment may appear in the same order as these CDR and frame sequences.

[0243] However, it should be noted that this technology is not limited to the origin of the ISVD sequence or the origin of the nucleotide sequence used to express the ISVD sequence, nor to the manner in which the ISVD sequence or nucleotide sequence is 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 a specific but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and particularly partially or fully humanized V... HH Immunoglobulin sequences, "camel-derived" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from a synthetic, random, or naturally occurring immunoglobulin sequence), 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.

[0244] 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, such as 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.

[0245] As mentioned above, ISVD can Or a suitable fragment thereof. For a general description, refer to the following further description and the prior art cited herein. However, in this regard, it should be noted that this description and the prior art primarily describe the so-called "V". H Category 3 That is, V such as DP-47, DP-51 or DP-29 H The three types of phylogenetic sequences have high sequence homology. However, it should be noted that this technology, in its broadest sense, can generally be used with any type of And for example, it also uses what is called "V" H "4 categories" That is, V such as DP-78 H The four types of phylogenetic sequences have high sequence homology. As described, for example, in WO 2007 / 118670.

[0246] generally, (especially V) HH Sequence, including (partially) humanized V HH Sequence and camel-derived V H The sequence can be characterized by the presence of one or more "marker residues" (as described herein) within one or more frame sequences (as further described herein). Therefore, it can generally be considered that... Defined as an immunoglobulin sequence having a (general) structure.

[0247] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0248] FR1 to FR4 refer to framework 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 further defined herein.

[0249] In particular, It can be an immunoglobulin sequence with a (general) structure.

[0250] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0251] FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. The frame sequence is further defined in this paper.

[0252] More specifically, It can be an immunoglobulin sequence with a (general) structure.

[0253] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0254] FR1 to FR4 refer to frame regions 1 to 4, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively.

[0255] One or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 of the Kabat number are selected from the marker residues mentioned in Table A-6 below.

[0256] Table A-6: Marker Residues in Nanobodies

[0257]

[0258]

[0259] This technique specifically utilizes ISVDs that can bind to the p19 subunit of TNFα or IL-23. In the context of this technique, "binding to a target molecule" has the common meaning understood in the art, as in the context of antibodies and their corresponding antigens.

[0260] The polypeptide of this technology may contain one or more ISVDs that bind to TNFα and two or more ISVDs that bind to IL-23. For example, the polypeptide may contain one ISVD that binds to TNFα and two ISVDs that bind to the p19 subunit of IL-23.

[0261] In some embodiments, at least one ISVD can functionally block its target molecule. For example, an ISVD can block the interaction between TNFα and TNFR (TNF receptor), or it can block the interaction between IL-23 and IL-23R (IL-23 receptor). In the polypeptide of the present invention, at least one ISVD can functionally block IL-23, for example by blocking the interaction between IL-23 and IL-23R and / or inhibiting IL-23-induced IL-22 release. Therefore, in one embodiment, the polypeptide of the present invention comprises one ISVD that binds to TNFα and functionally blocks TNFα, and two ISVDs that bind to IL-23, one of which can functionally block IL-23.

[0262] The ISVDs used in this technology form part of the polypeptide of this technology, the polypeptide containing or consisting of at least three ISVDs, such that the polypeptide can specifically bind to TNFα and IL-23.

[0263] Therefore, the target molecules of the at least three ISVDs used in the polypeptides of this technology are TNFα and IL-23. Examples are mammalian TNFα and IL-23. In addition to human TNFα (Uniprot accession number P01375) and human IL-23 (IL-23A, Uniprot accession number for the p19 subunit: Q9NPF7), forms from other species are also applicable to this technology, such as TNFα and IL-23 from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (such as cynomolgus monkeys (also referred to herein as "cyno")), or camelids (such as llamas or alpacas).

[0264] Specific examples of ISVDs that specifically bind to the p19 subunit of TNFα or IL-23 and can be used in this technique are described in items A through C below:

[0265] A. ISVDs that specifically bind to human TNFα and include the following:

[0266] i.CDR1, which is the amino acid sequence of SEQ ID NO:6 or an amino acid sequence that differs from SEQ ID NO:6 by 2 or 1 amino acids;

[0267] ii. CDR2, which is the amino acid sequence of SEQ ID NO:10 or an amino acid sequence differing from SEQ ID NO:10 by 2 or 1 amino acids; and

[0268] iii. CDR3, which is the amino acid sequence of SEQ ID NO:14 or an amino acid sequence that differs from SEQ ID NO:14 by 2 or 1 amino acids.

[0269] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:6, CDR2 of the amino acid sequence of SEQ ID NO:10, and CDR3 of the amino acid sequence of SEQ ID NO:14.

[0270] Examples of ISVDs that specifically bind to human TNFα have one or more or all of the frame regions (other than the CDRs defined in prior item A) as indicated in Table A-2 for construct 6C11. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct 6C11 (SEQ ID NO:2, see Tables A-1 and A-2) or composed thereof.

[0271] In another embodiment, the amino acid sequence of the ISVD that specifically binds to human TNFα 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 as defined in prior item A. In one embodiment, the ISVD that specifically binds to TNFα comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0272] When this TNFα-specific ISVD has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item A above) in at least one CDR, the ISVD has at least half or at least the same binding affinity for human TNFα compared to the construct 6C11 (SEQ ID NO:2), wherein the binding affinity is measured using the same method (such as SPR).

[0273] B. ISVDs that specifically bind to the p19 subunit of human IL-23 and contain the following:

[0274] i.CDR1, which is the amino acid sequence of SEQ ID NO:7 or an amino acid sequence that differs from SEQ ID NO:7 by 2 or 1 amino acids;

[0275] ii.CDR2, which is the amino acid sequence of SEQ ID NO:11 or an amino acid sequence differing from SEQ ID NO:11 by 2 or 1 amino acids; and

[0276] iii. CDR3, which is the amino acid sequence of SEQ ID NO:15 or an amino acid sequence that differs from SEQ ID NO:15 by 2 or 1 amino acids.

[0277] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:7, CDR2 of the amino acid sequence of SEQ ID NO:11, and CDR3 of the amino acid sequence of SEQ ID NO:15.

[0278] Examples of ISVDs that specifically bind to human IL-23 have one or more or all of the frame regions (in addition to the CDRs defined in prior item B) as indicated in Table A-2 for construct 119A03 / 1. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct 119A03 / 1 (SEQ ID NO:3, see Tables A-1 and A-2) or composed thereof.

[0279] In another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 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 as defined in prior item B. In one embodiment, the ISVD that binds to IL-23 comprises or is composed of the amino acid sequence of SEQ ID NO:3.

[0280] When such an IL-23-binding ISVD has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item B above) in at least one CDR, the ISVD has at least half or at least the same binding affinity to human IL-23 compared to construct 119A03 / 1 (SEQ ID NO:3), wherein the binding affinity is measured using the same method (such as SPR).

[0281] In one embodiment, an ISVD comprising a CDR2 differing from SEQ ID NO:11 (TIESGSRTN) by 2 or 1 amino acids, wherein there is no E to N substitution at amino acid position 3 of the CDR2 sequence and / or no N to Y substitution at amino acid position 9 of the CDR2 sequence. In another embodiment, such an ISVD does not have E to N substitution at amino acid position 3 of the CDR2 sequence, and does not have N to Y substitution at amino acid position 9 of the CDR2 sequence. In such embodiments comprising an ISVD comprising a CDR2 differing from SEQ ID NO:11 (TIESGSRTN) by 2 or 1 amino acids, E is maintained as the amino acid at position 3 of the CDR2 sequence and / or N is maintained as the amino acid at position 9 of the CDR2 sequence. In another embodiment, both E at amino acid position 3 and N at amino acid position 9 of the CDR2 sequence are maintained. Compared to the same polypeptide that does not contain N at amino acid position 3 of the CDR2 sequence, and particularly compared to the same polypeptide that contains E at that amino acid position, using an ISVD containing N at that amino acid position can, for example, lead to a decrease in amino acid sequence stability during the production of the polypeptide due to deamination. Compared to the same polypeptide that does not contain Y at amino acid position 9 of the CDR2 sequence, and particularly compared to the same polypeptide that contains N at that amino acid position, using an ISVD containing Y at that amino acid position can lead to an increase in protein aggregation of the polypeptide.

[0282] C. ISVDs that specifically bind to the p19 subunit of human IL-23 and include the following:

[0283] i.CDR1, which is the amino acid sequence of SEQ ID NO:9 or an amino acid sequence that differs from SEQ ID NO:9 by 2 or 1 amino acids;

[0284] ii. CDR2, which is the amino acid sequence of SEQ ID NO:13 or an amino acid sequence differing from SEQ ID NO:13 by 2 or 1 amino acids; and

[0285] iii. CDR3, which is the amino acid sequence of SEQ ID NO:17 or an amino acid sequence that differs from SEQ ID NO:17 by 2 or 1 amino acids.

[0286] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:9, CDR2 of the amino acid sequence of SEQ ID NO:13, and CDR3 of the amino acid sequence of SEQ ID NO:17.

[0287] Examples of ISVDs that specifically bind to human IL-23 have one or more or all of the frame regions (other than the CDRs defined in prior item C) as indicated in Table A-2 for construct 81A12. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct 81A12 (SEQ ID NO:5, see Tables A-1 and A-2) or composed thereof.

[0288] In another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 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 as defined in prior item C. In one embodiment, the ISVD that binds to IL-23 comprises or is composed of the amino acid sequence of SEQ ID NO:5.

[0289] When this IL-23-specific ISVD has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item C above) in at least one CDR, the ISVD has at least half or at least the same binding affinity for human IL-23 compared to construct 81A12 (SEQ ID NO:5), wherein the binding affinity is measured using the same method (such as SPR).

[0290] In one embodiment, each ISVD as defined in items A through C above is included in the polypeptide of the present technology. Compared to the polypeptide composed of the amino acids of SEQ ID NO:1, this polypeptide of the present technology containing each ISVD as defined in items A through C above has at least half or at least the same binding affinity for human TNFα and human IL-23, wherein the binding affinity is measured using the same method (such as SPR).

[0291] 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 defined according to the Kabat definition (see Table A-2.1) can also be used for items A through C above.

[0292] Therefore, the specific ISVD that can be used in this technique to specifically bind to the p19 subunit of TNFα or IL-23, as defined above using AbM, can also be described using the Kabat definition set forth in items A' through C' below:

[0293] A'. ISVD that specifically binds to human TNFα and includes the following:

[0294] i.CDR1, which is the amino acid sequence of SEQ ID NO:122 or an amino acid sequence that differs from SEQ ID NO:122 by 2 or 1 amino acids;

[0295] ii. CDR2, which is the amino acid sequence of SEQ ID NO:130 or an amino acid sequence differing from SEQ ID NO:130 by 2 or 1 amino acids; and

[0296] iii. CDR3, which is the amino acid sequence of SEQ ID NO:138 or an amino acid sequence that differs from SEQ ID NO:138 by 2 or 1 amino acids.

[0297] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence SEQ ID NO:122, CDR2 of the amino acid sequence SEQ ID NO:130, and CDR3 of the amino acid sequence SEQ ID NO:138.

[0298] Examples of ISVDs that specifically bind to human TNFα have one or more or all of the framework regions (other than the CDRs defined in prior item A) as indicated for construct 6C11 in Table A-2.1. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct 6C11 (SEQ ID NO:2, see Tables A-1 and A-2.1) or composed thereof.

[0299] In another embodiment, the amino acid sequence of the ISVD that specifically binds to human TNFα 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 as defined in the previous item A'. In one embodiment, the ISVD that specifically binds to TNFα comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0300] When this TNFα-specific ISVD has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item A' above) in at least one CDR, the ISVD has at least half or at least the same binding affinity for human TNFα compared to the construct 6C11 (SEQ ID NO:2), wherein the binding affinity is measured using the same method (such as SPR).

[0301] B'. ISVD that specifically binds to the p19 subunit of human IL-23 and contains the following:

[0302] i.CDR1, which is the amino acid sequence of SEQ ID NO:123 or an amino acid sequence that differs from SEQ ID NO:123 by 2 or 1 amino acids;

[0303] ii.CDR2, which is the amino acid sequence of SEQ ID NO:131 or an amino acid sequence differing from SEQ ID NO:131 by 2 or 1 amino acids; and

[0304] iii. CDR3, which is the amino acid sequence of SEQ ID NO:139 or an amino acid sequence that differs from SEQ ID NO:139 by 2 or 1 amino acids.

[0305] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence SEQ ID NO:123, CDR2 of the amino acid sequence SEQ ID NO:131, and CDR3 of the amino acid sequence SEQ ID NO:139.

[0306] Examples of ISVDs that specifically bind to human IL-23 have one or more or all of the frame regions (other than the CDRs defined in previous item B') as indicated in Table A-2.1 for construct 119A03 / 1. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct 119A03 / 1 (SEQ ID NO:3, see Tables A-1 and A-2.1) or composed thereof.

[0307] In another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 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 as defined in the previous item B'. In one embodiment, the ISVD that binds to IL-23 comprises or is composed of the amino acid sequence of SEQ ID NO:3.

[0308] When such an IL-23-binding ISVD has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item B' above) in at least one CDR, the ISVD has at least half or at least the same binding affinity to human IL-23 compared to construct 119A03 / 1 (SEQ ID NO:3), wherein the binding affinity is measured using the same method (such as SPR).

[0309] In one embodiment, the ISVD containing CDR2 (which comprises or is composed of an amino acid sequence differing from or consisting of SEQ ID NO:131 (TIESGSRTNYADSVKG) by 2 or 1 amino acids) does not have E to N substitutions at amino acid position 3 of the CDR2 sequence and / or does not have N to Y substitutions at amino acid position 9 of the CDR2 sequence. In another embodiment, such an ISVD does not have E to N substitutions at amino acid position 3 of the CDR2 sequence and does not have N to Y substitutions at amino acid position 9 of the CDR2 sequence. In such embodiments of the ISVD containing CDR2 (which comprises or is composed of an amino acid sequence differing from or consisting of SEQ ID NO:131 (TIESGSRTNYADSVKG) by 2 or 1 amino acids), E is maintained as the amino acid at position 3 of the CDR2 sequence and / or N is maintained as the amino acid at position 9 of the CDR2 sequence. In another embodiment, both E at amino acid position 3 and N at amino acid position 9 of the CDR2 sequence are maintained. Compared to the same polypeptide that does not contain N at amino acid position 3 of the CDR2 sequence, and particularly compared to the same polypeptide that contains E at that amino acid position, using an ISVD containing N at that amino acid position can, for example, lead to a decrease in amino acid sequence stability during the production of the polypeptide due to deamination. Compared to the same polypeptide that does not contain Y at amino acid position 9 of the CDR2 sequence, and particularly compared to the same polypeptide that contains N at that amino acid position, using an ISVD containing Y at that amino acid position can lead to an increase in protein aggregation of the polypeptide.

[0310] C'. ISVD that specifically binds to the p19 subunit of human IL-23 and contains the following:

[0311] i.CDR1, which is the amino acid sequence of SEQ ID NO:125 or an amino acid sequence that differs from SEQ ID NO:125 by 2 or 1 amino acids;

[0312] ii. CDR2, which is the amino acid sequence of SEQ ID NO:133 or an amino acid sequence differing from SEQ ID NO:133 by 2 or 1 amino acids; and

[0313] iii. CDR3, which is the amino acid sequence of SEQ ID NO:141 or an amino acid sequence that differs from SEQ ID NO:141 by 2 or 1 amino acids.

[0314] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence SEQ ID NO:125, CDR2 of the amino acid sequence SEQ ID NO:133, and CDR3 of the amino acid sequence SEQ ID NO:141.

[0315] Examples of ISVDs that specifically bind to human IL-23 have one or more or all of the frame regions (other than the CDRs defined in previous item C') as indicated in Table A-2.1 for construct 81A12. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct 81A12 (SEQ ID NO:5, see Tables A-1 and A-2.1) or composed thereof.

[0316] In another embodiment, the amino acid sequence of the ISVD that specifically binds to human IL-23 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 as defined in the previous item C'. In one embodiment, the ISVD that binds to IL-23 comprises or is composed of the amino acid sequence of SEQ ID NO:5.

[0317] When this IL-23-specific ISVD has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item C' above) in at least one CDR, the ISVD has at least half or at least the same binding affinity for human IL-23 compared to construct 81A12 (SEQ ID NO:5), wherein the binding affinity is measured using the same method (such as SPR).

[0318] In one embodiment, each ISVD as defined in items A' to C' above is included in the polypeptide of the present technology. Compared to the polypeptide composed of amino acids of SEQ ID NO:1, this polypeptide of the present technology containing each ISVD as defined in items A' to C' above has at least half or at least the same binding affinity for human TNFα and human IL-23, wherein the binding affinity is measured using the same method (such as SPR).

[0319] The percentage of “sequence identity” between the first amino acid sequence and the second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the corresponding amino acid residues in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of amino acid residues in the second amino acid sequence (compared to the first amino acid sequence) is considered a difference of a single amino acid residue (i.e., at a single position).

[0320] 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.

[0321] 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 one embodiment, "amino acid difference" is substitution.

[0322] In one embodiment, the amino acid substitution is a conservative substitution. 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) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0323] In one implementation, 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.

[0324] 5.2 Specificity

[0325] 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 sufficiently high affinity (see below). “Specific,” “specifically binding,” or “specifically binding” are used interchangeably herein with “selective,” “selectively binding,” or “selectively binding.” A binding unit (such as an ISVD) specifically binds to its designated target.

[0326] 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 units of mol / L (or M). Affinity can also be expressed as the association constant KA, which is equal to 1 / KD and has (mol / L) -1 (or M) -1 (Units)

[0327] Affinity is a measure of the strength of binding between a part of the target molecule and its binding site: the lower the KD value, the stronger the binding between the target molecule and the target part.

[0328] Typically, the bonding units (such as ISVD) used in this technology will be in the form of 10 -5 Up to 10 -12 mol / L or lower, 10 -7 Up to 10 -12 moles per liter or lower, or 10 -8 Up to 10 -12 Dissociation constant (KD) in mol / L (i.e., in terms of 10) 5 Up to 10 12 liters per mole or higher, 10 7 Up to 10 12 liters per mole or higher, or 10 8 Up to 10 12 The association constant (KA) of liters / moles binds to its target.

[0329] Greater than 10 -4 Any KD value in mol / L (or less than 10) 4 Any KA value (in liters / moles) is generally considered to indicate nonspecific binding.

[0330] KD, which is considered to have specific biological interactions (such as the binding of immunoglobulin sequences to antigens), is typically in the 10 range. -5 mol / L (10000 nM or 10 μM) to 10 -12 In the range of mol / L (0.001 nM or 1 pM) or lower.

[0331] Therefore, specific / selective binding may mean that, using the same measurement method, such as SPR, the binding unit (or the polypeptide containing it) is 10 -5 Up to 10 -12 KD values ​​of mol / L or lower bind to TNFα and / or IL-23, and at a concentration greater than 10 -4A KD value of mol / L binds to relevant cytokines. An example of a cytokine associated with IL-23 is IL-12, as it shares a p40 subunit with IL-23. Examples of cytokines associated with TNFα are TNF superfamily members FASL, TNFβ, LIGHT, TL-1A, and RANKL. Therefore, in one embodiment of this technology, at least one ISVD contained in the polypeptide is 10 -5 Up to 10 -12 Moles / L or lower KD values ​​bind to (human) TNFα and at a concentration greater than 10 -4 The KD value per mole / L is associated with FASL, TNFβ, LIGHT, TL-1A, and RANKL of the same species, and at least two ISVDs contained in the polypeptide are 10. -5 Up to 10 -12 KD values ​​of mol / L or lower bind to IL-23, and at a concentration greater than 10 -4 The molar / liter KD value is associated with IL-12 in the same species.

[0332] Therefore, compared with the polypeptide composed of the amino acids of SEQ ID NO:1, the polypeptide of this technology has at least half or at least the same binding affinity for human TNFα and human IL-23, wherein the binding affinity is measured using the same method (such as SPR).

[0333] Specific binding to a 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 TNFα does not preclude the binding unit or a peptide containing said binding unit from specifically binding to TNFα from cynomolgus monkeys. Similarly, for example, specific binding to human IL-23 does not preclude the binding unit or a peptide containing said binding unit from specifically binding to IL-23 from cynomolgus monkeys (“cyno”).

[0334] The specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays, as well as various variants of these known per se in the art; and other techniques mentioned herein.

[0335] As will be clear to those skilled in the art, the dissociation constant can be either actual or apparent. The methods used to determine 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 measurements greater than 10 may not be possible. -4 moles per liter or 10 -3 moles per liter (e.g., 10)-2 The dissociation constant (mol / L). Optionally, those skilled in the art will also know that the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) using the relationship [KD = 1 / KA].

[0336] The affinity of molecular interactions between two molecules can be measured using various known techniques, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al. 2001, Intern. Immunology 13:1551-1559). As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule passes through the immobilized molecule under flow conditions, thus yielding k 缔合 k 解离 The measured value, and thus K. D (or K) A ) value. For example, this can be done using well-known The system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscatave, New Jersey) was used. For further details, 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).

[0337] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the term “biolayer interferometry” or “BLI” refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an inner reference layer (reference beam) and a layer of immobilized proteins 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 in real time, association and dissociation rates and affinity can be determined. For example, BLI can use well-known... The system (ForteBio, a division of Pall LifeSciences, Menlo Park, USA) is used for this purpose.

[0338] Alternatively, one can use The platform (Sapidyne Instruments Inc., Boise, USA) measures affinity in the Kinetic Repulsion Assay (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem., 328:35-43). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a column containing beads pre-coated with 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).

[0339] Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).

[0340] 5.3 (In vivo) prolonged half-life

[0341] The polypeptide may further comprise 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 a polypeptide with an increased (in vivo) half-life compared to a corresponding polypeptide without said one or more other groups, residues, portions, or binding units. Increased in vivo half-life means, for example, that the polypeptide has an increased half-life in mammals (such as human subjects) after administration. Half-life may be expressed, for example, as t1 / 2β.

[0342] The types of groups, residues, parts or binding units are generally unrestricted and can be, for example, selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind serum proteins, Fc parts, and small proteins or peptides that can bind serum proteins.

[0343] More specifically, the one or more other groups, residues, portions, or binding units of the polypeptide providing an increased half-life may be selected from binding units capable of binding serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG). In one embodiment, the one or more other groups, residues, portions, or binding units of the polypeptide providing an increased half-life are binding units capable of binding human serum albumin. In one embodiment, the binding unit is ISVD.

[0344] For example, WO 2004 / 041865 describes an ISVD that binds to serum albumin (and particularly to human serum albumin) and can be linked to other proteins (such as one or more other ISVDs that bind to a desired target) to increase the half-life of said protein.

[0345] International application WO 2006 / 122787 describes several ISVDs targeting (human) serum albumin. These ISVDs include an ISVD called Alb-1 (SEQ ID NO:52 in WO 2006 / 122787) and its humanized variants, such as Alb-8 (SEQ ID NO:62 in WO 2006 / 122787). Similarly, these can be used to extend the half-life of therapeutic proteins and peptides, as well as other therapeutic entities or portions.

[0346] In addition, WO 2012 / 175400 describes a further improved form of Alb-1, called Alb-23.

[0347] 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 (WO 2006 / 122787), and Alb-23. In one embodiment, the serum albumin-binding moiety is Alb-8 or Alb-23 or a variant thereof, as shown on pages 7-9 of WO 2012 / 175400. In one embodiment, the serum albumin-binding moiety is selected from albumin binders described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, and WO 2018 / 134234. Table A-4 also shows some serum albumin binding agents. In one embodiment, another component of the technical polypeptide is described in item D below:

[0348] D. ISVD that binds to human serum albumin and includes the following:

[0349] i.CDR1, which is the amino acid sequence of SEQ ID NO:8 or an amino acid sequence that differs from SEQ ID NO:8 by 2 or 1 amino acids;

[0350] ii. CDR2, which is the amino acid sequence of SEQ ID NO:12 or an amino acid sequence differing from SEQ ID NO:12 by 2 or 1 amino acids; and

[0351] iii. CDR3, which is the amino acid sequence of SEQ ID NO:16 or an amino acid sequence that differs from SEQ ID NO:16 by 2 or 1 amino acids.

[0352] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence of SEQ ID NO:8, CDR2 of the amino acid sequence of SEQ ID NO:12, and CDR3 of the amino acid sequence of SEQ ID NO:16.

[0353] Examples of ISVDs that bind to human serum albumin have one or more or all of the frame regions (other than the CDRs defined in prior item D) as indicated in Table A-2 for construct ALB23002. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct ALB23002 (SEQ ID NO:4, see Tables A-1 and A-2) or composed thereof.

[0354] Alternatively, you can use Kabat definitions to describe project D as follows:

[0355] D'. ISVD that binds to human serum albumin and includes the following:

[0356] i.CDR1, which is the amino acid sequence of SEQ ID NO:124 or an amino acid sequence that differs from SEQ ID NO:124 by 2 or 1 amino acids;

[0357] ii. CDR2, which is the amino acid sequence of SEQ ID NO:132 or an amino acid sequence differing from SEQ ID NO:132 by 2 or 1 amino acids; and

[0358] iii. CDR3, which is the amino acid sequence of SEQ ID NO:140 or an amino acid sequence that differs from SEQ ID NO:140 by 2 or 1 amino acids.

[0359] In one embodiment, the ISVD comprises CDR1 of the amino acid sequence SEQ ID NO:124, CDR2 of the amino acid sequence SEQ ID NO:132, and CDR3 of the amino acid sequence SEQ ID NO:140.

[0360] Examples of ISVDs that bind to human serum albumin have one or more or all of the frame regions (other than the CDRs defined in previous item D') as indicated in Table A-2.1 for construct ALB23002. In one embodiment, it is an ISVD comprising the complete amino acid sequence of construct ALB23002 (SEQ ID NO:4, see Tables A-1 and A-2.1) or composed thereof.

[0361] In another embodiment, the amino acid sequence of the ISVD that binds to human serum albumin may also have greater than 90%, such as greater than 95% or greater than 99%, sequence identity with SEQ ID NO:4, wherein the CDR is as defined in the preceding item D or D'. In one embodiment, the ISVD that binds to human serum albumin comprises or is composed of the amino acid sequence of SEQ ID NO:4.

[0362] When such an ISVD that binds to human serum albumin has a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item D or D' above) in at least one CDR, the ISVD has at least half or at least the same binding affinity to human serum albumin compared to the construct ALB23002 (SEQ ID NO:4), wherein the binding affinity is measured using the same method (such as SPR).

[0363] In one embodiment, when such an ISVD that binds to human serum albumin has a C-terminal position, it exhibits C-terminal extension, such as a C-terminal alanine (A) or glycine (G) extension. In one embodiment, such an ISVD is selected from SEQ ID NO: 33, 34, 36, 38, 39, 40, 41, 42, 43, and 45 (see Table A-4 below). In another 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 a peptide of the present invention). In one embodiment, such an ISVD is selected from SEQ ID NO: 4, 31, 32, 35, and 37 (see Table A-4 below).

[0364] 5.4 Nucleic Acid Molecules

[0365] It also provides nucleic acid molecules encoding the polypeptides of this technology.

[0366] 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. A suitable host or host cell for production purposes will be clear to a person 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 polypeptides of this technology are also covered by this technology.

[0367] 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. In one embodiment, they are in the form of double-stranded DNA. For example, the nucleotide sequence of this technology can be genomic DNA or cDNA.

[0368] The nucleic acids of this technique 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 adapters.

[0369] The techniques used to generate nucleic acids will be clear to a technician 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 expression 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 a PCR reaction using one or more “mismatched” primers.

[0370] 5.5 Carrier

[0371] Vectors containing nucleic acid molecules encoding polypeptides of this technology are also provided. Vectors, as used herein, are suitable mediums 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.

[0372] In some embodiments, the vector comprises at least one nucleic acid optionally linked to one or more regulatory elements, such as one or more suitable promoters, enhancers, terminators, etc. In one embodiment, the vector is an expression vector, i.e., a vector suitable for expressing a encoded polypeptide or construct under suitable conditions (e.g., when said vector is introduced into cells, e.g., human cells). DNA-based vectors include the presence of elements for transcription (e.g., promoters and polyadenylate signals) and translation (e.g., Kozak sequences).

[0373] In one implementation, 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 oriented in the same direction and are usually also in the same reading frame. They are also usually substantially sequential, although this may not be necessary.

[0374] In one implementation, any regulatory elements of the vector enable it to provide its intended biological function in the intended host cell or host organism.

[0375] 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.

[0376] 5.6 Composition

[0377] This technology also provides compositions comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding the polypeptide of the present technology, or at least one carrier comprising such a nucleic acid molecule. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprise one or more other pharmaceutically active polypeptides and / or compounds.

[0378] 5.7 Host organism

[0379] This technology also relates to host cells or host organisms that contain polypeptides of this technology, nucleic acids encoding polypeptides of this technology, and / or carriers containing nucleic acid molecules encoding polypeptides of this technology.

[0380] A suitable host cell or host organism is clear to a person skilled in the art and is, 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 one embodiment, the host is *Pichia pastoris*.

[0381] 5.8 Methods and uses of the polypeptide described

[0382] This technology also provides a method for generating the polypeptide of this technology. 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, and optionally subsequently performing one or more isolation and / or purification steps. Specifically, the method may include:

[0383] a) Express the nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism or another suitable expression system; optionally, this is followed by:

[0384] b) Isolate and / or purify the polypeptide.

[0385] The suitable host cell or host organism for production purposes will be clear to a person skilled in the art, and may 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 one embodiment, the host is *Pichia pastoris*.

[0386] The polypeptides, nucleic acid molecules or carriers described herein, or compositions containing the polypeptides, nucleic acid molecules or carriers of this technology may be used as pharmaceuticals.

[0387] Therefore, this technology provides polypeptides of this technology, nucleic acid molecules or carriers as described, or compositions comprising polypeptides, nucleic acid molecules or carriers of this technology for use as pharmaceuticals.

[0388] Also provided are polypeptides of the present technology, nucleic acid molecules or carriers as described, or compositions comprising polypeptides, nucleic acid molecules or carriers of the present technology, for (preventive and / or therapeutic) treatment.

[0389] Also provided are polypeptides of the present technology, nucleic acid molecules or carriers as described, or compositions comprising polypeptides, nucleic acid molecules or carriers of the present technology for (preventive and / or therapeutic) treatment of autoimmune or inflammatory diseases.

[0390] Also provided are polypeptides of the present technology, nucleic acid molecules or carriers as described, or compositions comprising polypeptides, nucleic acid molecules or carriers of the present technology, for (preventive and / or therapeutic) treatment of inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis and hidradenitis suppurativa.

[0391] Further, methods for treating autoimmune or inflammatory diseases (preventive and / or therapeutic) are provided, wherein the methods include administering to a subject in need a pharmaceutically active amount of a polypeptide of the present technology, a nucleic acid molecule or carrier as described herein, or a composition comprising a polypeptide, nucleic acid molecule or carrier of the present technology.

[0392] Further, methods for treating inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, and hidradenitis suppurativa (preventive and / or therapeutic) are provided, wherein said methods include administering to a subject in need a pharmaceutically active amount of a polypeptide of the present technology, a nucleic acid molecule or carrier as described herein, or a composition comprising a polypeptide, nucleic acid molecule or carrier of the present technology.

[0393] The invention further provides the use of polypeptides, nucleic acid molecules or carriers as described herein, or compositions comprising said polypeptides, nucleic acid molecules or carriers in the preparation of pharmaceutical compositions for the treatment of autoimmune or inflammatory diseases.

[0394] Further, the use of the polypeptides, nucleic acid molecules or carriers as described herein, or compositions comprising the polypeptides, nucleic acid molecules or carriers, in the preparation of pharmaceutical compositions for the treatment of inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis or hidradenitis suppurativa.

[0395] Inflammatory bowel disease can be, for example, Crohn's disease or ulcerative colitis.

[0396] In the context of this technology, "subject" can refer to any animal. In one implementation, the subject is a mammal. Among mammals, a distinction can be made between humans and non-human mammals. 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 (such as cynomolgus monkeys), or camelids (such as llamas or alpacas).

[0397] In cases of preventative and / or therapeutic purposes, the subject can be any animal, and more specifically, any mammal. In one implementation, the subject is a human subject.

[0398] Substances including peptides, nucleic acid molecules and carriers, or compositions, may be administered to a 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). In one embodiment, the substance is administered via parenteral administration (e.g., intramuscular, subcutaneous, or intradermal). In one embodiment, subcutaneous administration is used.

[0399] An effective amount of the polypeptide, the nucleic acid molecule or carrier as described, or a composition containing the polypeptide, nucleic acid molecule or carrier may be administered to a subject to provide the desired therapeutic outcome.

[0400] 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.

[0401] Table A-1: ​​Different monovalent Vs identified in tetravalent polypeptide F027500069 HH The amino acid sequence of the building block (“ID” refers to SEQ ID NO as used herein)

[0402]

[0403] Table A-2: Sequence of CDRs and frames based on AbM numbers (“ID” refers to a given SEQ ID NO)

[0404]

[0405] Table A-2.1: Sequence of CDRs and frames based on Kabat numbers (“ID” refers to the given SEQ ID NO)

[0406]

[0407] Table A-3: Amino acid sequences of selected multivalent polypeptides (“ID” refers to the given SEQ ID NO)

[0408]

[0409] Table A-4: ISVD sequences of serum albumin (“ID” refers to SEQ ID NO as used herein)

[0410]

[0411]

[0412] Table A-5: Connector Sequences (“ID” refers to SEQ ID NO as used herein)

[0413]

[0414]

[0415] 6 Examples

[0416] 6.1 Example 1: Generation of Multispecific ISVD Constructs

[0417] The identification of the ISVD-containing peptide F027500069 (SEQ ID NO:1) that binds to TNFα and IL-23 stemmed from a data-driven, multispecific engineering and formatting activity that included building blocks based on anti-TNFαV HH Constructs (TNF06C11 (WO 2017 / 081320), TNF01C02 (WO 2015 / 173325, SEQ ID NO:327) and VHH#3E (WO2004 / 041862, SEQ ID NO:4)), anti-IL-23p19 V HH Components (23IL37D05, 23IL119A03 and 23IL81A12 (WO 2009 / 068627)) and HSA V resistance HH The building block ALB23002 (WO 2017085172, SEQ ID NO:10) was used. Different positions / orientations and different linker lengths (9GS vs. 35GS) of the building block were applied, and these were demonstrated to be crucial for different parameters (potency, cross-reactivity, expression, etc.). In this context, potency refers to the inhibition of TNFα-induced in vitro NFκB activation as determined in Example 6, and the inhibition of IL-23-induced in vitro mIL-22 production and the inhibition of IL-23-induced SIE promoter activation as determined in Examples 7 and 8.

[0418] A group containing 38 constructs (Table 1) was transformed into *Pichia pastoris* for small-scale production. ISVD construct expression was induced by stepwise addition of methanol. Clarified medium containing the secreted ISVD constructs was used as the starting material for purification via protein A affinity chromatography, followed by desalting. The purified samples were used for functional characterization and expression evaluation.

[0419] Some constructs exhibited impaired potency, depending on the connector length and the relative position of the ISVD building blocks. For example, the potency of bivalent VHH#3E against cynomolgus monkey TNFα was significantly impaired when connected to a short 9GS connector. Another example is that the position of the anti-IL-23 ISVD building block 37D05 within a multispecific construct is crucial for achieving maximum potency.

[0420] Table 1: List of 38 different multispecific ISVD formats evaluated. BB = building block, ALB = ALB23002.

[0421]

[0422]

[0423] Subsequently, the large group was reduced to a group of four multispecific constructs consisting of ISVD constructs F027500069, F027500093, F027500095, and F027500096. Based on preliminary yield estimates, these ISVD constructs have been shown to be effective against two targets (human and cynomolgus monkey) and have the potential for high expression levels.

[0424] Large-scale 2L production was conducted in *Pichia pastoris* to determine expression yield and assess biophysical properties and pre-existing antibody reactivity. Table 2 shows that a specific orientation of the building block is required to achieve high expression levels in *Pichia pastoris*. The expression yields of four formatted ISVD constructs with the same building block but different orientations and linker lengths, obtained from 5 ml cultures, clearly demonstrate that ISVD 6C11 requires an N-terminal position for good expression. This was confirmed in 2L fermentations of F027500069 and F027500070, where the ISVD construct with N-terminal 6C11 (F027500069) reached a titer of 6.4 g / L, which is 3.2 times that of F027500070 with C-terminal 6C11.

[0425] Table 2: Expression levels of four ISVD constructs with different orientations and joint lengths: 6C11, 119A03 / 1, and 81A12.

[0426]

[0427] Table 3 and Example 9 show that the pre-existing antibody reactivity is driven by the composition and titer of the corresponding ISVD construct.

[0428] Table 3: Binding of pre-existing antibodies present in 96 human serum samples to F027500069, F027500093, F027500095, and F027500096 compared to control ISVD constructs F027301099 and F027301186.

[0429]

[0430] Finally, ISVD construct F027500069 was selected based on potency, reduced binding to pre-existing antibodies, higher expression levels, and CMC characteristics. It exhibits a low viscosity of 3.3 cP at 100 mg / mL in a defined buffer and a low viscosity of 6.4 cP at 146 mg / mL.

[0431] 6.2 Example 2: Binding affinity of the multispecific ISVD construct for TNFα, IL-23 and serum albumin

[0432] The affinity of F027500069 for human and cynomolgus monkey TNFα, human and cynomolgus monkey IL-23, and human and cynomolgus monkey serum albumin (expressed as equilibrium dissociation constant (K)). D Quantification was performed using in-solution affinity measurements on the Gyrolab xP workstation (Gyros).

[0433] In K D Under controlled measurements, serially diluted TNFα or IL-23 (range 1 μM–0.1 pM) or serum albumin (range 10 μM–1 pM) and fixed amounts of F027500069 (50 pM for TNFα, 20 pM or 12.5 pM for IL-23, and 1 nM for serum albumin) were mixed to allow interaction and incubated for 24 or 48 hours (for IL-23 and TNFα) or 2 hours (for serum albumin) to reach equilibrium.

[0434] Under receptor-controlled measurements, serially diluted TNFα or IL-23 (range 1 μM–0.1 pM) or serum albumin (range 10 μM–1 pM) were mixed with fixed amounts of F027500069 (5 nM for TNFα, 1.25 nM for IL-23, and 50 nM for serum albumin) to allow for interaction, and incubated for 24 or 48 hours (for IL-23 and TNFα) or 2 hours (for serum albumin) to reach equilibrium.

[0435] Biotinylated human TNFα / IL-23 / serum albumin was captured in a microstructure of a Gyrolab Bioaffy 1000 CD containing a beaded column and used as a molecular probe to capture free F027500069 from equilibrium solution. A mixture of TNFα / IL-23 / serum albumin and F027500069 (containing free TNFα / IL-23 / serum albumin, free F027500069, and the TNFα / IL-23 / serum albumin-F027500069 complex) was passed through the beads, and a small fraction of free F027500069 was captured in proportion to the concentration of free ISVD. Fluorescently labeled anti-V antibodies were then injected. HH Antibodies were used to label any captured F027500069, and changes in fluorescence were determined after washing away excess fluorescent probe. Fitting of dilution series was performed using Gyrolab Analysis software, where K was analyzed. D Control and receptor control curves to determine K D value.

[0436] The results (Table 4) demonstrate that the multispecific ISVD construct binds human / cynomolgus monkey IL-23 and human / cynomolgus monkey TNFα with high affinity.

[0437] Table 4: Binding affinity of F027500069 with human and cynomolgus monkey IL-23, TNFα and serum albumin

[0438]

[0439] 6.3 Example 3: Binding of the multispecific ISVD construct to membrane-bound TNFα

[0440] The binding of F027500069 to membrane-bound TNFα was confirmed by flow cytometry in human HEK293H cells expressing membrane TNFα and in activated CD4+ cells isolated from PBMCs and stimulated with PMA and iomycin (data for HEK293H cells expressing TNFα are shown). In short, cells were loaded at 1 × 10⁻⁶ cells... 4 Cells were seeded at a density of 100 nM / well and incubated at 4°C for 1 hour with a series of F027500069 dilutions from 100 nM down to 0.5 pM. In parallel, cells were fixed prior to seeding with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (to enhance the detection of membrane-bound TNFα) and incubated at 4°C for 1 hour or at room temperature for 24 hours with an ISVD dilution series. Cells were washed three times and subsequently treated with anti-V HHThe mAb was incubated at 4°C for 30 min, washed again, and then incubated at 4°C for 30 min with a goat anti-mouse PE-labeled antibody. The sample was 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 a GraphPad Prism. After 1 hour of incubation, the EC50 values ​​for F027500069 were within the same range for both live and fixed cells, but cell fixation resulted in higher TNFα expression levels on the membrane (Table 5). After 24 hours of incubation, binding equilibrium was reached, with the EC50 increasing 6.6-fold.

[0441] Table 5: Binding affinity of F027500069 to membrane-expressed TNFα after 1 hour or 24 hours of incubation.

[0442]

[0443] 6.4 Example 4: Multispecific ISVD constructs selectively bind TNFα and IL-23

[0444] The deficiency of human cytokines associated with TNFα and IL-23 was assessed by SPR. hIL-12 was tested because it shares the p40 subunit with IL-23. TNF superfamily members human FASL, TNFβ, LIGHT, TL-1A, and RANKL were tested as TNFα-associated cytokines.

[0445] The target was immobilized at 10 μg / mL for 600 seconds using amine coupling, followed by a 420-second injection of EDC / NHS for activation and a 420-second injection of 1M ethanolamine HCl for inactivation (Sierra Sensor Amine Coupling Kit II, catalog number ACK-001-025). The flow rate for activation, inactivation, and ligand injection was set to 10 μl / min. The pH of the 10 mM acetate immobilization buffer was selected by subtracting approximately 1.5 from the pI of each ligand.

[0446] Next, 1 μM F027500069 was injected for 2 minutes and dissociated for 900 seconds at a flow rate of 45 μL / min. 1xHBS-EP+pH7.4 was used as the run buffer. As positive controls, 0.2 μM α-huIL-12Ab, 0.5 μM α-huFASLAb, 0.5 μM α-huTNFβAb, 0.5 μM α-huLIGHT Ab, 0.5 μM α-huTL-1A Ab, and 0.5 μM α-huRANKL V were injected. HHThe interaction between F027500069 and the positive control with the fixed target was measured by detecting the increase in refractive index due to changes in chip mass after binding.

[0447] All positive controls bound to their respective targets. No binding of F027500069 to human IL-12, FASL, TNFβ, LIGHT, TL-1A, or RANKL was detected.

[0448] 6.5 Example 5: Simultaneous binding of a multispecific ISVD construct to hIL-23 and hTNFα

[0449] The ProteOn XPR36 device was used to determine whether F027500069 could simultaneously bind to hTNFα and hIL-23. For this purpose, the HSA was immobilized on a GLC ProteOn sensor chip via amine coupling. 100 nM F027500069 was injected onto the HSA surface at 10 μL / min for 2 minutes to capture ISVD via the ALB23002 building block. Subsequently, 100 nM hIL-23, hTNFα, or hOX40L, or a mixture of 100 nM IL-23 + 100 nM TNFα, 100 nM IL-23 + 100 nM MOX40L, or 100 nM TNFα + 100 nM IL-13 was injected at a flow rate of 10 μL / min for 2 minutes, 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. (Sensing diagram) Figure 1 The results indicate that F027500069 can bind both human IL-23 and human TNFα simultaneously, as shown by the increase in reaction units: approximately 500 RU from TNFα alone, approximately 880 RU from IL-23 alone, and approximately 1300 RU for a mixture of IL-23 and TNFα.

[0450] 6.6 Example 6: In vitro inhibition of TNFα-induced NFkB activation by a multispecific ISVD construct

[0451] HEK293_NFkB-NLucP cells are TNF receptor-expressing cells that have been stably transfected with a reporter construct encoding Nanoluciferase under NFkB-dependent promoter control. Incubation of cells with soluble human and cynomolgus monkey TNFα 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 minutes to achieve complete lysis.

[0452] Glo responseTM HEK293_NFkB-NLucP cells were seeded at 20,000 cells / well in normal growth medium in 96-well plates with clear bottoms treated with white tissue culture (TC). Serial dilutions of F027500069 or the reference compound (anti-TNFα reference mAb) were added to 25 pM human or 70 pM cynomolgus monkey TNFα and incubated with the cells at 37°C for 5 h in the presence of 30 μM HSA.

[0453] F027500069 inhibited TNFα-induced NFκB activation in humans and cynomolgus monkeys in a concentration-dependent manner, with IC50 values ​​of 38.8 pM (for human TNFα) and 128 pM (for cynomolgus monkey TNFα), comparable to the anti-TNFα reference mAb (Table 6, Figure 2).

[0454] Table 6: Compared to the reference mAb for anti-hTNFα, in Glo response TM In the HEK293_NFκB-NLucP reporter assay, the IC50 values ​​of F027500069-mediated neutralization of TNFα in humans and cynomolgus monkeys were determined.

[0455]

[0456] 6.7 Example 7: In vitro inhibition of IL-23-induced mIL-22 production by a multispecific ISVD construct

[0457] Human (and cynomolgus monkey) IL-23 stimulates mouse splenocytes to secrete mIL-17 and mIL-22 (Aggarwal et al. 2003, J. Biol. Chem. 278(3):1910-4). It has been demonstrated that F027500069 blocks IL-23-induced mIL-22 expression in vitro. The spleens of five C57BL / 6 mice were removed, splenocytes were harvested, and single-cell suspensions were prepared. Cells were cultured in the presence of 20 ng / ml recombinant mIL-2 and seeded at 400,000 cells / well in 96-well plates. Serial dilutions of F027500069 or reference compounds (anti-hIL-23 reference mAb1 and anti-hIL-23 reference mAb2) were pre-incubated with recombinant hIL-23 (36 pM) or recombinant IL-23 (36 pM) from cynomolgus monkeys in culture medium at room temperature for 30 min, and then incubated with spleen cells at 37°C in the presence of 30 μM HSA for 3 days. The supernatant was collected and the level of mIL-22 was measured using ELISA.

[0458] The results shown in Table 7 indicate that F027500069 inhibits hIL-23- and cynomolgus monkey IL-23-induced mIL-22 production in a concentration-dependent manner, with IC50 values ​​of 43 pM (for human IL-23) and 31 pM (for cynomolgus monkey IL-23). ​​This inhibitory effect is stronger than that of the reference compounds against hIL-23 reference mAb1 and reference mAb2.

[0459] 6.8 Example 8: In vitro inhibition of IL-23-induced SIE promoter activation by a multispecific ISVD construct

[0460] Gloresponse was stably transfected with a reporter construct containing the luciferase gene controlled by the SIE response promoter. TM HEK293 human IL-23R / IL-12Rb1-Luc2P cells. These cells also constitutively overexpress both subunits of the human IL-23 receptor, IL-12Rb1 and IL-23R. When IL-23 triggers these cells, they express a luciferase reporter protein, which, upon addition of the substrate 5'-fluoroluciferin (Bio-Glo), expresses this protein. TM The luciferase assay system quantifies luciferase based on its enzymatic activity.

[0461] Cells were cultured in normal growth medium and seeded at 15,000 cells / well in 96-well plates with clear bottoms treated with white tissue culture. Serial dilutions of F027500069 or reference compounds (anti-hIL-23 reference mAb1 and anti-hIL-23 reference mAb2) were added to the cells, followed by the addition of recombinant hIL-23 (10 pM) or cynomolgus monkey IL-23 (40 pM). Cells were incubated at 37 °C for 4 h 15 min in the presence of 30 μM HSA. Subsequently, Bio-Glo was added to the cells in each well, and luciferase luminescence was measured. F027500069 inhibited IL-23-dependent signaling in humans and cynomolgus monkeys with IC50 values ​​of 250 pM and 323 pM, respectively (Table 7 and Figure 3).

[0462] Table 7: Compared with reference compounds anti-hIL-23 reference mAb1 and anti-hIL-23 reference mAb2, in mouse spleen cell assays and Glo response TM IC50 values ​​of human and cynomolgus monkey IL-23 neutralization mediated by F027500069 in the HEK293_human IL-23R / IL-12Rb1-Luc2P reporter assay.

[0463]

[0464] 6.9 Example 9: Binding of a multispecific ISVD construct to a pre-existing antibody

[0465] The reactivity of the pre-existing antibody against ISVD construct F027500069 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 25°C.

[0466] ISVD was captured on the chip via binding of the ALB23002 building block to HSA immobilized on the chip. To immobilize the HSA, the ligand lanes of the ProteOn GLC sensor chip were activated with 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 3200 RU. After immobilization, the surface was inactivated with ethanolamine HCl (flow rate 30 μL / min).

[0467] 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 cycle (i.e., before a new ISVD capture and blood sample injection step), 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 double referencing by subtracting 1) ISVD-HSA dissociation and 2) non-specific binding with the reference ligand lane. The binding level of the pre-existing antibody was determined by setting the reporter point at 125 sec (5 sec after association ended). The percentage reduction in pre-existing antibody binding was calculated relative to the binding level of the reference ISVD at 125 sec.

[0468] Compared to the unoptimized tetravalent ISVD construct F027301099 (control), the tetravalent ISVD construct F027500069, optimized for reduced binding to pre-existing antibodies by introducing mutants L11V and V89L and C-terminal alanine in each building block, showed a significant reduction in binding to pre-existing antibodies (Table 8 and 10). Figure 4 ).

[0469] Table 8: Binding of pre-existing antibodies present in 96 human serum samples to F027500069 compared to the control ISVD construct F027301099.

[0470]

[0471] Pre-existing antibody binding depends on the titer and composition of the multispecific construct. Table 3 and Figure 5 It was demonstrated that construct F027500069 exhibited lower pre-existing antibody reactivity than constructs F027500095 and F027500096.

[0472] 6.10 Example 10: Evaluation of F027500069 in a human TNFα transgenic Tg197 polyarthritis model.

[0473] Analysis of F027500069 (Keffer et al., 1991, EMBO J., 10:4025-4031) in a TNF-driven progressive polyarthritis Tg197 mouse model. In these mice, a modified human TNFα 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 TNFα and spontaneous progressive arthritis with 100% penetrance in all four paws. Without treatment, signs and symptoms became visible at approximately 6 weeks of age and continued to increase until they led to marked mortality and death from approximately 10 weeks of age. The severity of the arthritis was clinically assessed using a scoring system, detailed below:

[0474]

[0475]

[0476] 1 Arthritis score Figure 6 As shown on the y-axis.

[0477] Arthritis is sensitive to treatment with agents that inhibit human TNFα (Shealy et al., 2002, Arthritis Res. 4(5): R7).

[0478] To establish dose-dependent efficacy, different doses of F027500069 were administered therapeutically via intraperitoneal injection twice weekly to 6-week-old animals with obvious signs and symptoms of arthritis (n=8 animals per group). Human IgG1 purified from human myeloma serum (BioXcell#BE0297) was used as a negative control, and an anti-hTNFα reference mAb was used as a positive control for arthritis inhibition. F027500069 was administered at three different dose strengths: 1.3 mg / kg body weight, 4 mg / kg, and 13.5 mg / kg. Treatment continued until 11 weeks of age. Clinical arthritis scores were determined weekly. Figure 6 As shown, treatment with F027500069 resulted in a dose-dependent inhibition of clinical arthritis scores over time.

[0479] By week 11, the mean arthritis score of animals treated with the human IgG1 negative control antibody had progressed to 1.099 ± 0.1071. The anti-hTNFα reference mAb completely inhibited arthritis progression, with a mean score of 0.4844 ± 0.0594 by week 11. By week 11, F027500069 reduced arthritis progression, with mean scores of 0.8047 ± 0.0929 (1.3 mg / kg), 0.7969 ± 0.0585 (4 mg / kg), and 0.6016 ± 0.0349 (13.5 mg / kg). (The data were obtained by measuring the area under the curve (AUC)...) Figure 7 The overall inhibition of arthritis was analyzed. In the Tg197 arthritis model, all doses of F027500069 significantly inhibited arthritis progression, comparable to the anti-hTNFα reference mAb.

[0480] After treatment, the hindlimb ankle joint was processed for histological analysis, and the arthritic structural signs of the sections were evaluated using the following scoring system:

[0481]

[0482] 1 Arthritis score Figure 8 As shown on the y-axis.

[0483] The results of histological scoring in Figure 8 As shown in the figure, at higher doses, F027500069 significantly inhibited structural arthritis and joint destruction.

[0484] In summary, the results indicate that F027500069 has a dose-dependent inhibitory effect on signs and symptoms of arthritis, as well as an inhibitory effect on structural progression, comparable to that of the anti-TNFα reference mAb.

[0485] 6.11 Example 11: Evaluation of F027500069 in a human IL-23-induced skin inflammation model.

[0486] Intradermal injection of recombinant IL-23 in mice induced acute skin inflammation, characterized by redness and swelling around the injection site. Histologically, hallmarks of psoriatic skin inflammation were visible, such as epidermal thickening due to keratinocyte proliferation, keratosis, and infiltration of immune cells such as T cells and macrophages. At the molecular level, the transcriptomic changes in the model largely overlapped with those observed in human psoriatic lesions compared to normal skin (Gauld et al., 2018, Journal of Dermatological Science 92:45–53). Therefore, the IL-23 skin inflammation model is analogous to a mechanical model of psoriasis.

[0487] To investigate the inhibitory effect on IL-23-mediated inflammation, F027500069 was tested in a skin inflammation model adjusted from Rizzo et al., 2011, J Immunol; 186:1495-1502. On days 1, 2, 3, and 4, 1 μg of recombinant human IL-23 (total volume 20 μl) was injected intradermally into the right ear of female C57BL / 6 mice. PBS was injected into the ears of a control group of mice. Ear skin thickening was measured daily using calipers. On days 1 and 3, F027500069 and the control compound were administered via intraperitoneal injection. On day 5, mice were sacrificed, and skin biopsies were performed. The biopsies were homogenized in PBS supplemented with a mixture of protease inhibitors, and the levels of the downstream effector cytokine IL-22 were determined.

[0488] To establish dose-dependent efficacy, different doses of the ISVD construct were administered therapeutically via twice-weekly intraperitoneal injection to 6-week-old animals with obvious signs and symptoms of arthritis (n=10 animals per group). Human IgG1 purified from human myeloma serum (BioXcell#BE0297) was used as a negative control, and anti-hIL-23 reference mAb1 was used as a positive control for inhibiting skin inflammation. F027500069 was administered at four different dose intensities: 0.13 mg / kg body weight, 0.4 mg / kg, 1 mg / kg, and 4 mg / kg. Figure 9 As shown, treatment with F027500069 resulted in a dose-dependent suppression of skin swelling, which was depicted as a change in ear thickness from baseline on day 5.

[0489] Skin biopsies were performed on day 5, and tissue homogenates were prepared. The IL-22 assay was performed using the Mesoscale Discovery V-plex Mouse IL-22 Assay Kit (#K152WVD). Figure 10 IL-22 levels were measured in mice. Administration of IL-23 resulted in measurable IL-22 levels, as all samples from skin injected with PBS were below the lower limit of quantitation (LLOQ) of the assay used. All doses of F027500069 and anti-hIL-23 reference mAb1 significantly inhibited IL-22 tissue levels (IL-22 levels were not determined for the 1 mg / kg dose group).

[0490] In addition, the feasibility of subcutaneous administration was evaluated in another experiment in an IL-23-induced skin inflammation model. Two doses of F027500069 (0.1 mg / kg and 1 mg / kg) were administered intraperitoneally or subcutaneously on days 1 and 3, and changes in ear thickness were obtained. Figure 11 In this experiment, an irrelevant dose of 1 mg / kg V was used. HH Used as a negative control (Nab ctrl), and a 3 mg / kg dose of anti-hIL-23 reference mAb1 (IP) was used as a positive control.

[0491] In addition, the tissue IL-22 level was measured from the skin biopsy homogenate on day 5. Figure 12 ).

[0492] In summary, the results demonstrated that IL-23-induced skin inflammation was suppressed in terms of both skin thickening and the level of tissue effector cytokines. Both intraperitoneal and subcutaneous administration routes are feasible.

[0493] 6.12 Example 12: Evaluation of F027500069 in a collagen antibody-induced arthritis model in human TNFα / TNFR1 knock-in mice.

[0494] F027500069 was analyzed in a proprietary mouse model of collagen antibody-induced arthritis (CAIA), in which both the TNFα and TNF receptor 1 (TNFR1) loci were replaced by their respective human loci.

[0495] On day 0, eight animals in each group were injected with a mixture of monoclonal antibodies against chondroitin 2 (ArthritoMab, MDbioscience, CIA-MAB-2C). On day 1, arthritis development was triggered by injection of 25 μg of bacterial lipopolysaccharide (LPS). Six hours after LPS injection, the test compound was administered as a single injection. The development of signs and symptoms of arthritis was assessed daily until day 7 based on the arthritis score detailed below:

[0496]

[0497] 1 Arthritis score Figure 13 As shown on the y-axis.

[0498] F027500069 was administered at 1.3 mg / kg body weight, and the anti-hTNFα reference mAb was administered at 0.5 mg / kg as a positive control. Figure 13 The study demonstrated the progression of signs and symptoms of arthritis over time. A single prophylactic administration of either F027500069 or an anti-hTNFα reference mAb resulted in complete suppression of arthritis progression.

[0499] 6.13 Example 13: Evaluation of F027500069 in a human IL-23-induced skin inflammation model in human TNFα knock-in mice.

[0500] Because F027500069 binds to and inhibits only human or primate targets (both IL-23 and TNFα), it replicated human IL-23-induced skin inflammation in TNFα-humanized mice. In this proprietary strain, the entire TNFα locus in the mouse genome was replaced by the human locus. Exon-intron structures and regulatory elements are conserved between mice and humans. The true expression of human TNFα in mice and its functional ability to elicit a response had been previously evaluated.

[0501] Intradermal injection of hIL-23 resulted in a modest increase in TNFα expression (own data and Gauld et al. 2018, Journal of Dermatological Science 92:45–53). In this model, equimolar doses of F027500069 and the corresponding monospecific ISVD building blocks F027500101 (anti-TNFα) and F027500017 (anti-IL-23) were administered to address the potential additive effect of dual targeting. The dose intensity administered was 3.6 nmol / kg, corresponding to approximately 0.1 mg / kg. This low dose was chosen to allow some residual free IL-23 and thus allow downstream TNFα secretion. Additionally, a high dose of F027500069 and an anti-hIL-23 reference mAb1 were administered as positive controls. Changes in ear thickness were normalized against high (Nab negative control) and low (no IL-23 injection) controls (sample minus low control divided by high control minus low control). Figure 14The results are shown. The single-specific TNFα inhibition of 3.6 nmol / kg F027500101 did not inhibit skin swelling, while the single-specific IL-23 inhibition of 3.6 nmol / kg F027500017 had a moderate but significant effect. The dual targeting of both TNFα and IL-23 by 3.6 nmol / kg F027500069 resulted in numerically superior inhibition of skin swelling.

[0502] Skin biopsies were performed on day 5, and mRNA was prepared using standard methods. (In IluminaNovaSeq) TM Paired-end, ontology-based whole transcriptome sequencing of mRNA was performed on the 6000 platform. The overlap of differentially expressed genes (DEG, fold change > 2, p < 0.001) compared to NAb controls was analyzed between treatment groups. Figure 15 As shown, although there is significant overlap between the monospecific and multispecific treatment groups, 199 out of 769 DEGs were specific for F027500069 treatment. This suggests that, in this model, dual targeting of both TNFα and IL-23 leads to a unique molecular response and may synergistically improve human disease.

[0503] In summary, the results indicate that multispecific inhibition of both IL-23 and TNFα in IL-23-induced skin inflammation leads to a numerically cumulative effect on skin thickening and induces a unique transcriptomic profile.

[0504] 7 Industrial Applicability

[0505] The polypeptides described herein, the nucleic acid molecules encoding the polypeptides, the carriers containing the nucleic acids, and the compositions thereof can be used, for example, for the treatment of subjects suffering from inflammatory bowel disease, psoriasis, psoriatic arthritis, or hidradenitis suppurativa. sequence list <110> Alberlinx <110> Sanofi <120> A polypeptide containing a single variable domain of an immunoglobulin targeting TNFα and IL-23 <130> 228667 <150> US 62 / 944,619 <151> 2019-12-06 <150> EP 20 305 056.2 <151> 2020-01-23 <150> EP 20 000 090.9 <151> 2020-02-28 <150> EP 20 305 216.2 <151> 2020-03-02 <160> 157 <170> BiSSAP 1.3.6 <210> 1 <211> 517 <212> PRT <213> Artificial Sequence <220> <223> F027500069 <400> 1 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala 20 25 30 Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val 35 40 45 Ala Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro 130 135 140 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser 145 150 155 160 Leu Pro Ala Ser Gly Asn Ile Phe Asn Leu Leu Thr Ile Ala Trp Tyr 165 170 175 Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Glu Ser 180 185 190 Gly Ser Arg Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 195 200 205 Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr Leu Gln Met Asn Ser Leu 210 215 220 Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Gln Thr Ser Gly Ser Gly 225 230 235 240 Ser Pro Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 260 265 270 Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 275 280 285 Ser Gly Phe Thr Phe Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala 290 295 300 Pro Gly Lys Gly Pro Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser 305 310 315 320 Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 325 330 335 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro 340 345 350 Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg 355 360 365 Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 370 375 380 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val 385 390 395 400 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr 405 410 415 Leu Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 420 425 430 Arg Glu Phe Val Ala Arg Ile Ser Gln Gly Gly Thr Ala Ile Tyr Tyr 435 440 445 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 450 455 460 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala 465 470 475 480 Leu Tyr Tyr Cys Ala Lys Asp Pro Ser Pro Tyr Tyr Arg Gly Ser Ala 485 490 495 Tyr Leu Leu Ser Gly Ser Tyr Asp Ser Trp Gly Gln Gly Thr Leu Val 500 505 510 Light Val Ser Ser Ala 515 <210> 2 <211> 121 <212> PRT <213> artificial sequence <220> <223> 6C11 <400> 2 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Ala 20 25 30 Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val 35 40 45 Ala Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Arg Ser Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 125 <212> PRT <213> artificial sequence <220> <223> 119A03 / 1 <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Leu Pro 20 25 30 Ala Ser Gly Asn Ile Phe Asn Leu Leu Thr Ile Ala Trp Tyr Arg Gln 35 40 45 Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Thr Ile Glu Ser Gly Ser 50 55 60 Arg Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 65 70 75 80 Asp Asn Ser Lys Lys Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro 85 90 95 Glu Asp Thr Ala Leu Tyr Tyr Cys Gln Thr Ser Gly Ser Gly Ser Pro 100 105 110 Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125<l <210> 4 [ <211> 115 <212> PRT <213> Artificial Sequence [[ID=Z6]]<220> <223> ALB23002 <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe [ 20 25 3a Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 5 <211> 128 <212> PRT <213> artificial sequence <220> <223> 81A12 <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Arg Ile Ser Gln Gly Gly Thr Ala Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Pro Ser Pro Tyr Tyr Arg Gly Ser Ala Tyr Leu Leu Ser 100 105 110 Gly Ser Tyr Asp Ser Trp Gly Gln Gly Thr Leu Val Lys Val Ser Ser 115 120 125 <210> 6 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR1 <400> 6 Gly Phe Thr Phe Ser Thr Ala Asp Met Gly 1 5 10 <210> 7 <211> 19 <212> PRT <213> artificial sequence <220> <223> CDR1 <400> 7 Gly Arg Ile Phe Ser Leu Pro Ala Ser Gly Asn Ile Phe Asn Leu Leu 1 5 10 15 Thr Ile Ala <210> 8 <211> 10 <212> PRT <213> artificial sequence <220> <223> CDR1 <400> 8 Gly Phe Thr Phe Arg Ser Phe Gly Met Ser 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 9 Gly Arg Thr Leu Ser Ser Tyr Ala Met Gly 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 10 Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr 1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 11 Thr Ile Glu Ser Gly Ser Arg Thr Asn 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 12 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 13 Arg Ile Ser Gln Gly Gly Thr Ala Ile Tyr 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 14 Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr 1 5 10 <210> 15 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 15 Ser Gly Ser Gly Ser Pro Asn Phe 1 5 <210> 16 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 16 Gly Gly Ser Leu Ser Arg 1 5 <210> 17 <211> 19 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 17 Asp Pro Ser Pro Tyr Tyr Arg Gly Ser Ala Tyr Leu Leu Ser Gly Ser 1 5 10 15 Tyr Asp Ser <210> 18 <211> 25 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 18 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> 19 <211> 25 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 20 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 20 Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val Ala 1 5 10 <210> twenty one <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> twenty one Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 22 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 22 Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser 1 5 10 <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> FR2 <400> 23 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 24 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> FR3 <400> 24 Tyr Asp Glu Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Arg Ser 35 <210> 25 <211> 39 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 25 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Lys Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Gln Thr 35 <210> 26 <211> 39 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 26 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Thr Ile 35 <210> 27 <211> 39 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 27 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 1 5 10 15 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30 Ala Leu Tyr Tyr Cys Ala Lys 35 <210> 28 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 28 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 29 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 29 Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 30 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 30 Trp Gly Gln Gly Thr Leu Val Lys Val Ser Ser 1 5 10 <210> 31 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Alb8 <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 32 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Alb23 <400> 32 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 33 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Alb129 <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95[[ID=!15]] Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 34 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Alb132 <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 35 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Alb11 <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 36 <211> 116 <212> PRT <213> artificial sequence <220> <223> Alb11 (S112K)‑A <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Lys 100 105 110 Val Ser Ser Ala 115 <210> 37 <211> 115 <212> PRT <213> artificial sequence <220> <223> Alb82 <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 38 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Alb82‑A <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 39 <211> 117 <212> PRT <213> artificial sequence <220> <223> Alb82‑AA <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala 115 <210> 40 <211> 118 <212> PRT <213> artificial sequence <220> <223> Alb82‑AAA <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala Ala 115 <210> 41 <211> 116 <212> PRT <213> artificial sequence <220> <223> Alb82‑G <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly 115 <210> 42 <211> 117 <212> PRT <??> Artificial Sequence <220> <223> Alb82‑GG <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Note: There is a typo in the original text, the "wraped" should be "wrapped". And for the tag <213> which was "人工序列" in Chinese, I translated it as "Artificial Sequence" as it seems to be a more appropriate English term for such a context in patent texts. If there are specific requirements for this term, it can be adjusted accordingly.Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly 115 <210> 43 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Alb82‑GGG <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly 115 <210> 44 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Alb23002 <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 45 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Alb223 <400> 45[[ID=�3]] Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 <210> 46 <211> 3 <212> PRT <213> Artificial sequence <220> <223> 3A <400> 46 Ala Ala Ala 1 <210> 47 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 5GS <400> 47 Gly Gly Gly Gly Ser 1 5 <210> 48 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 7GS <400> 48 Ser Gly Gly Ser Gly Gly Ser 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 8GS <400> 49 Gly Gly Gly Gly Ser Gly Gly Ser 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 9GS <400> 50 Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 51 <211> 10 <212> PRT <213> Artificial sequence <2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Ser <210> 54 <211> 20 <212> PRT <213> Synthetic sequence <220> <223> 20GS <400> 54 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 55 <211> 25 <212> PRT <213> Synthetic sequence <220> <223> 25GS <400> 55 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 56 <211> 30 <212> PRT <213> Synthetic sequence <220> <223> 30GS <400> 56 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <210> 57 <211> 35 <212> PRT <213> Artificial sequence <220> <223> 35GS <400> 57 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser 35 <210> 58 <211> 40 <212> PRT <213> Artificial sequence <220> <223> 40GS <400> 58 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser 35 40 <210> 59 <211> 15 <212> PRT <213> Artificial sequence <220> <223> G1 hinge <400> 59 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 60 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> 9GS-G1 hinge <400> 60 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Pro Lys Ser Cys Asp Lys 1 5 10 15 Thr His Thr Cys Pro Pro Cys Pro 20 <210> 61 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Upper hinge area of ​​the llama <400> 61 Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala 1 5 10 <210> 62 <211> 62 <212> PRT <213> Artificial sequence <220> <223> G3 hinge <400> 62 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu 35 40 45 Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 50 55 60 <210> 63 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 63 Lys Glu Arg Glu 1 <210> 64 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 64 Lys Gln Arg Glu 1 <210> 65 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 65 Gly Leu Glu Trp 1 <210> 66 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 66 Lys Glu Arg Glu Leu 1 5 <210> 67 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 67 Lys Glu Arg Glu Phe 1 5 <210> 68 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 68 Lys Gln Arg Glu Leu 1 5 <210> 69 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 69 Lys Gln Arg Glu Phe 1 5 <210> 70 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 70 Lys Glu Arg Glu Gly 1 5 <210> 71 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 71 Lys Gln Arg Glu Trp 1 5 <210> 72 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 72 Lys Gln Arg Glu Gly 1 5 <210> 73 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 73 Thr Glu Arg Glu 1 <210> 74 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 74 Thr Glu Arg Glu Leu 1 5 <210> 75 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 75 Thr Gln Arg Glu 1 <210> 76 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 76 Thr Gln Arg Glu Leu 1 5 <210> 77 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 77 Lys Glu Cys Glu 1 <210> 78 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 78 Lys Glu Cys Glu Leu 1 5 <210> 79 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 79 Lys Glu Cys Glu Arg 1 5 <210> 80 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 80 Lys Gln Cys Glu 1 <210> 81 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 81 Lys Gln Cys Glu Leu 1 5 <210> 82 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 82 Arg Glu Arg Glu 1 <210> 83 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 83 Arg Glu Arg Glu Gly 1 5 <210> 84 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 84 Arg Gln Arg Glu 1 <210> 85 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 85 Arg Gln Arg Glu Leu 1 5 <210> 86 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 86 Arg Gln Arg Glu Phe 1 5 <210> 87 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 87 Arg Gln Arg Glu Trp 1 5 <210> 88 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 88 Gln Glu Arg Glu 1 <210> 89 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 89 Gln Glu Arg Glu Gly 1 5 <210> 90 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 90 Gln Gln Arg Glu 1 <210> 91 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 91 Gln Gln Arg Glu Trp 1 5 <210> 92 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 92 Gln Gln Arg Glu Leu 1 5 <210> 93 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 93 Gln Gln Arg Glu Phe 1 5 <210> 94 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 94 Lys Gly Arg Glu 1 <210> 95 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 95 Lys Gly Arg Glu Gly 1 5 <210> 96 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 96 Lys Asp Arg Glu 1 <210> 97 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 97 Lys Asp Arg Glu Val 1 5 <210> 98 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 98 Asp Glu Cys Lys Leu 1 5 <210> 99 <211> 5 <212> PRT <213> Artificial sequence <220> <223> motif <400> 99 Asn Val Cys Glu Leu 1 5 <210> 100 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 100 Gly Val Glu Trp 1 <210> 101 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 101 Glu Pro Glu Trp 1 <210> 102 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 102 Gly Leu Glu Arg 1 <210> 103 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 103 Asp Gln Glu Trp 1 <210> 104 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 104 Asp Leu Glu Trp 1 <210> 105 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 105 Gly Ile Glu Trp 1 <210> 106 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 106 Glu Leu Glu Trp 1 <210> 107 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 107 Gly Pro Glu Trp 1 <210> 108 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 108 Glu Trp Leu Pro 1 <210> 109 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 109 Gly Pro Glu Arg 1 <210> 110 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 110 Gly Leu Glu Arg 1 <210> 111 <211> 4 <212> PRT <213> Artificial sequence <220> <223> motif <400> 111 Glu Leu Glu Trp 1 <210> 112 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 112 Val Thr Val Ser Ser 1 5 <210> 113 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 113 Val Lys Val Ser Ser 1 5 <210> 114 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 114 Val Gln Val Ser Ser 1 5 <210> 115 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 115 Val Thr Val Ser Ser Ala 1 5 <210> 116 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 116 Val Lys Val Ser Ser Ala 1 5 <210> 117 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 117 Val Gln Val Ser Ser Ala 1 5 <210> 118 <211> 30 <212> PRT <213> Artificial sequence <220> <223> FR1 <400> 118 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 119<00026*89><211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 119 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser 20 25 30 <210> 120 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 120 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg 20 25 30 <210> 121 <211> 30<00*02712><212> PRT <213> Artificial Sequence <220> <223> FR1 <400> 121 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser 20 25 30 <210> 122 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 122 Thr Ala Asp Met Gly 1 5 <210> 123 <211> 14 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 123 Leu Pro Ala Ser Gly Asn Ile Phe Asn Leu Leu Thr Ile Ala 1 5 10 <210> 124 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 124 Ser Phe Gly Met Ser 1 5 <210> 125 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDR1 <400> 125 Ser Tyr Ala Met Gly 1 5 <210> 126 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 126 Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe Val Ala 1 5 10 <210> 127 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 127 Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 1 5 10 <210> 128 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 128 Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser 1 5 10 <210> 129 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2 <400> 129 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 130 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 130 Arg Ile Ser Gly Ile Asp Gly Thr Thr Tyr Tyr Asp Glu Pro Val Lys 1 5 10 15 Gly <210> 131 <211> 16 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 131 Thr Ile Glu Ser Gly Ser Arg Thr Asn Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 132 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 132 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 133 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDR2 <400> 133 Arg Ile Ser Gln Gly Gly Thr Ala Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 134 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 134 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Arg Ser 20 25 30 <210> 135 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 135 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Gln Thr 20 25 30 <210> 136 <211> 32[[ID=五十]] [ <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 136 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr Ile 20 25 30 <210> 137 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3 <400> 137 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys 20 25 30 <210> 138 <211> 12 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 138 Pro Arg Tyr Ala Asp Gln Trp Ser Ala Tyr Asp Tyr 1 5 10 <210> 139 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 139 Ser Gly Ser Gly Ser Pro Asn Phe 1 5 <210> 140 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 140 Gly Gly Ser Leu Ser Arg 1 5 <210> 141 <211> 19 <212> PRT <213> Artificial sequence <220> <223> CDR3 <400> 141 Asp Pro Ser Pro Tyr Tyr Arg Gly Ser Ala Tyr Leu Leu Ser Gly Ser 1 5 10 15 Tyr Asp Ser <210> 142 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 142 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 143 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 143 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 144 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 144 Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 145 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4 <400> 145 Trp Gly Gln Gly Thr Leu Val Lys Val Ser Ser 1 5 10 <210> 146 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 146 Val Thr Val Lys Ser 1 5 <210> 147 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 147 Val Thr Val Gln Ser 1 5 <210> 148 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 148 Val Lys Val Lys Ser 1 5 <210> 149 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 149 Val Lys Val Gln Ser 1 5 <210> 150 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 150 Val Gln Val Lys Ser 1 5 <210> 151 <211> 5 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 151 Val Gln Val Gln Ser 1 5 <210> 152 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 152 Val Thr Val Lys Ser Ala 1 5 <210> 153 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 153 Val Thr Val Gln Ser Ala 1 5 <210> 154 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 154 Val Lys Val Lys Ser Ala 1 5 <210> 155 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 155 Val Lys Val Gln Ser Ala 1 5 <210> 156 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 156 Val Gln Val Lys Ser Ala 1 5 <210> 157 <211> 6 <212> PRT <213> Artificial sequence <220> <223> C-terminus <400> 157 Val Gln Val Gln Ser Ala 1 5

Claims

1. A polypeptide, said polypeptide comprising, starting from its N-terminus, the following items in sequence: The immunoglobulin ISVD that specifically binds to TNFα, the first linker, the first ISVD that specifically binds to IL-23, the second linker, the ISVD that binds to human serum albumin, the third linker, and the second ISVD that specifically binds to IL-23, wherein: a) The amino acid sequence of the ISVD that specifically binds to TNFα has greater than 90% sequence identity with SEQ ID NO: 2, and is characterized by having CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 10 and CDR3 of SEQ ID NO: 14; b) The amino acid sequence of the first ISVD that specifically binds to IL-23 has greater than 90% sequence identity with SEQ ID NO: 3, and is characterized by having CDR1 of SEQ ID NO: 7, CDR2 of SEQ ID NO: 11 and CDR3 of SEQ ID NO: 15; c) The amino acid sequence of the ISVD bound to human serum albumin has greater than 90% sequence identity with SEQ ID NO: 4, and is characterized by having CDR1 of SEQ ID NO: 8, CDR2 of SEQ ID NO: 12, and CDR3 of SEQ ID NO: 16; and d) The amino acid sequence of the second ISVD that specifically binds to IL-23 has greater than 90% sequence identity with SEQ ID NO: 5, and is characterized by having CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 17; and The first, second, and third connectors each have an amino acid sequence selected from SEQ ID NOs 50-57.

2. The polypeptide according to claim 1, wherein: a) The ISVD that specifically binds to TNFα consists of the amino acid sequence of SEQ ID NO: 2; b) The first ISVD that specifically binds to IL-23 consists of the amino acid sequence of SEQ ID NO: 3; c) The ISVD bound to human serum albumin consists of the amino acid sequence of SEQ ID NO: 4; and d) The second ISVD that specifically binds to IL-23 consists of the amino acid sequence of SEQ ID NO:

5.

3. The polypeptide according to claim 1 or 2, wherein the first adapter, the second adapter, and the third adapter independently have an amino acid sequence selected from SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO:

57.

4. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

5. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of claims 1 to 4.

6. A composition comprising a polypeptide according to any one of claims 1 to 4 or a nucleic acid according to claim 5.

7. Use of the polypeptide according to any one of claims 1 to 4 or the composition according to claim 6 for the preparation of a medicament for the treatment of an autoimmune or inflammatory disease, wherein the autoimmune or inflammatory disease is selected from inflammatory bowel disease, psoriasis, psoriatic arthritis, and hidradenitis suppurativa.

8. The use according to claim 7, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

9. A host cell comprising the nucleic acid according to claim 5.

10. A method for generating a polypeptide according to any one of claims 1 to 4, the method comprising at least the following steps: a) Express the nucleic acid according to claim 5 in a suitable host cell or host organism or another suitable expression system to produce the polypeptide according to any one of claims 1 to 4; subsequently: b) Isolate and / or purify the polypeptide according to any one of claims 1 to 4.

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