TL1A binding molecules, IL-23p19 binding molecules, and bispecific antigen binding molecules, and their uses

Abstract

This invention provides a means to simultaneously block TL1A and IL-23 as a novel therapeutic pathway in the treatment of inflammatory and autoimmune diseases. [Solution] The present invention provides a TL1A-binding molecule, an IL-23p19-binding molecule, and a bispecific antigen-binding molecule having a specific sequence that targets both TL1A and IL-23p19. The present invention further provides nucleic acid molecules encoding the antigen-binding molecule, expression vectors and host cells, compositions thereof, and their use for treating diseases.

Description

[Technical Field]

[0001] This application relates to the field of immunology. More specifically, it relates to TL1A-binding molecules, IL-23p19-binding molecules, and bispecific antigen-binding molecules that target both TL1A and IL-23p19. [Background technology]

[0002] Inflammatory and autoimmune diseases typically involve abnormal activation of the immune system, resulting in the immune system attacking the body's own tissues and triggering a persistent inflammatory response. Imbalances in the cytokine network are a key factor in these diseases, and tumor necrosis factor-like ligand 1A (TL1A) and interleukin-23 (IL-23) are important cytokines involved in immunomodulation and inflammatory responses.

[0003] TL1A and its functional receptor, DR3 (death receptor 3), both belong to the TNF / TNFR protein superfamily. TL1A, also known as TNFSF15, is a type 2 transmembrane protein that exists either in a membrane-bound form (mTL1A) or is cleaved by matrix metalloproteinases and released as a soluble secreted form (sTL1A). TL1A is expressed in various cell types, including antigen-presenting cells (e.g., dendritic cells and macrophages), lymphocytes, plasma cells, and fibroblasts. On the other hand, DR3 expression is mainly limited to activated lymphocytes. When TL1A binds to DR3, the TRADD pathway is activated, regulating TRAF2 and RIP1, thereby promoting pro-inflammatory effects by PI3K, MAPK, NF-κB, and other proteins. This process promotes lymphocyte proliferation, stimulates T cell polarization into Th1 and Th17 cells, and induces the secretion of Th1, Th2, and Th17 cytokines. TL1A also synergistically promotes the production of IL-4, IL-12, and IL-23, further promoting inflammation by increasing DR3 expression in Th1, Th2, and Th17 cells. Furthermore, TL1A is involved in apoptosis and necrotizing cell death by activating the FADD, RIP3, and Caspase-8 / 3 / 7 pathways.

[0004] IL-23 is a heterodimer cytokine consisting of its own p19 subunit (IL-23p19) and a p40 subunit shared with IL-12. IL-23 is primarily produced by activated dendritic cells and macrophages, and binds to the heterodimer IL-23 receptor (composed of an IL-23R chain and an IL-12Rβ1 chain), activating the JAK-STAT signaling pathway to promote IL-17A secretion and Th17 cell proliferation and maintenance, further enhancing the Th17 cell-mediated inflammatory response. IL-23 also induces Th17 cell production, further exacerbating the inflammatory response.

[0005] Studies have shown that TL1A and IL-23 can synergistically promote the Th17-mediated immune response. After Th17 cells differentiate, TL1A / DR3 and IL-23 synergistically enhance the IL-23-mediated Th17 cell proliferation-promoting effect, inducing IL-17A production and exacerbating the inflammatory response.

[0006] Considering that TL1A and IL-23 play crucial roles and exert synergistic effects in the pro-inflammatory pathway, simultaneously blocking both TL1A and IL-23 may more effectively mitigate inflammatory responses than blocking either pathway individually. This strategy could represent a new therapeutic pathway in the treatment of inflammatory and autoimmune diseases. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Through extensive research and creative efforts, the inventors obtained monoclonal antibodies capable of specifically binding to human TL1A and human IL-23p19 using phage surface display and alpaca immunoscreening. Compared to conventional anti-TL1A antibodies (e.g., Duvakitug, 1D1, and PRA-023) and anti-IL-23p19 antibodies (e.g., risankizumab), these antibodies can more effectively block the binding of human TL1A or human IL-23p19 to their receptors and inhibit their signaling. Furthermore, based on the anti-TL1A and anti-IL-23p19 antibodies, the inventors also constructed bispecific antibodies targeting both human TL1A and human IL-23p19. These bispecific antibodies can more effectively inhibit or block the biological activity of TL1A and IL-23p19 and inhibit inflammatory responses compared to the use of either TL1A or IL-23p19 antibodies alone. Therefore, the monoclonal antibodies and bispecific antibodies according to the present invention are expected to treat, prevent, or alleviate inflammatory diseases. [Means for solving the problem]

[0008] Accordingly, in the first aspect, the present invention provides a tumor necrosis factor-like protein 1A (TL1A) binding molecule comprising at least one antigen-binding unit targeting TL1A, wherein the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 6. 2) The VH comprises the amino acid sequence HCDR1 shown in SEQ ID NO: 9, the amino acid sequence HCDR2 shown in SEQ ID NO: 10, and the amino acid sequence HCDR3 shown in SEQ ID NO: 11, and the VL comprises the amino acid sequence LCDR1 shown in SEQ ID NO: 12, the amino acid sequence LCDR2 shown in SEQ ID NO: 5, and the amino acid sequence LCDR3 shown in SEQ ID NO: 13. 3) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 19. 4) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 23. 5) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29. 6) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 33. 7) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 37, or 8) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 40, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 41.

[0009] In some embodiments, the VH includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NOs: 7, 14, 20, 24, 30, 34, 38, or 42.

[0010] In some embodiments, the VL includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NOs: 8, 15, 21, 25, 31, 35, 39, or 43.

[0011] In some embodiments, here 1) The VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8. 2) The VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15. 3) The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO: 21. 4) The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO: 25. 5) The VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 31. 6) The VH comprises the amino acid sequence shown in SEQ ID NO: 34, and the VL comprises the amino acid sequence shown in SEQ ID NO: 35. 7) The VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39, or 8) The VH comprises the amino acid sequence shown in SEQ ID NO: 42, and the VL comprises the amino acid sequence shown in SEQ ID NO: 43.

[0012] In some embodiments, the VH and / or VL further comprises substitutions, additions, and / or deletions of one or more amino acid residues and maintains specific binding to TL1A, where the substitutions, additions, and / or deletions of one or more amino acid residues are present in the VH and / or VL sequences but not in either CDR sequence.

[0013] In some embodiments, the TL1A-binding molecule is an anti-TL1A antibody or its antigen-binding fragment.

[0014] In some embodiments, the TL1A-binding molecule is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, or an antigen-binding fragment thereof.

[0015] In some embodiments, the TL1A-binding molecule is F(ab), F(ab'), F(ab')2, scFab, Fd, Fv, dsFv, dAb, double-chain antibody (diabody), or scFv.

[0016] In some embodiments, the TL1A-binding molecule further comprises an immunoglobulin heavy chain constant domain.

[0017] In some embodiments, the immunoglobulin heavy chain constant domain is a human immunoglobulin heavy chain constant domain, preferably a heavy chain constant domain derived from human IgG, and more preferably a heavy chain constant domain derived from human IgG1 or IgG4.

[0018] In some embodiments, the immunoglobulin heavy chain constant domain further comprises one or more amino acid mutations, the one or more amino acid mutations being L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 Selected from Del.

[0019] In some embodiments, the immunoglobulin heavy chain constant domain sequence includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 80, 81, or 114.

[0020] In some embodiments, the TL1A-binding molecule further comprises an immunoglobulin light chain constant domain.

[0021] In some embodiments, the immunoglobulin light chain constant domain is derived from a human λ light chain constant domain or a κ light chain constant domain.

[0022] In some embodiments, the immunoglobulin light chain constant domain sequence includes the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 82.

[0023] In some embodiments, the TL1A-binding molecule includes multiple antigen-binding units that target the TL1A.

[0024] In some embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain, the heavy chain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, or 97.

[0025] In some embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin light chain, the light chain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NOs: 84, 86, 88, 90, 92, 94, 96, or 98.

[0026] In some embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain and an immunoglobulin light chain, where, 1) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 83, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 84. 2) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 85, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 86. 3) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 87, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 88. 4) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 89, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 90. 5) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 91, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 92. 6) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 93, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 94. 7) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 95, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 96, or 8) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 97, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 98.

[0027] In a second aspect, the present invention provides an interleukin-23p19 subunit (IL-23p19) binding molecule comprising at least one immunoglobulin monovariable domain that specifically binds to IL-23p19, wherein the immunoglobulin monovariable domain that specifically binds to IL-23p19 is 1) CDR1 of the amino acid sequence shown in SEQ ID NO: 44, CDR2 of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 of the amino acid sequence shown in SEQ ID NO: 46 2) CDR1 of the amino acid sequence shown in SEQ ID NO: 48, CDR2 of the amino acid sequence shown in SEQ ID NO: 49, and CDR3 of the amino acid sequence shown in SEQ ID NO: 50 3) CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54 4) CDR1 of the amino acid sequence shown in SEQ ID NO: 56, CDR2 of the amino acid sequence shown in SEQ ID NO: 57, and CDR3 of the amino acid sequence shown in SEQ ID NO: 58 5) CDR1 of the amino acid sequence shown in SEQ ID NO: 60, CDR2 of the amino acid sequence shown in SEQ ID NO: 61, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 6) CDR1 of the amino acid sequence shown in SEQ ID NO: 64, CDR2 of the amino acid sequence shown in SEQ ID NO: 65, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 7) CDR1 of the amino acid sequence shown in SEQ ID NO: 67, CDR2 of the amino acid sequence shown in SEQ ID NO: 68, and CDR3 of the amino acid sequence shown in SEQ ID NO: 69, or 8) Includes CDR1 of the amino acid sequence shown in SEQ ID NO: 71, CDR2 of the amino acid sequence shown in SEQ ID NO: 72, and CDR3 of the amino acid sequence shown in SEQ ID NO: 73.

[0028] In some embodiments, the immunoglobulin monovariate domain that specifically binds to IL-23p19 includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 99%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78, or 79, and that maintains specific binding to IL-23p19.

[0029] In some embodiments, the immunoglobulin monovariable domain further comprises substitutions, additions, and / or deletions of one or more amino acid residues and retains specific binding to IL-23p19, wherein the substitutions, additions, and / or deletions of one or more amino acid residues are present in the immunoglobulin monovariable domain sequence but not in any CDR sequence.

[0030] In some embodiments, the immunoglobulin single variable domain is VHH.

[0031] In some embodiments, the immunoglobulin monovariate domain is a VHH derived from a camelid, preferably an alpaca or a llama.

[0032] In some embodiments, the immunoglobulin monovariate domain is a humanized VHH.

[0033] In some embodiments, the IL-23p19 binding molecule is an anti-IL-23p19 antibody or an antigen-binding fragment thereof.

[0034] In some embodiments, the IL-23p19 binding molecule is a heavy-chain antibody, a heavy-chain single-domain antibody, a chimeric antibody or a humanized antibody.

[0035] In some embodiments, the IL-23p19 binding molecule further comprises an immunoglobulin Fc domain.

[0036] In some embodiments, the immunoglobulin Fc domain is a human immunoglobulin Fc domain, preferably an Fc domain derived from human IgG, more preferably an Fc domain derived from human IgG1 or IgG4.

[0037] In some embodiments, the immunoglobulin Fc domain further comprises one or more amino acid mutations, and the one or more amino acid mutations are selected from C 220 A, L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 del.

[0038] In some embodiments, the immunoglobulin Fc domain has an amino acid sequence represented by SEQ ID NO: 99, 100, 118 or 119, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence represented by SEQ ID NO: 99, 100, 118 or 119.

[0039] In some embodiments, the immunoglobulin Fc domain and the immunoglobulin monovariate domain are optionally linked via an immunoglobulin hinge region or linker, preferably the linker being (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is 1, 2, 3, 4, or 5.

[0040] In some embodiments, the IL-23p19 binding molecule comprises a plurality of immunoglobulin monovariable domains that specifically bind to the IL-23p19.

[0041] In some embodiments, the immunoglobulin monovariate domains that specifically bind to the plurality of IL-23p19 are optionally linked via a linker, preferably (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is selected from 1, 2, 3, 4, or 5.

[0042] In some embodiments, the IL-23p19 binding molecule includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any of SEQ ID NOs: 101 to 113, and that retains specific binding to IL-23p19.

[0043] In a third aspect, the present invention provides a multispecific antigen-binding molecule comprising a TL1A-binding molecule according to a first aspect of the present invention and / or an IL-23p19-binding molecule according to a second aspect of the present invention, and one or more other antigen-binding functional regions, wherein the one or more other antigen-binding functional regions bind to an antigen different from the TL1A-binding molecule and / or the IL-23p19-binding molecule, or to a different epitope of the same antigen.

[0044] In a fourth aspect, the present invention provides a bispecific antigen-binding molecule comprising a TL1A-binding molecule according to a first aspect of the present invention and an IL-23p19-binding molecule according to a second aspect of the present invention.

[0045] In some embodiments, the bispecific antigen-binding molecule according to the present invention comprises a first antigen-binding functional region and a second antigen-binding functional region, wherein the first antigen-binding functional region comprises at least one antigen-binding unit targeting TL1A, and the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 6. 2) The VH comprises the amino acid sequence HCDR1 shown in SEQ ID NO: 9, the amino acid sequence HCDR2 shown in SEQ ID NO: 10, and the amino acid sequence HCDR3 shown in SEQ ID NO: 11, and the VL comprises the amino acid sequence LCDR1 shown in SEQ ID NO: 12, the amino acid sequence LCDR2 shown in SEQ ID NO: 5, and the amino acid sequence LCDR3 shown in SEQ ID NO: 13. 3) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 19. 4) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 23. 5) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29. 6) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 33. 7) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 37, or 8) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 40, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 41. Furthermore, the second antigen-binding functional region includes at least one immunoglobulin monovariate domain that specifically binds to IL-23p19, and the immunoglobulin monovariate domain that specifically binds to IL-23p19 is 1) CDR1 of the amino acid sequence shown in SEQ ID NO: 44, CDR2 of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 of the amino acid sequence shown in SEQ ID NO: 46 2) CDR1 of the amino acid sequence shown in SEQ ID NO: 48, CDR2 of the amino acid sequence shown in SEQ ID NO: 49, and CDR3 of the amino acid sequence shown in SEQ ID NO: 50 3) CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54 4) CDR1 of the amino acid sequence shown in SEQ ID NO: 56, CDR2 of the amino acid sequence shown in SEQ ID NO: 57, and CDR3 of the amino acid sequence shown in SEQ ID NO: 58 5) CDR1 of the amino acid sequence shown in SEQ ID NO: 60, CDR2 of the amino acid sequence shown in SEQ ID NO: 61, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 6) CDR1 of the amino acid sequence shown in SEQ ID NO: 64, CDR2 of the amino acid sequence shown in SEQ ID NO: 65, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 7) CDR1 of the amino acid sequence shown in SEQ ID NO: 67, CDR2 of the amino acid sequence shown in SEQ ID NO: 68, and CDR3 of the amino acid sequence shown in SEQ ID NO: 69, or 8) Includes CDR1 of the amino acid sequence shown in SEQ ID NO: 71, CDR2 of the amino acid sequence shown in SEQ ID NO: 72, and CDR3 of the amino acid sequence shown in SEQ ID NO: 73.

[0046] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding functional region and a second antigen-binding functional region, wherein the first antigen-binding functional region comprises at least one antigen-binding unit targeting TL1A, and the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8. 2) The VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15. 3) The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO: 21. 4) The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO: 25. 5) The VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 31. 6) The VH comprises the amino acid sequence shown in SEQ ID NO: 34, and the VL comprises the amino acid sequence shown in SEQ ID NO: 35. 7) The VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39. 8) The VH comprises the amino acid sequence shown in SEQ ID NO: 42, and the VL comprises the amino acid sequence shown in SEQ ID NO: 43, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the above amino acid sequence and retaining specific binding to TL1A. Furthermore, the second antigen-binding functional region includes at least one immunoglobulin monovariate domain that specifically binds to IL-23p19, and the immunoglobulin monovariate domain that specifically binds to IL-23p19 is less than the amino acid sequence shown in SEQ ID NOs. 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78 or 79, or less than the amino acid sequence shown in SEQ ID NOs. 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78 or 79 Each contains an amino acid sequence that has 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity and retains specific binding to IL-23p19.

[0047] In some embodiments, VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29.

[0048] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO: 30, and VL comprises the amino acid sequence shown in SEQ ID NO: 31, or comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 30 or 31, and that maintains specific binding to TL1A.

[0049] In some embodiments, the immunoglobulin single variable domain includes CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54.

[0050] In some embodiments, the immunoglobulin monovariable domain comprises the amino acid sequence shown in SEQ ID NO: 77, or comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 77, and that retains specific binding to IL-23p19.

[0051] In some embodiments, VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29. Furthermore, the immunoglobulin single variable domain includes CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54.

[0052] In some embodiments, VH comprises the amino acid sequence shown in SEQ ID NO: 30, and VL comprises the amino acid sequence shown in SEQ ID NO: 31, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 30 or 31, and retaining specific binding to TL1A. Furthermore, the immunoglobulin monovariable domain includes the amino acid sequence shown in SEQ ID NO: 77, or includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 77, and that maintains specific binding to IL-23p19.

[0053] In some embodiments, the VH, VL, and / or immunoglobulin monovariable domains further comprise substitutions, additions, and / or deletions of one or more amino acid residues and maintain specific binding to TL1A and / or IL-23p19, wherein the substitutions, additions, and / or deletions of one or more amino acid residues are present in the VH, VL, and / or immunoglobulin monovariable domain sequences but not in any of the CDR sequences.

[0054] In some embodiments, the immunoglobulin monovariate domain further comprises one or more amino acid mutations that reduce immunogenicity.

[0055] In some embodiments, the immunoglobulin single variable domain is VHH.

[0056] In some embodiments, the immunoglobulin monovariate domain is a VHH derived from a camelid, preferably an alpaca or a llama.

[0057] In some embodiments, the immunoglobulin monovariate domain is a humanized VHH.

[0058] In some embodiments, the antigen-binding unit targeting TL1A is F(ab), F(ab'), F(ab')2, scFab, Fd, Fv, dsFv, dAb, double-chain antibody (diabody), or scFv.

[0059] In some embodiments, the antigen-binding unit targeting TL1A is F(ab), and VH is linked to the immunoglobulin CH1 domain, and VL is linked to the constant domain of the immunoglobulin light chain.

[0060] In some embodiments, the immunoglobulin CH1 domain is a human immunoglobulin CH1 domain, preferably a CH1 domain derived from human IgG, more preferably a CH1 domain derived from human IgG1 or IgG4, and / or the immunoglobulin light chain constant domain is derived from a human λ light chain constant domain or a κ light chain constant domain.

[0061] In some embodiments, the immunoglobulin CH1 domain sequence is an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 117. The immunoglobulin light chain constant domain sequence includes, and / or, the amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 82.

[0062] In some embodiments, the bispecific antigen-binding molecule further comprises an immunoglobulin Fc domain.

[0063] In some embodiments, the immunoglobulin Fc domain is a human immunoglobulin Fc domain, preferably a human IgG-derived Fc domain, and more preferably a human IgG1 or IgG4-derived Fc domain.

[0064] In some embodiments, the immunoglobulin Fc domain further comprises one or more amino acid mutations, and the one or more amino acid mutations are L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 Selected from Del.

[0065] In some embodiments, the immunoglobulin Fc domain is an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence represented by sequence number 99, 100, 118, or 119.

[0066] In some embodiments, the first antigen-binding functional region comprises a plurality of antigen-binding units targeting the TL1A, and / or the second antigen-binding functional region comprises a plurality of immunoglobulin monovariable domains that specifically bind to the IL-23p19.

[0067] In some embodiments, the first antigen-binding functional region and the second antigen-binding functional region are each independent of one, two, or more.

[0068] In some embodiments, the plurality of antigen-binding units, the plurality of immunoglobulin monovariable domains, and / or the plurality of antigen-binding functional regions are optionally linked via linkers, preferably the linkers being (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is selected from 1, 2, 3, 4, or 5.

[0069] In some embodiments, the bispecific antigen-binding molecule is From the N-terminus to the C-terminus, the immunoglobulin heavy chain comprises the VH, immunoglobulin CH1 domain, Fc domain, an optional linker, and the immunoglobulin single variable domain, The immunoglobulin light chain includes the VL and the constant region of the immunoglobulin light chain, extending from the N-terminus to the C-terminus.

[0070] In some embodiments, the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGS)n, where n is selected from 1, 2, 3, 4, or 5.

[0071] In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NO: 115 or 116, and The immunoglobulin light chain includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence that is at least 92% identical to the amino acid sequence that is at least 92% identical to the amino acid sequence that is at least 92% identical to the amino acid sequence that is at least 93% identical to the amino acid sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence that is at least 923% identical to the amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence that is at least 92% identical to the amino acid sequence that is at least 92% identical to the amino acid sequence that is at least 92% identical to the amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least

[0072] In a fifth aspect, the present invention provides a fusion protein comprising a TL1A binding molecule according to the first aspect of the present invention, an IL-23p19 binding molecule according to the second aspect of the present invention, a multispecific antigen binding molecule according to the third aspect of the present invention, and / or a bispecific antigen binding molecule according to the fourth aspect of the present invention, and a polypeptide or protein expressed in fusion with the same.

[0073] In a sixth aspect, the present invention provides a conjugate comprising a TL1A binding molecule according to a first aspect of the present invention, an IL-23p19 binding molecule according to a second aspect of the present invention, a multispecific antigen binding molecule according to a third aspect of the present invention, a bispecific antigen binding molecule according to a fourth aspect of the present invention, and / or a fusion protein according to a fifth aspect of the present invention, and a conjugate conjugated thereto.

[0074] In the seventh aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a TL1A binding molecule according to the first aspect of the present invention, an IL-23p19 binding molecule according to the second aspect of the present invention, a multispecific antigen binding molecule according to the third aspect of the present invention, a bispecific antigen binding molecule according to the fourth aspect of the present invention, and / or a fusion protein according to the fifth aspect of the present invention.

[0075] In its eighth aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to its seventh aspect, operably linked to an expression regulatory element.

[0076] In the ninth aspect, the present invention provides a host cell comprising a nucleic acid molecule according to the seventh aspect of the present invention and / or an expression vector according to the eighth aspect of the present invention.

[0077] In the tenth aspect, the present invention provides a method for producing a TL1A binding molecule according to the first aspect of the present invention, an IL-23p19 binding molecule according to the second aspect of the present invention, a multispecific antigen binding molecule according to the third aspect of the present invention, a bispecific antigen binding molecule according to the fourth aspect of the present invention, and / or a fusion protein according to the fifth aspect of the present invention, the method being The present invention comprises culturing host cells according to the ninth aspect of the present invention under conditions suitable for the expression of a vector according to the eighth aspect of the present invention, and optionally isolating and / or purifying the TL1A binding molecule, the IL-23p19 binding molecule, the multispecific antigen binding molecule, the bispecific antigen binding molecule, and / or the fusion protein from the host cells or the host cell culture.

[0078] In the eleventh aspect, the present invention provides a composition comprising a TL1A binding molecule according to the first aspect of the present invention, an IL-23p19 binding molecule according to the second aspect of the present invention, a multispecific antigen binding molecule according to the third aspect of the present invention, a bispecific antigen binding molecule according to the fourth aspect of the present invention, a fusion protein according to the fifth aspect of the present invention, and / or a conjugate according to the sixth aspect of the present invention.

[0079] In some embodiments, the composition is a pharmaceutical composition, which further comprises at least one pharmaceutically acceptable carrier and / or excipient, and optionally one or more therapeutic agents having other pharmaceutically active properties.

[0080] In the twelfth aspect, the present invention provides the use of a TL1A binding molecule according to the first aspect of the present invention, an IL-23p19 binding molecule according to the second aspect of the present invention, a multispecific antigen binding molecule according to the third aspect of the present invention, a bispecific antigen binding molecule according to the fourth aspect of the present invention, a fusion protein according to the fifth aspect of the present invention, a conjugate according to the sixth aspect of the present invention, and / or a composition according to the eleventh aspect of the present invention in the manufacture of a drug for treating, preventing or alleviating inflammatory diseases and / or autoimmune diseases.

[0081] In some embodiments, the inflammatory and / or autoimmune disease is selected from asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, or psoriasis.

[0082] In some embodiments, the inflammatory bowel disease is selected from Crohn's disease or ulcerative colitis.

[0083] In some embodiments, the drug further comprises one or more other therapeutic agents for treating inflammatory diseases and / or autoimmune diseases.

[0084] In the 13th aspect, the present invention provides a method for treating, preventing or alleviating inflammatory diseases and / or autoimmune diseases, the method comprising administering a therapeutically effective amount of a TL1A-binding molecule according to the 1st aspect of the present invention, an IL-23p19-binding molecule according to the 2nd aspect of the present invention, a multispecific antigen-binding molecule according to the 3rd aspect of the present invention, a bispecific antigen-binding molecule according to the 4th aspect of the present invention, a fusion protein according to the 5th aspect of the present invention, a conjugate according to the 6th aspect of the present invention, and / or a composition according to the 11th aspect of the present invention to a subject in need thereof.

[0085] In some embodiments, the inflammatory and / or autoimmune disease is selected from asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, or psoriasis.

[0086] In some embodiments, the inflammatory bowel disease is selected from Crohn's disease or ulcerative colitis.

[0087] In some embodiments, the method further includes being used in combination with one or more other therapeutic agents for treating inflammatory diseases and / or autoimmune diseases.

[0088] In the fourteenth aspect, the present invention provides the use of a TL1A-binding molecule according to the first aspect of the present invention, an IL-23p19-binding molecule according to the second aspect of the present invention, a multispecific antigen-binding molecule according to the third aspect of the present invention, a bispecific antigen-binding molecule according to the fourth aspect of the present invention, a fusion protein according to the fifth aspect of the present invention, a conjugate according to the sixth aspect of the present invention, and / or a composition according to the eleventh aspect of the present invention for in vivo or in vitro inhibition or blockade of TL1A and / or IL-23p19 signaling. [Brief explanation of the drawing]

[0089] [Figure 1] This is the detection result of blockade of human TL1A-DR3 binding by crude anti-TL1A-F(ab) extract, showing that eight clones (4, 5, 8, 9, 11, 12, 24, and 30) exhibit a significant blocking effect. [Figure 2] This shows the results of detecting the inhibitory effect of an anti-TL1A IgG antibody on the caspase3 / 7 apoptotic signaling pathway in TF-1 cells mediated by TL1A (single duplicate well). [Figure 3] This is the ELISA detection result of blocking anti-TL1A IgG antibody against TL1A-DR3 binding (two overlapping wells). [Figure 4] This is the ELISA detection result of the binding of anti-TL1A IgG antibody to cynomolgus monkey TL1A. [Figure 5] This is the ELISA detection result (single duplicate well) of the blockade of TL1A-DcR3 binding by anti-TL1A IgG antibody. [Figure 6] These are the results of detecting signaling pathway inhibition based on huTL1A / CHO cells and DR3 reporter gene cells. 1D1 represents a single duplicate well, while all others are two duplicate wells. [Figure 7] This study involved ELISA detection of the binding of anti-IL-23p19 IgG antibodies to monkey IL-23A and mouse IL-12B heterodimeric antigen proteins, with risankizumab used as a positive control. [Figure 8] This is a result of detecting the blocking effect of anti-IL-23p19 IgG antibodies against the binding of human IL-23R-Fc and IL-23-Avi. Each molecule showed a clear blocking effect, and among them, HYB1903 exhibited the most superior blocking effect. [Figure 9] This shows the inhibition of human IL-23-induced mRNA release from mouse splenocytes by an anti-IL-23p19 IgG antibody. The experiment was performed in a single duplicate well, and HYB1903 was found to have the best inhibitory effect among all candidate molecules. [Figure 10]This shows the inhibition of human IL-23-induced mRNA release from mouse splenocytes by a humanized anti-IL-23p19 IgG antibody; the experiment was performed in two overlapping wells. [Figure 11] This study describes the inhibition of human IL-23-induced mRNA release from mouse splenocytes using a bispecific antibody. The experiment was performed in three duplicate wells, with risankizumab used as a positive control. [Figure 12] This study detected the inhibitory effect of a bispecific antibody on the caspase 3 / 7 apoptotic signaling pathway in TF-1 cells via TL1A, and the experiment was performed in three overlapping wells. [Modes for carrying out the invention]

[0090] While the present invention can be implemented in various forms, disclosure herein provides only specific, illustrative, explanatory embodiments that demonstrate the principles of the present invention. It should be emphasized that the present invention is not limited to the specific embodiments described as examples. Those skilled in the art will see that various equivalent forms, modifications, or alterations are possible without departing from the scope of the disclosure of the present invention, and such equivalent embodiments are also included in the present invention. All references cited herein, including patent publications, patents, and patent applications, are incorporated herein by reference in their entirety. Furthermore, any section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0091] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, the singular terms “kind,” “one,” and “the” as used herein and in the appended claims shall include plural forms unless otherwise specified in the context. In this application, unless otherwise specified, the word “or” means “and / or.” In this application, “first” and “second” have no substantive meaning and are used solely to distinguish the same term. Furthermore, the use of the term “includes” and other forms (e.g., “equips” and “contains”) is not restrictive. Furthermore, the scope defined in the specification and the appended claims includes all values ​​at and between endpoints. The term “about” when used in combination with a number means covering a range of numbers having a lower limit 10% lower than the specified number and an upper limit 10% higher than the specified number.

[0092] The terms and techniques used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein, nucleic acid chemistry, and hybridization are generally well known and commonly used in the art. Unless otherwise noted, the methods and techniques of the present invention are generally carried out in accordance with conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see Sambrook J. & Russell D., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Abbas et al., Cellular and Molecular Immunology, 6th edition, WBSaunders Company (2010); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terminology, experimental procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, drugs, and medicinal chemistry described herein are well known and commonly used in the art. definition

[0093] To better understand the present invention, definitions and interpretations of relevant terms are provided below.

[0094] As used herein, the term “antigen-binding molecule” may include any molecule that specifically binds to a particular antigen. In some cases, “antigen-binding molecule” may also include “antigen antagonists.” An “antigen antagonist” is any compound or biomolecule that blocks a particular antigen from binding to its innate receptor. An antigen-binding molecule or antigen antagonist may also be an antigen-binding protein. An “antigen-binding protein” is typically a protein molecule that includes an antigen-binding portion and a scaffold or skeletal portion that optionally allows the antigen-binding portion to assume a conformation that facilitates the binding of the antigen-binding molecule to the antigen. Antigen-binding proteins typically include the light chain variable domain (VL) of an antibody, the heavy chain variable domain (VH) of an antibody, or both, and their functional fragments. The variable domains of the heavy and light chains include a binding domain that interacts with the antigen. In this specification, the term “antigen-binding molecule” also includes proteins containing heavy chain single-domain antibodies and immunoglobulin single-variable domains. Examples of antigen-binding molecules include, but are not limited to, antibodies, antigen-binding fragments, heavy chain single-domain antibodies, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, and fusion proteins, as long as they exhibit the desired antigen-binding activity.

[0095] As used herein, the terms “antibody” or “immunoglobulin” are used as general terms encompassing full-length antibodies, their individual chains, and all their parts, domains, or fragments (including, but not limited to, antigen-binding domains or fragments such as the VHH domain or VH / VL domain, respectively), whether referring to heavy-chain antibodies or typical four-chain antibodies. A typical four-chain antibody comprises a glycoprotein, or its antigen-binding fragment, consisting of at least two heavy chains (H) and two light chains (L) linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of the domain CL. Furthermore, the term “sequence” as used herein (for example, “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence”) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence that encodes that sequence, unless a more specific interpretation is required herein.

[0096] As used herein, the term “domain” (of a polypeptide or protein) refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally speaking, a domain is responsible for a single functional property of a protein and can often be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain itself.

[0097] As used herein, the term “immunoglobulin domain” refers to a globular region of an antibody chain (e.g., the chain of a typical quadruple-chain antibody or the chain of a heavy-chain antibody), or a polypeptide essentially composed of such globular regions. Immunoglobulin domains are characterized by maintaining the immunoglobulin folding features of the antibody molecule.

[0098] As used herein, the term “immunoglobulin variable domain” or “variable domain” refers to an immunoglobulin domain essentially composed of four “framework regions” referred to in the art and context as “framework region 1” or “FR1,” “framework region 2” or “FR2,” “framework region 3” or “FR3,” and “framework region 4” or “FR4,” which are separated by three “complementarity-determining regions” or “CDRs” referred to in the art and context as “complementarity-determining region 1” or “CDR1,” “complementarity-determining region 2” or “CDR2,” and “complementarity-determining region 3” or “CDR3.” Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid sequences of FR1, FR2, FR3, and FR4 all constitute the “non-CDR region” of the VH or VL as described herein. The immunoglobulin variable domain confers antigen specificity to antibodies through its antigen-binding site.

[0099] Methods for numbering amino acid residues within VH and VL domains are known in the art, and these methods can be similarly applied to VHH domains. For example, Chothia numbering based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al, “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), and Kabat numbering based on the variability of the antibody sequence (Kabat et al, Sequences of Proteins of Immunological Interest, 4 th Examples of numbering schemes include the US Department of Health and Human Services, National Institutes of Health (1987 edition), AbM numbering (University of Bath), Contact numbering (University College London), the International ImMunoGeneTics Database (IMGT numbering) (1999 Nucleic Acids Research, 27, 209-212), and North numbering based on affinity propagation clustering based on numerous crystal structures. Variable domains and CDRs in antibody sequences can be identified according to common rules developed in the art (such as the AbM numbering schemes mentioned above) or by aligning the sequence with a database of known variable domains. The precise amino acid sequence boundaries of the variable domain CDRs of antibodies according to the present invention can be determined by those skilled in the art according to any scheme in the art (e.g., different numbering schemes or combinations thereof).

[0100] Furthermore, the CDR boundaries of the variable domain of the same antibody may differ based on different numbering schemes. That is, the CDR sequences of the same antibody variable domain defined by different numbering schemes will be different. Therefore, when referring to antibodies limited by a particular CDR sequence as defined in this invention, the range of such antibodies also includes antibodies whose variable domain sequence contains the particular CDR sequence, but whose claimed CDR boundary differs from the particular CDR boundary defined in this invention due to the application of a different scheme (e.g., a different numbering scheme or a combination thereof). Unless otherwise specified, the numbering and position of CDR amino acid residues within immunoglobulin variable domains described herein are based on the AbM numbering scheme.

[0101] Antibodies are classified based on their genetic constant region (also called isotype). Human constant light chains are classified into κ (Cκ) and λ (Cλ) light chains. Heavy chains are classified into μ, δ, γ, α, and ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Thus, as used herein, “isotype” refers to any class and / or subclass of immunoglobulin defined by the chemical and antigenic properties of its constant region. Known human immunoglobulin isotypes are IgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3), IgG4 (IGHG4), IgA1 (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD (IGHD), and IgE (IGHE). IgG is the most commonly used human immunoglobulin isotype for therapeutic purposes. In humans, this class includes the subclasses IgG1, IgG2, IgG3, and IgG4.

[0102] As used herein, the term “chimeric antibody” includes antibodies in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, antibodies in which the variable region sequence originates from the camelid family and the constant region sequence originates from humans.

[0103] As used herein, the term "Fab" includes polypeptides containing VH, CH1, VL, and CL immunoglobulin domains. Fab refers to an isolated region or a region in the environment of a full-length antibody or antibody fragment. Fab includes F(ab) fragments produced by a complete antibody digested with papain, F(ab') fragments produced by reduction after treatment of a complete antibody with pepsin, or F(ab')2 fragments produced without reduction after treatment of a complete antibody with pepsin, where F(ab')2 is a dimer of two F(ab') fragments linked by two disulfide bonds.

[0104] As used herein, the terms “Fc” or “Fc domain” include polypeptides comprising the constant region of an antibody heavy chain, excluding the first constant region immunoglobulin domain (CH1). Therefore, Fc refers to the immunoglobulin domains of the last two constant regions of IgA, IgD, and IgG, the immunoglobulin domains of the last three constant regions of IgE and IgM, and the flexible hinges at the N-terminus of these domains. Fc may refer to an isolated region or to that region in the environment of an Fc polypeptide (e.g., an antibody).

[0105] As used herein, the term “hinge region” refers to a flexible polypeptide containing amino acids between the first and second constant domains of an antibody. As used herein, the “hinge region” is a sequence region of 6 to 62 amino acids in length that is present only in IgA, IgD, and IgG and contains cysteine ​​residues that cross-link the two heavy chains.

[0106] Unless otherwise noted, all human immunoglobulin heavy chain constant domains described herein are numbered according to the EU numbering system (Edelman GM et al., (1969) Proc Natl Acad Sci USA, 63(1): 78-85).

[0107] As used herein, the terms “immunoglobulin monovariate domain” or “monovariate domain” refer to an immunoglobulin monovariate domain that can specifically bind to an antigen epitope without pairing with other immunoglobulin monovariate domains. Typically, in conventional immunoglobulins, the heavy chain monovariate domain (VH) and the light chain monovariate domain (VL) interact to form an antigen-binding site. In contrast, an immunoglobulin monovariate domain can specifically bind to an antigen epitope without pairing with other immunoglobulin monovariate domains. Therefore, an example of an immunoglobulin monovariate domain as described herein is a “monodomain antibody,” for example, an immunoglobulin monovariate domain VH and VL (VH domain and VL domain). Another example of an immunoglobulin monovariate domain is the “VHH domain” (or simply “VHH”) of the camelid family, as defined below.

[0108] The "VHH domain," also known as a heavy-chain single-domain antibody, VHH, VHH antibody fragment, VHH antibody, or sdAb, is a variable domain of an antigen-binding immunoglobulin called a "heavy-chain antibody (HCab)" (i.e., an antibody lacking a light chain). The term "VHH domain" is used to distinguish this variable domain from the heavy-chain variable domain (referred to herein as the "VH domain") and the light-chain variable domain (referred to herein as the "VL domain") present in typical quadruple-chain antibodies. The VHH domain specifically binds to an epitope without the assistance of other antigen-binding domains (unlike the VH domain or VL domain of a typical quadruple-chain antibody, in this case the epitope is recognized by both the VL domain and the VH domain). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain.

[0109] In the context of this invention, the terms "heavy chain single-domain antibody," "VHH domain," "VHH," "VHH antibody fragment," "VHH antibody," and "sdAb" are used interchangeably.

[0110] VHH domains (which lack a light chain variable domain and are naturally "designed" to functionally bind to an antigen without interacting with the light chain variable domain) can be used as a single, relatively small, functional antigen-binding structural unit, domain, or polypeptide. This property distinguishes VHH domains from the VH and VL domains of typical four-chain antibodies. These VH and VL domains are generally not practically suitable for use as a single antigen-binding protein or immunoglobulin monovariate domain. Therefore, they need to be combined in some way to provide a functional antigen-binding unit (e.g., in the form of a typical antibody fragment such as a Fab fragment, or in the form of an scFv consisting of a VH domain covalently bonded to a VL domain).

[0111] These unique properties make using VHH domains alone or as part of a larger polypeptide several significant advantages compared to using conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., F(ab) or F(ab')2 fragments): Only one domain is needed to bind to the antigen with high affinity and selectivity, eliminating the need for two separate domains, nor the need for those two domains to be in the correct spatial conformation and configuration (e.g., scFv typically requires a specially designed linker). VHH domains can be expressed from a single gene and do not require post-translational folding or modification. VHH domains can be readily modified into multivalent and multispecific formats. VHH domains are highly soluble and do not tend to aggregate. VHH domains are very stable to heat, pH, proteases, and other denaturing agents and conditions, allowing them to be manufactured, stored, and transported without refrigeration, saving costs, time, and being environmentally friendly. VHH domains are easy to manufacture and relatively inexpensive, even at the scale required for production. Because the VHH domain is relatively small compared to conventional quadruple-chain antibodies and their antigen-binding fragments (approximately 15 kDa, or 1 / 10 the size of conventional IgG), it has higher tissue permeability and can be administered at relatively higher doses. The VHH domain can exhibit so-called cavity-binding properties (particularly due to its longer CDR3 loop compared to conventional VH domains), thereby allowing it to reach targets and epitopes that conventional quadruple-chain antibodies and their antigen-binding fragments cannot access.

[0112] The heavy chain single-domain antibodies disclosed herein can be manufactured by those skilled in the art using methods known or future methods in the art. Methods for obtaining VHHs that bind to specific antigens or epitopes have already been disclosed, for example, in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology, Vol. 8,(12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0113] The present invention is not limited with respect to the origin of the immunoglobulin monovariable domain sequence (or the nucleotide sequence used to express it), nor is it limited with respect to the method by which the immunoglobulin monovariable domain sequence or nucleotide sequence is generated or obtained (or the method by which it is generated or obtained). The present invention may use immunoglobulin sequences of different origins, such as immunoglobulin sequences derived from camelids. The present invention also includes fully human sequences, humanized sequences, or chimeric sequences. For example, the present invention includes immunoglobulin sequences derived from camelids, humanized immunoglobulin sequences derived from camelids, and chimeric sequences in which the monovariable domain sequence is derived from a camelid immunoglobulin and the constant region sequence (e.g., the Fc domain) is derived from human immunoglobulin. Furthermore, the present invention also uses fusion immunoglobulin sequences to form, for example, polyvalent and / or multispecific antigen-binding molecules, or immunoglobulin sequences derived from the immunoglobulin sequences of the present invention that include markers or other functional parts (e.g., biologically active compounds or polypeptides, detectable markers, water-soluble polymers, etc.).

[0114] Once the most effective animal-derived immunoglobulins are identified, their amino acid sequences can be optimized, for example, through affinity maturation or humanization.

[0115] Camelid-derived VHH domains can be “humanized” (also referred to herein as “sequence optimization”; in addition to humanization, “sequence optimization” also includes other modifications to the sequence, such as introducing one or more mutations that improve the properties of the VHH, including improving transient transfection expression yield) by substituting one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a normal human quadruple-chain antibody. Humanization can prevent a human immune response to animal-derived VHH. The humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be carried out by methods known to the art, for example, based on further descriptions herein and prior art (e.g., WO2008 / 020079). Furthermore, it should be noted that such humanized VHH can be obtained by any suitable method known to the art, and is not strictly limited to polypeptides obtained using polypeptides containing naturally occurring VHH domains as starting materials.

[0116] As used herein, the term “full-length antibody” encompasses the structure of an antibody that forms a natural biological form, including a variable region and a constant region. For example, in most mammals, including humans and mice, a full-length antibody of the IgG class is a tetramer consisting of two pairs of identical immunoglobulin chains, each pair containing one light chain and one heavy chain. Each light chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, CH1(Cγ1), CH2(Cγ2), and CH3(Cγ3). In some camelids, such as alpacas and llamas, an IgG antibody may consist of only two heavy chains, each heavy chain containing a single immunoglobulin variable domain linked to an Fc region.

[0117] Those skilled in the art will understand that once the VH and VL domain sequences of an antibody are obtained, these domains can be further manipulated using conventional molecular biology methods, for example, by reconstituting the VH and VL domains into an antibody fragment having antigen-binding activity. Antibody fragments include Fab fragments consisting of a VL domain, VH domain, CL domain, and CH1 domain, such as F(ab), F(ab'), F(ab')2, and scFab linked via a peptide linker (e.g., a long-chain flexible linker); Fd fragments consisting of the VH domain and CH1 domain of a single antibody; Fv fragments consisting of the VL domain and VH domain of a single antibody, such as disulfide-stable Fv fragments (dsFv) linked by disulfide bonds, or single-chain Fv fragments (scFv) linked via a peptide linker (e.g., a long-chain flexible linker), in which both variable domains can associate to form an antigen-binding site; and double-chain antibodies (diabodies) where two VH domains of a single antibody linked via a short linker form a dimer, generating two antigen-binding sites (Holliger P et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-6448; EP 404,097; WO 93 / 11161); This includes, but is not limited to, dAb fragments consisting of a single variable region.

[0118] As used herein, the term “specificity” refers to a non-random binding reaction between two molecules, such as between a particular antigen and its innate receptor, and / or between a particular antigen and an antigen-binding molecule (e.g., the immunoglobulin single variable domain, heavy chain single-domain antibody, TL1A binding molecule, or IL-23p19 binding molecule of the present invention). Its specificity can be determined based on the affinity and / or avidity of the antigen-binding molecule. The equilibrium dissociation constant (K) between the antigen and the antigen-binding molecule is also used. D The affinity expressed by ) is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding molecule. DThe smaller the value, the stronger the binding strength between the epitope and the antigen-binding molecule (or affinity is the binding constant (K)). A )(1 / K D (It can also be expressed as )). As those skilled in the art will understand, affinity can be determined by known methods depending on the specific antigen in question. Affinity is a measure of the strength of binding between an antigen-binding molecule (e.g., immunoglobulin, antibody, immunoglobulin monovariate domain, or polypeptide containing such domain) and its associated antigen. Affinity is related to two factors: the affinity between the antigen-binding site on the antigen-binding molecule and its associated antigen, and the number of associated binding sites present on the antigen-binding molecule.

[0119] As used herein, the term “blockage” refers to the ability of an antigen-binding molecule to inhibit a specific binding interaction between two molecules (e.g., a particular target antigen and its innate receptor) to a detectable degree. In some embodiments, an antigen-binding molecule that blocks a specific binding interaction between two molecules inhibits the binding interaction between the two molecules by at least 50%. In some embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0120] As used herein, the term "TL1A" includes variants, isotypes, and species homologs of TL1A. Therefore, the TL1A-binding molecules of the present invention can bind to human TL1A and can cross-react with TL1A of non-human species, such as cynomolgus monkeys. The use of the term "TL1A" herein encompasses all known and undiscovered alleles and polymorphic forms of TL1A, preferably including human TL1A.

[0121] In the present invention, "TL1A-binding molecule" refers to any molecule that can specifically bind to tumor necrosis factor-like ligand 1A (TL1A) and block the binding interaction between TL1A and DR3. In some cases, the "TL1A-binding molecule" may include at least one antigen-binding domain that binds to TL1A and blocks the binding interaction between TL1A and DR3.

[0122] As used herein, the term "IL-23p19" includes variants, isotypes, and species homologs of IL-23p19. Therefore, the IL-23p19-binding molecules of the present invention can bind to human IL-23p19 and cross-react with IL-23p19 from non-human species such as cynomolgus monkeys. The use of "IL-23p19" herein encompasses all known and undiscovered alleles and polymorphic forms of IL-23p19, preferably including human IL-23p19.

[0123] In the present invention, the term "IL-23p19 binding molecule" refers to any molecule that can specifically bind to interleukin-23 (IL-23p19) and block the binding interaction between IL-23p19 and IL-23R and / or IL-12Rβ1. In some cases, the "IL-23p19 binding molecule" may include at least one immunoglobulin monovariable domain, such as a VHH as defined herein, that binds to IL-23p19 and blocks the binding interaction between IL-23p19 and IL-23R and / or IL-12Rβ1.

[0124] In some embodiments, the "TL1A binding molecule" or "IL-23p19 binding molecule" in the present invention may each independently contain two, three, or four or more antigen-binding domains that bind to a target antigen.

[0125] As used herein, the term "valence" refers to the presence of a specific number of antigen-binding sites in a particular molecule. The term "monovalent" refers to an antigen-binding molecule having only one single antigen-binding site, and the term "polyvalent" refers to an antigen-binding molecule having multiple antigen-binding sites. Thus, the terms "divalent," "tetravalent," and "hexavalent" indicate the presence of two, four, and six binding sites in the antigen-binding molecule, respectively. In some embodiments, the TL1A-binding molecule or IL-23p19-binding molecule as described herein is divalent.

[0126] In some embodiments, the “TL1A binding molecule” or “IL-23p19 binding molecule” of the present invention also includes multispecific antigen-binding molecules comprising at least one antigen-binding domain that specifically binds to TL1A and blocks the binding interaction between TL1A and DR3, and / or at least one immunoglobulin monovariable domain that specifically binds to IL-23p19 and blocks the binding interaction between IL-23p19 and IL-23R and / or IL-12Rβ1, and further comprising antigen-binding domains that bind to different antigens or different regions of the same antigen (e.g., different epitopes).

[0127] In some embodiments, the present invention also encompasses a "TL1A / IL-23p19 bispecific antigen-binding molecule" comprising at least one immunoglobulin monovariable domain that specifically binds to TL1A and blocks the binding interaction between TL1A and DR3, and at least one immunoglobulin monovariable domain that specifically binds to IL-23p19 and blocks the binding interaction between IL-23p19 and IL-23R and / or IL-12Rβ1.

[0128] The "TL1A binding molecule," "IL-23p19 binding molecule," and / or "TL1A / IL-23p19 bispecific antigen binding molecule" in the present invention may include a linker and / or a functional moiety in addition to the immunoglobulin monovariable domain that binds to the target antigen, such as a biologically active compound or polypeptide (e.g., an Fc domain), and / or a conjugate that can modulate (e.g., increase or decrease) the intrinsic activity of the antigen binding molecule or confer novel activity to the molecule.

[0129] As used herein, the term “isolated” typically refers to a biological material (e.g., a virus, nucleic acid, or protein) that is substantially free from components that typically accompany or interact with it in its natural environment. The isolated biological material may optionally include other materials (e.g., nucleic acids or proteins) not found in the biological material in its natural environment. When referring to a protein herein, “isolated” typically means that the molecule has been isolated or separated from the whole organism in which it naturally exists, or that there are substantially no other biological macromolecules of the same kind. When referring to a nucleic acid molecule, it means that the nucleic acid has been completely or partially isolated from the sequence to which it naturally binds, that the nucleic acid has a heterologous sequence to which it binds, or that the nucleic acid has been isolated from a chromosome.

[0130] As used herein, the terms “polypeptide” and “protein” are interchangeable and typically refer to polymers of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimics of corresponding natural amino acids, as well as natural amino acid polymers. The term may also include amino acid polymers modified, for example, by adding sugar residues to form glycoproteins or by phosphorylation. Polypeptides and proteins may be produced by naturally occurring non-recombinant cells or by genetically modified or recombinant cells, and may include molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acid deletions, additions, and / or substitutions of the natural sequence. The terms “polypeptide” and “protein” specifically include sequences of one or more amino acid deletions, additions, and / or substitutions of antigen-binding proteins described in this invention.

[0131] Amino acid residues are represented according to standard three-letter or one-letter amino acid codes known and agreed in the art. When comparing two amino acid sequences, the term “amino acid difference” refers to the insertion, deletion, or substitution of a specified number of amino acid residues at specific positions in the reference sequence compared to the other sequence. In the case of substitutions, such substitutions are preferably conservative amino acid substitutions, meaning that an amino acid residue is replaced by another amino acid residue having a similar chemical structure and has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are known in the art. For example, a conservative amino acid substitution is preferably the substitution of one amino acid from the following groups (i) to (v) with another amino acid residue from the same group: (i) relatively small aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) polar positively charged residues: His, Arg, and Lys; (iv) relatively large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; (v) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Gln, or Glu; Met is substituted with Leu, Tyr, or Ile; Phe is substituted with Met, Leu, or Tyr; Ser is substituted with Thr; Thr is substituted with Ser; Trp is substituted with Tyr; Tyr is substituted with Trp or Phe; Val is substituted with Ile or Leu.

[0132] "Sequence identity" between two polypeptide sequences refers to the proportion of identical amino acids between the sequences. "Sequence similarity" refers to the proportion of amino acids that are identical or represent conserved amino acid substitutions. Methods for evaluating the degree of sequence homology between amino acids or nucleotides are well known to those skilled in the art. For example, amino acid sequence homology is usually measured using sequence analysis software. For example, homology can be determined using the BLAST program in the NCBI database. For determining sequence homology, you can refer to, for example, the following references: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of SequenceData, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0133] When used herein, "EC 50 The term "half-maximal effective concentration" is also called the "half-maximal effective concentration" and refers to the concentration of a drug, antibody, or toxin that elicits a 50% response between baseline and maximum after a specific exposure time. In the context of this application, EC 50 The unit is "μg / mL".

[0134] As used herein, the term “operatably linked” typically means that at least one nucleic acid molecule and at least one regulatory element have a functional relationship with each other. For example, a promoter is considered “operatably linked” to a coding sequence if it can initiate the transcription and / or expression of the coding sequence or otherwise control / regulate it (in which case the coding sequence should be understood to be “under the control” of the promoter). Typically, when two nucleotide sequences are operatably linked, they are oriented in the same direction and usually within the same reading frame. They are also usually substantially contiguous.

[0135] As used herein, the term “subject” includes all animals, more specifically mammals. Among mammals, humans and non-human mammals can be distinguished. Non-human animals include, for example, companion animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals commonly used for research purposes and / or antibody production (e.g., rats, mice, guinea pigs, non-human primates (e.g., cynomolgus macaques), camelids, etc.). For preventive and / or therapeutic purposes, the subject is preferably human.

[0136] As used herein, the term “prevention” means a method performed to prevent or delay the development of a disease, illness, or symptom (e.g., an inflammatory disease) in a subject. The term “treatment” means to achieve a desired pharmacological and / or physiological effect. Such effect may be preventive in that it is the complete or partial prevention of a disease or its symptoms, and / or therapeutic in that it is the partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, “treatment” encompasses diseases of mammals, in particular humans, and includes: (a) prevention of the development of a disease or symptom in an individual that is susceptible to the disease but has not yet been diagnosed; (b) inhibition of the disease, such as stopping the progression of the disease; or (c) mitigation of the disease, such as reducing the symptoms associated with the disease. As used herein, “treatment” encompasses the administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or inhibit a disease of that individual, and includes, but is not limited to, administering a drug containing a compound described herein to an individual in need.

[0137] As used herein, the term “effective dose” means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective dose for disease prevention means an amount sufficient to prevent, inhibit, or delay the onset of the disease. On the other hand, an effective dose for disease treatment means an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient who already has the disease. Determining such an effective dose is within the capabilities of those skilled in the art. For example, an effective dose for therapeutic purposes will vary depending on the severity of the disease being treated, the overall state of the patient’s immune system, the patient’s general circumstances such as age, weight, and sex, the method of drug administration, and any combination therapies.

[0138] As used herein, “pharmaceutical composition” refers to a composition used for the treatment of a disease or for in vitro cell culture experiments. When used for the treatment of a disease, the term “pharmaceutical composition” usually refers to a composition in the form of a unit dose, which can be prepared by any method well known in the pharmaceutical industry. Any method involves mixing an active ingredient with pharmaceutical excipients comprising one or more auxiliary components. Typically, compositions are prepared by homogeneously and appropriately mixing an active compound with a liquid pharmaceutical excipient, a finely crushed solid pharmaceutical excipient, or both.

[0139] The present invention aims to provide novel drugs for the treatment, prevention, or alleviation of inflammatory diseases and / or autoimmune diseases (e.g., inflammatory bowel disease, typically Crohn's disease and ulcerative colitis).

[0140] In some embodiments, the present invention relates to TL1A-binding molecules, IL-23p19-binding molecules, and bispecific antigen-binding molecules that target both TL1A and IL-23p19. Compared to prior art anti-TL1A antibodies (e.g., Duvakitug, 1D1, and PRA-023) and anti-IL-23p19 antibodies (e.g., risankizumab), the TL1A and IL-23p19-binding molecules can more effectively block the binding of human TL1A or human IL-23p19 to their receptors and inhibit their signaling. Compared to TL1A or IL-23p19 antibodies alone, the bispecific antigen-binding molecules can more effectively inhibit or block the biological activity of TL1A and IL-23p19 and inhibit inflammatory responses. TL1A binding molecule

[0141] Compared to conventional anti-TL1A antibodies (e.g., Duvakitug, 1D1, and PRA-023), the TL1A-binding molecule according to the present invention more effectively blocks the binding of membrane-bound and secreted human TL1A to DR3, and can exert biological activities such as inhibiting the production of inflammatory cytokines and mitigating inflammatory responses. Furthermore, since the TL1A-binding molecule according to the present invention does not completely block the binding of human TL1A, which has a negative regulatory effect on the TL1A-DR3 signaling pathway, to DcR3, it is expected to have excellent potential in clinical development.

[0142] Accordingly, in some embodiments, the present invention provides a TL1A-binding molecule comprising at least one antigen-binding unit targeting TL1A, wherein the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL).

[0143] In some embodiments, the antigen-binding unit targeting at least one TL1A may include HCDR1, HCDR2, and HCDR3 in VH as shown in SEQ ID NOs: 7, 14, 20, 24, 30, 34, 38, or 42, and LCDR1, LCDR2, and LCDR3 in VL as shown in SEQ ID NOs: 8, 15, 21, 25, 31, 35, 39, or 43, where the CDRs may be defined by any of the Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR definition systems.

[0144] In some embodiments, the antigen-binding unit targeting at least one TL1A is, 1) HCDR1, HCDR2 and HCDR3 in VH indicated by Sequence ID 7, and LCDR1, LCDR2 and LCDR3 in VL indicated by Sequence ID 8, 2) HCDR1, HCDR2 and HCDR3 in VH as indicated by Sequence ID 14, and LCDR1, LCDR2 and LCDR3 in VL as indicated by Sequence ID 15, 3) HCDR1, HCDR2 and HCDR3 in VH as shown in Sequence ID No. 20, and LCDR1, LCDR2 and LCDR3 in VL as shown in Sequence ID No. 21, 4) HCDR1, HCDR2 and HCDR3 in VH as shown in Sequence ID No. 24, and LCDR1, LCDR2 and LCDR3 in VL as shown in Sequence ID No. 25, 5) HCDR1, HCDR2 and HCDR3 in VH indicated by Sequence ID 30, and LCDR1, LCDR2 and LCDR3 in VL indicated by Sequence ID 31, 6) HCDR1, HCDR2 and HCDR3 in VH as shown in Sequence ID 34, and LCDR1, LCDR2 and LCDR3 in VL as shown in Sequence ID 35, 7) HCDR1, HCDR2 and HCDR3 in VH as shown in Sequence ID 38, and LCDR1, LCDR2 and LCDR3 in VL as shown in Sequence ID 39, or 8) This may include HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID No. 42, and LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID No. 43. The aforementioned CDR may be defined by any of the following definition systems: Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR.

[0145] In some embodiments, the immunoglobulin heavy chain variable domain (VH) and immunoglobulin light chain variable domain (VL) include one or more sets selected from the following CDR1, CDR2, and CDR3 as defined by the AbM numbering system: 1) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 6. 2) The VH comprises the amino acid sequence HCDR1 shown in SEQ ID NO: 9, the amino acid sequence HCDR2 shown in SEQ ID NO: 10, and the amino acid sequence HCDR3 shown in SEQ ID NO: 11, and the VL comprises the amino acid sequence LCDR1 shown in SEQ ID NO: 12, the amino acid sequence LCDR2 shown in SEQ ID NO: 5, and the amino acid sequence LCDR3 shown in SEQ ID NO: 13. 3) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 19. 4) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 23. 5) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29. 6) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 33. 7) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 37, or 8) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 40, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 41.

[0146] In some preferred embodiments, the antigen-binding unit targeting at least one TL1A is, 1) HCDR1, HCDR2, and HCDR3 in VH as shown in Sequence ID No. 20, and LCDR1, LCDR2, and LCDR3 in VL as shown in Sequence ID No. 21, or 2) This may include HCDR1, HCDR2, and HCDR3 in VH indicated by Sequence ID 30, and LCDR1, LCDR2, and LCDR3 in VL indicated by Sequence ID 31. The aforementioned CDR may be defined by any of the following definition systems: Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR.

[0147] In some preferred embodiments, the immunoglobulin heavy chain variable domain (VH) and immunoglobulin light chain variable domain (VL) include one or more sets selected from the following CDR1, CDR2, and CDR3 as defined by the AbM numbering system: 1) The VH comprises the amino acid sequence HCDR1 shown in SEQ ID NO: 1, the amino acid sequence HCDR2 shown in SEQ ID NO: 2, and the amino acid sequence HCDR3 shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence LCDR1 shown in SEQ ID NO: 17, the amino acid sequence LCDR2 shown in SEQ ID NO: 18, and the amino acid sequence LCDR3 shown in SEQ ID NO: 19, or 2) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29.

[0148] In some embodiments, the TL1A binding molecule according to the present invention comprises an immunoglobulin heavy chain variable domain, wherein the VH comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 99%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NOs: 7, 14, 20, 24, 30, 34, 38, or 42.

[0149] In some embodiments, the TL1A binding molecule according to the present invention comprises an immunoglobulin heavy chain variable domain, wherein the VH comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence that is at least 900% identical to the amino acid sequence that is at least 90% identical to the amino acid sequence that is at least 90% identical to the amino acid sequence that is at least 90% identical to the amino acid sequence that is at least 90% identical to the amino acid sequence that is at least 90% identical to the amino acid sequence that is at least 90% identical

[0150] In some embodiments, the TL1A binding molecule according to the present invention comprises an immunoglobulin light chain variable domain, the VL comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NOs: 8, 15, 21, 25, 31, 35, 39, or 43.

[0151] In some preferred embodiments, the TL1A binding molecule according to the present invention comprises an immunoglobulin light chain variable domain, the VL comprising an amino acid sequence represented by SEQ ID NO: 21 or 31, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NO: 21 or 31.

[0152] In some embodiments, the TL1A binding molecule according to the present invention comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL). 1) The VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8. 2) The VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15. 3) The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO: 21. 4) The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO: 25. 5) The VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 31. 6) The VH comprises the amino acid sequence shown in SEQ ID NO: 34, and the VL comprises the amino acid sequence shown in SEQ ID NO: 35. 7) The VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39, or 8) The VH comprises the amino acid sequence shown in SEQ ID NO: 42, and the VL comprises the amino acid sequence shown in SEQ ID NO: 43.

[0153] In some preferred embodiments, the TL1A-binding molecule provided by the present invention comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL). 1) The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO: 21, or 2) The VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 31.

[0154] In some embodiments, the TL1A-binding molecule is an anti-TL1A antibody or its antigen-binding fragment.

[0155] In some embodiments, the TL1A-binding molecule is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, or an antigen-binding fragment thereof.

[0156] In some embodiments, the TL1A-binding molecule is F(ab), F(ab'), F(ab')2, scFab, Fd, Fv, dsFv, dAb, double-chain antibody (diabody), or scFv. IL-23p19 binding molecule

[0157] The IL-23p19 binding molecule provided by the present invention can more effectively block the binding of human IL-23p19 to the IL-23R and IL-12Rβ1 complex compared to conventional anti-IL-23p19 antibodies (e.g., risankizumab), thereby providing biological activity such as suppressing the production of inflammatory cytokines and mitigating inflammatory responses.

[0158] Therefore, in some embodiments, the present invention provides an IL-23p19 binding molecule comprising at least one immunoglobulin monovariable domain that specifically binds to IL-23p19.

[0159] In some embodiments, the immunoglobulin monovariate domains that specifically bind to at least one IL-23p19 may include CDR1, CDR2, and CDR3 in the VHH represented by SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, or 74. The CDRs may be defined by any of the Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR definition systems.

[0160] In some embodiments, the immunoglobulin monovariate domains that specifically bind to at least one IL-23p19 include one or more sets selected from the following CDR1, CDR2, and CDR3 as defined by the AbM numbering system: 1) CDR1 of the amino acid sequence shown in SEQ ID NO: 44, CDR2 of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 of the amino acid sequence shown in SEQ ID NO: 46 2) CDR1 of the amino acid sequence shown in SEQ ID NO: 48, CDR2 of the amino acid sequence shown in SEQ ID NO: 49, and CDR3 of the amino acid sequence shown in SEQ ID NO: 50 3) CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54 4) CDR1 of the amino acid sequence shown in SEQ ID NO: 56, CDR2 of the amino acid sequence shown in SEQ ID NO: 57, and CDR3 of the amino acid sequence shown in SEQ ID NO: 58 5) CDR1 of the amino acid sequence shown in SEQ ID NO: 60, CDR2 of the amino acid sequence shown in SEQ ID NO: 61, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 6) CDR1 of the amino acid sequence shown in SEQ ID NO: 64, CDR2 of the amino acid sequence shown in SEQ ID NO: 65, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 7) CDR1 of the amino acid sequence shown in SEQ ID NO: 67, CDR2 of the amino acid sequence shown in SEQ ID NO: 68, and CDR3 of the amino acid sequence shown in SEQ ID NO: 69, or 8) CDR1 of the amino acid sequence shown in SEQ ID NO: 71, CDR2 of the amino acid sequence shown in SEQ ID NO: 72, and CDR3 of the amino acid sequence shown in SEQ ID NO: 73.

[0161] In some embodiments, the immunoglobulin monovariate domain that specifically binds to at least one IL-23p19 includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, or 74, and that retains specific binding to IL-23p19.

[0162] In some preferred embodiments, the immunoglobulin monovariate domains that specifically bind to at least one IL-23p19 may include CDR1, CDR2, and CDR3 in the VHH represented by SEQ ID NOs: 47, 55, 63, 66, or 70. The CDRs may be defined by any of the Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR definition systems.

[0163] In some preferred embodiments, the immunoglobulin monovariate domains that specifically bind to at least one IL-23p19 include one or more sets selected from the following CDR1, CDR2, and CDR3 as defined by the AbM numbering system: 1) CDR1 of the amino acid sequence shown in SEQ ID NO: 44, CDR2 of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 of the amino acid sequence shown in SEQ ID NO: 46 2) CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54 3) CDR1 of the amino acid sequence shown in SEQ ID NO: 60, CDR2 of the amino acid sequence shown in SEQ ID NO: 61, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 4) CDR1 of the amino acid sequence shown in SEQ ID NO: 64, CDR2 of the amino acid sequence shown in SEQ ID NO: 65, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62, or 5) CDR1 of the amino acid sequence shown in SEQ ID NO: 67, CDR2 of the amino acid sequence shown in SEQ ID NO: 68, and CDR3 of the amino acid sequence shown in SEQ ID NO: 69.

[0164] In some preferred embodiments, the immunoglobulin monovariate domain that specifically binds to at least one IL-23p19 comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NO: 47, 55, 63, 66, or 70, and that retains specific binding to IL-23p19.

[0165] In some embodiments, the immunoglobulin monovariate domain that specifically binds to IL-23p19 is a VHH, for example, a VHH derived from a camelid, preferably an alpaca or a llama.

[0166] In some preferred embodiments, the immunoglobulin monovariable domain that specifically binds to IL-23p19 is a humanized VHH, the humanized VHH comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to SEQ ID NO: 55. In some embodiments, the amino acid sequence of the humanized VHH comprises one or more amino acid substitutions with respect to SEQ ID NO: 55, and conserved amino acid substitutions are preferred. For example, the amino acid sequence of the humanized immunoglobulin monovariable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions with respect to SEQ ID NO: 55. In some preferred embodiments, the amino acid sequence of the humanized VHH includes the amino acid sequence shown in any of SEQ ID NOs. 75 to 79, or includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any of SEQ ID NOs. 75 to 79, and that retains specific binding to IL-23p19.In some more preferred embodiments, the amino acid sequence of the humanized VHH includes the amino acid sequence shown in any of SEQ ID NOs. 75-78, or includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any of SEQ ID NOs. 75-78, and that retains specific binding to IL-23p19. In some more preferred embodiments, the amino acid sequence of the humanized VHH includes the amino acid sequence shown in SEQ ID NO: 77, or includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 77, and that retains specific binding to IL-23p19.

[0167] In some embodiments, the IL-23p19-binding molecule is an anti-IL-23p19 antibody or its antigen-binding fragment. In further embodiments, the IL-23p19-binding molecule is a heavy chain antibody, a heavy chain single-domain antibody, a chimeric antibody, or a humanized antibody.

[0168] In some embodiments, the TL1A-binding molecule according to the present invention comprises a plurality of antigen-binding units that specifically bind to TL1A. In some embodiments, the IL-23p19-binding molecule according to the present invention comprises a plurality of immunoglobulin monovariable domains that specifically bind to IL-23p19. The plurality of antigen-binding units or immunoglobulin monovariable domains can be directly or indirectly linked to form a polyvalent antigen-binding molecule.

[0169] In some embodiments, the TL1A-binding molecule according to the present invention further comprises an immunoglobulin Fc domain in addition to at least one antigen-binding unit that specifically binds to TL1A. In some embodiments, the IL-23p19-binding molecule according to the present invention further comprises an immunoglobulin Fc domain in addition to at least one immunoglobulin monovariate domain that specifically binds to IL-23p19. The Fc domains that may be used in the present invention may be derived from various subtypes of immunoglobulins, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM. The immunoglobulin Fc domain is preferably a human immunoglobulin Fc domain, e.g., an Fc domain of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the TL1A-binding molecule and / or IL-23p19-binding molecule according to this specification comprises an Fc domain derived from human IgG1 (its amino acid sequence is shown in SEQ ID NO: 99). In some embodiments, the TL1A-binding molecule and / or IL-23p19-binding molecule according to this specification comprises an Fc domain variant derived from human IgG1. In some embodiments, the Fc domain variant derived from human IgG1 comprises C 220 A, L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 It includes one or more mutations selected from del. In some embodiments, the Fc domain variant derived from human IgG1 is L 234 A, L 235Includes A mutation. In some embodiments, the Fc domain variant derived from human IgG1 is C 220 A, L 234 A, L 235 Includes A mutation. In some embodiments, the Fc domain variant derived from human IgG1 is L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 This includes del mutations. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc domain variant is shown, for example, in SEQ ID NOs: 100, 118, or 119.

[0170] In some embodiments, in the TL1A-binding molecule and / or IL-23p19-binding molecule according to the present invention, the immunoglobulin Fc domain (e.g., the Fc domain of human IgG1) can be directly or indirectly linked to the C-terminus and / or N-terminus of the antigen-binding unit or immunoglobulin monovariate domain via a hinge region or linker. The inclusion of the immunoglobulin Fc domain in the TL1A-binding molecule and / or IL-23p19-binding molecule according to the present invention allows the binding molecule to form a dimer molecule. In some embodiments, the dimer is a homodimer, and in some embodiments, the dimer is a heterodimer.

[0171] In some embodiments, if the C-terminus of the IL-23p19-targeting immunoglobulin monovariable domain according to the present invention is not ligated to another domain (e.g., an Fc domain), the C-terminus of the immunoglobulin monovariable domain may be modified to reduce the immunogenicity of the immunoglobulin monovariable domain by adding 1 to 5 amino acids, for example, a single alanine.

[0172] In some embodiments, multiple immunoglobulin variable domains within the TL1A-binding molecule and / or IL-23p19-binding molecule that specifically bind to the target antigen bind to different regions or different epitopes of the target antigen, respectively.

[0173] In the present invention, the antigen-binding units, the immunoglobulin monovariable domains, and / or the antigen-binding units or immunoglobulin monovariable domains and immunoglobulin Fc domains can be directly linked or indirectly linked via a linker. The linker may be a non-functional amino acid sequence of 1 to 20 amino acids in length or longer that does not have a secondary structure or higher. For example, the linker may be (GS) n (GGS) n (GGGS) n , or (GGGGS) n These are flexible linkers, where n is 1, 2, 3, 4, or 5. In some embodiments, the linker is (GGGGS)3. In some embodiments, the linker is (GGGGS)4.

[0174] In some embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain, the heavy chain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, or 97.

[0175] In some embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin light chain, and the light chain comprises an amino acid sequence represented by SEQ ID NO: 84, 86, 88, 90, 92, 94, 96 or 98, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence represented by SEQ ID NO: 84, 86, 88, 90, 92, 94, 96 or 98.

[0176] In some embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain and an immunoglobulin light chain, 1) the immunoglobulin heavy chain comprises the amino acid sequence represented by SEQ ID NO: 83, and the immunoglobulin light chain comprises the amino acid sequence represented by SEQ ID NO: 84; 2) the immunoglobulin heavy chain comprises the amino acid sequence represented by SEQ ID NO: 85, and the immunoglobulin light chain comprises the amino acid sequence represented by SEQ ID NO: 86; 3) the immunoglobulin heavy chain comprises the amino acid sequence represented by SEQ ID NO: 87, and the immunoglobulin light chain comprises the amino acid sequence represented by SEQ ID NO: 88; 4) the immunoglobulin heavy chain comprises the amino acid sequence represented by SEQ ID NO: 89, and the immunoglobulin light chain comprises the amino acid sequence represented by SEQ ID NO: 90; 5) the immunoglobulin heavy chain comprises the amino acid sequence represented by SEQ ID NO: 91, and the immunoglobulin light chain comprises the amino acid sequence represented by SEQ ID NO: 92; 6) the immunoglobulin heavy chain comprises the amino acid sequence represented by SEQ ID NO: 93, and the immunoglobulin light chain comprises the amino acid sequence represented by SEQ ID NO: 94; 7) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 95, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 96, or 8) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 97, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 98.

[0177] In some preferred embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain and an immunoglobulin light chain. 1) The immunoglobulin heavy chain contains the amino acid sequence shown in SEQ ID NO: 87, and the immunoglobulin light chain contains the amino acid sequence shown in SEQ ID NO: 88, or 2) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 91, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO: 92.

[0178] In some embodiments, the IL-23p19 binding molecule includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in any of SEQ ID NOs: 101 to 113, and that retains specific binding to IL-23p19.

[0179] In some preferred embodiments, the IL-23p19 binding molecule includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NOs: 101, 103, 105, 106, 107, 109, 110, 111, or 112, and that retains specific binding to IL-23p19. In some more preferred embodiments, the IL-23p19 binding molecule includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 103, 109, 110, 111, or 112, and that retains specific binding to IL-23p19.In some more preferred embodiments, the IL-23p19 binding molecule includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 111, and that retains specific binding to IL-23p19. Multispecific antigen binding molecules

[0180] In another embodiment, the present invention further provides a multispecific antigen-binding molecule, the multispecific antigen-binding molecule comprising a TL1A-binding molecule and / or an IL-23p19-binding molecule according to the present invention.

[0181] The term "multispecificity" refers to the ability of an antigen-binding molecule to specifically bind to multiple different target antigens or epitopes. For example, "bispecificity" refers to the ability of an antigen-binding molecule to specifically bind to two different target antigens or epitopes.

[0182] In some embodiments, the multispecific antigen-binding molecule further comprises one or more additional antigen-binding functional regions in addition to the TL1A-binding molecule and / or IL-23p19-binding molecule, wherein the one or more additional antigen-binding functional regions bind to an antigen different from the TL1A-binding molecule and / or IL-23p19-binding molecule, or to a different epitope of the same antigen. Bispecific antigen binding molecules

[0183] In another embodiment, the present invention further provides a TL1A × IL-23p19 bispecific antigen-binding molecule that targets both TL1A and IL-23p19 and can inhibit or block their signaling.

[0184] In some embodiments, the bispecific antigen-binding molecule includes the TL1A-binding molecule and the IL-23p19-binding molecule according to the present invention.

[0185] In some embodiments, the bispecific antigen-binding molecule comprises a first antigen-binding functional region and a second antigen-binding functional region, wherein the first antigen-binding functional region comprises at least one antigen-binding unit targeting TL1A, and the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 6. 2) The VH comprises the amino acid sequence HCDR1 shown in SEQ ID NO: 9, the amino acid sequence HCDR2 shown in SEQ ID NO: 10, and the amino acid sequence HCDR3 shown in SEQ ID NO: 11, and the VL comprises the amino acid sequence LCDR1 shown in SEQ ID NO: 12, the amino acid sequence LCDR2 shown in SEQ ID NO: 5, and the amino acid sequence LCDR3 shown in SEQ ID NO: 13. 3) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 19. 4) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 23. 5) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29. 6) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 32, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 33. 7) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 37, or 8) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 40, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 41. Furthermore, the second antigen-binding functional region includes at least one immunoglobulin monovariate domain that specifically binds to IL-23p19, and the immunoglobulin monovariate domain that specifically binds to IL-23p19 is 1) CDR1 of the amino acid sequence shown in SEQ ID NO: 44, CDR2 of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 of the amino acid sequence shown in SEQ ID NO: 46 2) CDR1 of the amino acid sequence shown in SEQ ID NO: 48, CDR2 of the amino acid sequence shown in SEQ ID NO: 49, and CDR3 of the amino acid sequence shown in SEQ ID NO: 50 3) CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54 4) CDR1 of the amino acid sequence shown in SEQ ID NO: 56, CDR2 of the amino acid sequence shown in SEQ ID NO: 57, and CDR3 of the amino acid sequence shown in SEQ ID NO: 58 5) CDR1 of the amino acid sequence shown in SEQ ID NO: 60, CDR2 of the amino acid sequence shown in SEQ ID NO: 61, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 6) CDR1 of the amino acid sequence shown in SEQ ID NO: 64, CDR2 of the amino acid sequence shown in SEQ ID NO: 65, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 7) CDR1 of the amino acid sequence shown in SEQ ID NO: 67, CDR2 of the amino acid sequence shown in SEQ ID NO: 68, and CDR3 of the amino acid sequence shown in SEQ ID NO: 69, or 8) Includes CDR1 of the amino acid sequence shown in SEQ ID NO: 71, CDR2 of the amino acid sequence shown in SEQ ID NO: 72, and CDR3 of the amino acid sequence shown in SEQ ID NO: 73.

[0186] In some embodiments, the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO: 8. 2) The VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15. 3) The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO: 21. 4) The VH contains the amino acid sequence shown in SEQ ID NO: 24, and the VL contains the amino acid sequence shown in SEQ ID NO: 25. 5) The VH contains the amino acid sequence shown in SEQ ID NO: 30, and the VL contains the amino acid sequence shown in SEQ ID NO: 31. 6) The VH contains the amino acid sequence shown in SEQ ID NO: 34, and the VL contains the amino acid sequence shown in SEQ ID NO: 35. 7) The VH contains the amino acid sequence shown in SEQ ID NO: 38, and the VL contains the amino acid sequence shown in SEQ ID NO: 39. 8) The VH contains the amino acid sequence shown in SEQ ID NO: 42, and the VL contains the amino acid sequence shown in SEQ ID NO: 43, or contains an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the above amino acid sequences and retaining specific binding to TL1A. Furthermore, the immunoglobulin single variable domain that specifically binds to the IL-23p19 contains the amino acid sequence shown in SEQ ID NO: 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78 or 79, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least �3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78 or 79 and retaining specific binding to IL-23p19.

[0187] In some preferred embodiments, VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29.

[0188] In some preferred embodiments, VH comprises the amino acid sequence shown in SEQ ID NO: 30, and VL comprises the amino acid sequence shown in SEQ ID NO: 31, or comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 30 or 31, and retaining specific binding to TL1A.

[0189] In some preferred embodiments, the immunoglobulin monovariate domain that specifically binds to IL-23p19 includes CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54.

[0190] In some preferred embodiments, the immunoglobulin monovariate domain that specifically binds to IL-23p19 comprises the amino acid sequence shown in SEQ ID NO: 77, or comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 77, and that retains specific binding to IL-23p19.

[0191] In some more preferred embodiments, the VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 29, and furthermore, the immunoglobulin single variable domain comprises CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54.

[0192] In some more preferred embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 31, or has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 30 or 31, and has specific binding with TL1A. The immunoglobulin monovariable domain comprises an amino acid sequence that maintains a specific binding with IL-23p19, and further comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence that maintains a specific binding with IL-23p19.

[0193] In some embodiments, the immunoglobulin monovariate domain is VHH, for example, VHH derived from a camelid, preferably an alpaca or llama. In some preferred embodiments, the immunoglobulin monovariate domain that specifically binds to IL-23p19 is humanized VHH.

[0194] In some embodiments, the antigen-binding unit targeting TL1A is F(ab), F(ab'), F(ab')2, scFab, Fd, Fv, dsFv, dAb, double-chain antibody (diabody), or scFv.

[0195] In some preferred embodiments, the antigen-binding unit targeting TL1A is F(ab), and the VH is linked to the immunoglobulin CH1 domain, and the VL is linked to the constant domain of the immunoglobulin light chain.

[0196] In some embodiments, the immunoglobulin CH1 domain is a human immunoglobulin CH1 domain, preferably a CH1 domain derived from human IgG, more preferably a CH1 domain derived from human IgG1 or IgG4, and / or the immunoglobulin light chain constant domain is derived from a human λ light chain constant domain or a κ light chain constant domain.

[0197] In some embodiments, the immunoglobulin CH1 domain sequence is an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 117. The immunoglobulin light chain constant domain sequence includes, and / or, the amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO: 82.

[0198] In some embodiments, the first antigen-binding functional region of the TL1A×IL-23p19 bispecific antigen-binding molecule according to the present invention comprises a plurality of antigen-binding units that specifically bind to TL1A, and / or the second antigen-binding functional region comprises a plurality of immunoglobulin monovariable domains that specifically bind to IL-23p19, wherein the plurality of immunoglobulin monovariable domains can be directly or indirectly linked.

[0199] In some embodiments, the first antigen-binding functional region and the second antigen-binding functional region are each independently one, two, or two or more.

[0200] In some embodiments, the TL1A×IL-23p19 bispecific antigen-binding molecule according to the present invention further comprises an immunoglobulin Fc domain in addition to the antigen-binding functional domain. The Fc domain that may be used in the present invention may be derived from various subtypes of immunoglobulins, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM. The immunoglobulin Fc domain is preferably a human immunoglobulin Fc domain, e.g., the Fc domain of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the TL1A×IL-23p19 bispecific antigen-binding molecule according to this specification comprises an Fc domain derived from human IgG1 (its amino acid sequence is shown in SEQ ID NO: 99). In some embodiments, the TL1A×IL-23p19 bispecific antigen-binding molecule according to this specification comprises an Fc domain variant derived from human IgG1. In some embodiments, the Fc domain variant derived from human IgG1 is C 220 A, L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 It includes one or more mutations selected from del. In some embodiments, the Fc domain variant derived from human IgG1 is L 234 A, L 235Includes A mutation. In some embodiments, the Fc domain variant derived from human IgG1 is C 220 A, L 234 A and L 235 Includes A mutation. In some embodiments, the Fc domain variant derived from human IgG1 is L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 This includes del mutations. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc domain variant is, for example, represented by SEQ ID NOs. 100, 118, or 119.

[0201] In some embodiments, in the TL1A × IL-23p19 bispecific antigen-binding molecule according to the present invention, the immunoglobulin Fc domain (e.g., the Fc domain of human IgG1) can be directly or indirectly linked to the C-terminus and / or N-terminus of the first antigen-binding functional region and / or the second antigen-binding functional region via a hinge region or linker. The inclusion of the immunoglobulin Fc domain in the TL1A-binding molecule and / or IL-23p19-binding molecule according to the present invention allows the binding molecule to form a dimer molecule. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0202] In some specific embodiments, the TL1A×IL-23p19 bispecific antigen-binding molecule comprises an immunoglobulin heavy chain from the N-terminus to the C-terminus, comprising the VH, immunoglobulin CH1 domain, Fc domain, optional linker, and immunoglobulin monovariable domain, and an immunoglobulin light chain from the N-terminus to the C-terminus, comprising the VL and immunoglobulin light chain constant region.

[0203] In some embodiments, if the C-terminus of the IL-23p19-targeting immunoglobulin monovariable domain according to the present invention is not ligated to another domain (e.g., an Fc domain), the C-terminus of the immunoglobulin monovariable domain may be modified to reduce the immunogenicity of the immunoglobulin monovariable domain by adding 1 to 5 amino acids, for example, a single alanine.

[0204] The antigen-binding units, immunoglobulin monovariable domains, antigen-binding functional regions, and / or the antigen-binding units, immunoglobulin monovariable domains, antigen-binding functional regions, and immunoglobulin Fc domains of the present invention may be directly linked or indirectly linked via linkers. The linkers may be non-functional amino acid sequences of 1 to 20 amino acids in length or longer that do not have a secondary structure or higher. For example, the linkers may be (GS) n (GGS) n (GGGS) n , or (GGGGS) n These are flexible linkers, where n is 1, 2, 3, 4, or 5. In some embodiments, the linker is (GGGGS)3. In some embodiments, the linker is (GGGGS)4.

[0205] In some embodiments, the TL1A×IL-23p19 bispecific antigen-binding molecule comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the immunoglobulin heavy chain comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or less of the amino acid sequence shown in SEQ ID NO: 115 or 116. The immunoglobulin light chain contains an amino acid sequence having at least 99% identity, and further, the immunoglobulin light chain contains an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 92, and that maintains specific binding to TL1A and IL-23p19. Fusion protein

[0206] In another embodiment, the present invention further provides a fusion protein comprising a TL1A binding molecule, an IL-23p19 binding molecule, a multispecific antigen binding molecule, and / or a bispecific antigen binding molecule as described herein, and a polypeptide or protein expressed in fusion with the same.

[0207] In some embodiments, the fusion protein further comprises one or more other bioactive proteins in addition to the TL1A binding molecule, the IL-23p19 binding molecule, the multispecific antigen binding molecule, and / or the bispecific antigen binding molecule. The bioactive protein may be any protein having biological, therapeutic, prophylactic, or diagnostic significance or function that, when administered to a subject, mediates biological activity for preventing or mitigating a disease, disorder, or pathology. Specifically, the bioactive protein may be an agonist, antagonist, regulator, ligand, cytokine, enzyme, or hormone, and may be a bioactive protein that can be used in particular for the treatment, prevention, and / or mitigation of inflammatory diseases. Conjugate

[0208] In another embodiment, the present invention provides conjugates comprising TL1A-binding molecules, IL-23p19-binding molecules, multispecific antigen-binding molecules, bispecific antigen-binding molecules, and / or fusion proteins described herein, and conjugates conjugated thereto.

[0209] In this context, the term “conjugate” refers to any method known in the art for linking functional protein domains, including, but not limited to, recombinant fusion, intein-mediated fusion, non-covalent bonding, and covalent bonding such as disulfide bonds, peptide bonds, hydrogen bonds, electrostatic bonds, and conformational bonds such as biotin-avidin bonds, with or without linkers. In some embodiments, conjugation with an effector may be carried out by chemical or recombinant methods, the chemical method of forming a covalent bond between two molecules to form a single molecule.

[0210] In this context, the term “conjugate” refers to a component or functional group that can modulate (e.g., increase or decrease) the intrinsic activity of a molecule to which it is conjugated, or confer novel activity to that molecule. In some embodiments, the effector is a biologically active compound or polypeptide, or a non-protein module such as a detectable label or a water-soluble polymer.

[0211] In some embodiments, the conjugate may be a therapeutic portion, which refers to a compound or polypeptide that can be used as a therapeutic agent. In some embodiments, the therapeutic portion includes a therapeutic agent or drug for treating inflammatory diseases and / or autoimmune diseases. In some embodiments, the conjugate portion includes immunosuppressants, cytokine inhibitors, metabolic inhibitors, antioxidants, corticosteroids, and the like.

[0212] In some embodiments, the conjugate may be a detectable label. The detectable label described in the present invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase), radionuclides (e.g., 3H), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC)), luminescent substances (e.g., acridine ester compounds), magnetic beads, and biotin (e.g., streptavidin) for binding to avidin modified with the above label.

[0213] In some embodiments, the conjugate portion may be a water-soluble polymer that helps extend the half-life of the antibody. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone. The polymer can have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody varies, and if multiple polymers are attached, they may be the same molecule or different molecules.

[0214] In some embodiments, the antigen-binding molecules described herein are used as the base of the conjugate. antigen-binding molecule variant

[0215] In some embodiments, the antigen-binding molecules described herein also include variants of the amino acid sequences described herein.

[0216] In some embodiments, the variant comprises one or more CDR sequences described herein and / or one or more non-CDR sequences of an immunoglobulin light chain variable domain, an immunoglobulin heavy chain variable domain, or an immunoglobulin single variable domain, and / or one or more amino acid modifications or substitutions of a constant region (e.g., an Fc domain). These variants retain the binding specificity of the parent antibody but possess one or more desired properties conferred by the modifications or substitutions. For example, the antibody variant may have improved antigen-binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination activity, reduced or eliminated effector function, improved FcRn receptor binding, improved pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugates (e.g., one or more introduced cysteine ​​residues).

[0217] The nucleotide sequence encoding the antibody can be modified using methods known in the art. Alternatively, amino acid sequence variants of the antibody can be produced by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, as long as the final construct has the desired properties. Variant substitution, insertion, and deletion

[0218] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites targeted for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are as described above. By introducing amino acid substitutions into the target antibody, the resulting product can be screened for desired activities such as retention / improvement of antigen binding, reduction of immunogenicity, and improvement of ADCC or CDC.

[0219] Some substitution variants involve substituting one or more complementarity-determining region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, variants resulting from selection for further study have certain changes (e.g., improvements) in biological properties compared to the parent antibody (e.g., increased affinity, decreased immunogenicity), and / or substantially retain some of the biological properties of the parent antibody. For example, exemplary substitution variants are affinity-mature antibodies, which can be readily generated using affinity-mature techniques based on phage display, such as those described herein. In short, one or more CDR residues are mutated, the variant antibody is displayed on a phage, and its specific biological activity (e.g., binding affinity) is screened.

[0220] For example, to improve antibody affinity, changes (e.g., substitutions) can be made to the CDR. Such changes can be made to "hot spots" in the CDR, i.e., residues encoded by codons that frequently mutate during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that come into contact with the antigen, where the binding affinity to the resulting variant VH or VL is tested. Affinity maturation by secondary library construction and reselection is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1_37 (O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation using various methods (e.g., error-prone PCR, strand shuffling, oligonucleotide-induced mutagenesis, etc.). A secondary library is then constructed. Next, the library is screened to identify any antibody variant with the desired affinity. Another method for introducing diversity is CDR induction, which randomizes multiple CDR residues (e.g., 4-6 residues at a time). CDR residues involved in antigen binding can be specifically identified using methods such as alanine scan mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0221] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that these changes do not significantly reduce the antibody's ability to bind to the antigen. For example, conservative changes that do not significantly reduce binding affinity (e.g., conservative substitutions as described herein) may be made to the CDR. For example, such changes may occur at residues other than those in contact with the antigen within the CDR.

[0222] Furthermore, in some embodiments, the antibodies disclosed herein (e.g., anti-TL1A antibodies, anti-IL-23p19 antibodies, and multispecific antibodies based thereon) may include conservative substitutions or modifications of amino acids in heavy chain variable domains, light chain variable domains, and / or immunoglobulin monovariate domains. In this field, it is understood that certain conservative sequence modifications can be performed without excluding antigen binding (see, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot.Eng.10:835-41; Komissarov et al. (1997) J.Biol.Chem. 272:26864-26870; Hall et al. (1992) J.Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int.Immunol. 10:341-6, and Beers et al. (2000) Clin.Can.Res. 6:2835-43).

[0223] In some embodiments, the heavy chain variable domain, light chain variable domain, and / or immunoglobulin single variable domain of the antigen-binding molecule relating to this specification include substitutions, additions, and / or deletions of one or more amino acid residues in one or more CDR sequences and / or one or more FR sequences. In some embodiments, the variant includes a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or one or fewer substitutions, additions, and / or deletions in the CDR sequence and / or FR sequence. Glycosylated variant

[0224] In some embodiments, the antibodies according to this specification have been modified to increase or decrease the degree of glycosylation of the antibody. Adding or deleting glycosylation sites on an antibody can be easily achieved by modifying the amino acid sequence to generate or delete one or more glycosylation sites.

[0225] If an antibody contains an Fc domain, the attached carbohydrate can be modified. Natural antibodies produced from mammalian cells typically contain branched oligosaccharides, which are generally linked to Asn297 of the CH2 domain of the Fc domain via an N-bond. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, shearic acid, and fucose of GlcNAc attached to the "backbone" of the branched oligosaccharide structure. In some embodiments, modifying the oligosaccharide in the antibody of the present invention can produce antibody variants with improved specific properties. Cysteine-modified antibody variant

[0226] In some embodiments, it may be desirable to produce cysteine-modified antibodies such as "thioMAb," in which case one or more residues of the antibody are substituted with cysteine ​​residues. In specific embodiments, the substituted residues are located at accessible sites on the antibody. By substituting these residues with cysteine, a reactive thiol group is positioned at an accessible site on the antibody, and the antibody can be used to produce an immunoconjugate by conjugating it with other modules, such as a drug module or a linker drug module, as further described herein. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541. Fc domain variant

[0227] In some embodiments, Fc domain variants can be generated by introducing one or more amino acid modifications to the Fc domain and / or hinge region of the antibody according to this specification. The Fc domain variant may include a human Fc domain sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc domain) with one or more amino acid modifications (e.g., substitutions) at one or more amino acid positions for purposes such as extending the half-life of the antigen-binding molecule, reducing the effector function of the antigen-binding molecule, promoting polymerization of a bispecific antibody, and / or improving the manufacturing stability and pharmacovigilance of the bispecific antibody.

[0228] In some embodiments, the present invention encompasses antibody variants having some, but not all, effector functions, which are desirable candidates for applications where the in vivo half-life of the antibody is critical and certain effector functions (e.g., CDC and ADCC) are unnecessary or detrimental.

[0229] In some embodiments, where effector function is not required, the antibodies disclosed herein may be further manipulated to introduce at least one mutation into the antibody Fc domain that reduces antibody binding to the activated Fcγ receptor (FcγR) and / or reduces Fc effector function (such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP)).

[0230] The locations of the Fc domain where mutations can occur that reduce antibody binding to activated FcγR, and consequently reduce effector function, are described in the following literature: Xu, Alegre et al. (2000) (Vafa, Gilliland et al. (2014) (Bolt, Routledge et al. (1993) (Chu, Vostia et al. (2008) (Shields, Namenuk et al. (2001)). Fc mutations that minimize ADCC, ADCP, CDC, and Fc-mediated cell activation have also been described as σ mutations in IgG1, IgG2, and IgG4 (Tam, McCarthy et al. (2017)).

[0231] Exemplary mutations that can be performed singly or in combination are the K of IgG1, IgG2, IgG3 or IgG4 214 T, E 233 P, L 234 V, L 234 A, G 236 Deletion, V 234 A, F 234 A, L 235 A, G 237 A, P2 38 A, P 238 S, D 265 A, S 267 E, H 268 A, H 268 Q, Q 268 A, N​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​327 G / P 331 A / D 365 E / L 358 M, IgG2 H 268 Q / V 309 L / A 330 S / P 331 S, IgG1 S 267 E / L 328 F, IgG1 L 234 F / L 235 Ending 265 A, IgG1 L 234 A / L 235 A / G 237 A / P 238 S / H 268 A / A 330 S / P 331 S, IgG4 S 228 P / F 234 A / L 235 A / G 237 A / P 238 S, and S of IgG4 228 P / F 234 A / L 235 A / G 236 Missing / G 237 A / P 238 It is S. A hybrid IgG2 / 4Fc domain, for example, an Fc (EU numbered) having residues 117-260 from IgG2 and residues 261-447 from IgG4 can also be used.

[0233] In some embodiments, the present invention encompasses modifications of naturally occurring Fc regions to extend the half-life of an antibody compared to a native antibody in a biological environment, such as a serum half-life or half-life measured by an in vitro assay.

[0234] Exemplary mutations that can occur individually or in combination include: T 250 Q, M 252 Y, I 253 A, S 254 T, T 256 E, P 257 I, T 307 A, D 376 V, E 380 A, M 428 L, H 433 K, N434 S, N 434 A, N 434 H, N 434 F, H 435 A and H 435 This is an R (EU numbering) mutation.

[0235] An exemplary combination mutation that can extend the half-life is M in IgG1. 252 Y / S 254 T / T 256 E, M 428 L / N 434 S, T 250 Q / M 428 L, N 434 A, T 307 A / E 380 A / N 434 It is A (EU numbering).

[0236] In some embodiments, to avoid light chain mismatches, the present invention also includes mutations of one or more amino acids in the hinge region. An example of such mutation is, for example, C in the IgG1 hinge region (EU numbering retrospectively). 220 This includes the A mutation.

[0237] In some embodiments, to avoid protease digestion, the present invention provides an Fc domain variant in which glycine and / or lysine at the C-terminus of the Fc domain is deleted (in the case of EU numbering, G 446 del and / or K 447 It also includes del.

[0238] In some embodiments, the present invention encompasses assisting and / or promoting heterodimerization of the Fc domain by including one or more amino acid substitutions at the interface of the Fc region. These modifications include introducing protrusions to the first Fc polypeptide and cavities to the second Fc polypeptide. By positioning these protrusions within the cavities, the interaction between the first and second Fc polypeptides is promoted, forming a heterodimer or complex. Methods for producing antibodies having these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0239] In some embodiments, the antigen-binding molecule relating to this specification has an IgG1 isotype and includes one or more mutations in its Fc domain selected from:C 220 A, L 234 A, L 235 A, M 252 Y, S 254 T, T 256 E and K 447 del, or any combination thereof. In some embodiments, the antigen-binding molecule according to this specification has the IgG1 isotype and L 234 A / L 235 Includes A(LALA) amino acid substitution. In some embodiments, the antigen-binding molecule according to this specification has an IgG1 isotype and C 220 A and L 234 A / L 235 Includes A(LALA) amino acid substitution. In some embodiments, the antigen-binding molecule according to this specification has an IgG1 isotype and M 252 Y / S 254 T / T 256 Includes E(YTE) amino acid substitution. In some embodiments, the antigen-binding molecule according to this specification has the IgG1 isotype and L 234 A / L 235 A(LALA), M 252 Y / S 254 T / T 256 E(YTE) and K 447This includes del amino acid substitutions. In some embodiments, the antigen-binding molecules according to this specification include an amino acid sequence of the Fc domain represented by SEQ ID NOs. 99, 100, 118, or 119, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence represented by SEQ ID NOs. nucleic acid molecules, vectors, host cells

[0240] In another embodiment, the present invention also relates to nucleic acid molecules encoding TL1A-binding molecules, IL-23p19-binding molecules, multispecific antigen-binding molecules, bispecific antigen-binding molecules, and / or fusion proteins as described in the present invention.

[0241] The nucleic acid molecule may be, for example, DNA, RNA, or a hybrid thereof, or it may contain (e.g., chemically) modified nucleotides such as PNA. The nucleic acid molecule may be single-stranded or double-stranded. In some embodiments, the nucleic acid molecule is in the form of double-stranded DNA. For example, the nucleic acid molecule of the present invention may be genomic DNA or cDNA. In some embodiments, the nucleic acid molecule of the present invention is an isolated nucleic acid molecule.

[0242] The nucleic acid molecules of the present invention may be in the form of a vector, present within a vector such as a plasmid, cosmid, or YAC, and / or part of a vector. The vector may be an expression vector in particular, i.e., a vector for expressing a TL1A-binding molecule, an IL-23p19-binding molecule, a multispecific antigen-binding molecule, a bispecific antigen-binding molecule, and / or a fusion protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). This expression vector typically contains at least one nucleic acid of the present invention, which is operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). The selection of these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the TL1A-binding molecule, IL-23p19-binding molecule, multispecific antigen-binding molecule, bispecific antigen-binding molecule, and / or fusion protein of the present invention include, for example, promoters, enhancers, terminators, integrators, selection markers, reader sequences, and reporter genes.

[0243] The nucleic acids of the present invention can be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA technology) and / or isolated from suitable natural sources based on information relating to the amino acid sequences of the polypeptides of the present invention as described herein.

[0244] In another aspect, the present invention relates to a host cell that expresses, or can express, one or more of the TL1A-binding molecule, IL-23p19-binding molecule, multispecific antigen-binding molecule, bispecific antigen-binding molecule, fusion protein, and / or nucleic acids or vectors comprising the present invention. Suitable host cells or host organisms will be apparent to those skilled in the art, and preferred host cells in the present invention are bacterial cells, fungal cells, or mammalian cells.

[0245] Suitable bacterial cells include Gram-negative bacterial strains (e.g., Escherichia coli, Proteus, and Pseudomonas) as well as Gram-positive bacterial strains (e.g., Bacillus, Streptomyces, Staphylococcus, and Lactococcus).

[0246] Suitable fungal cells include those of the genera Trichoderma, Neurospora, and Aspergillus, or those of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0247] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, and COS cells.

[0248] However, the present invention may also use known cells such as amphibian cells, insect cells, and any other cells in the art used to express heterologous proteins.

[0249] The production of the TL1A-binding molecule, IL-23p19-binding molecule, multispecific antigen-binding molecule, bispecific antigen-binding molecule, and / or fusion protein of the present invention can be achieved by transforming / transfecting host cells with a nucleic acid molecule encoding the TL1A-binding molecule, IL-23p19-binding molecule, multispecific antigen-binding molecule, bispecific antigen-binding molecule, and / or fusion protein, or a vector containing the nucleic acid molecule, culturing the host cells under conditions suitable for the expression of the nucleic acid molecule or the vector, and optionally isolating and / or purifying the host cells or the host cell culture once or multiple times.

[0250] Methods and reagents used for production purposes, such as specific suitable expression vectors, transformation or transfection methods, selection markers, protein expression induction methods, and culture conditions, are known in the art. Similarly, techniques for the isolation and purification of proteins in methods applied to the production of the TL1A-binding molecule, IL-23p19-binding molecule, multispecific antigen-binding molecule, bispecific antigen-binding molecule, and / or fusion protein of the present invention are also known to those skilled in the art. composition

[0251] In some embodiments, the present invention relates to compositions comprising at least one of the TL1A binding molecules, IL-23p19 binding molecules, multispecific antigen binding molecules, bispecific antigen binding molecules, fusion proteins, and / or conjugates disclosed herein.

[0252] In some embodiments, the composition is a pharmaceutical composition, which further comprises at least one pharmaceutically acceptable carrier and / or excipient.

[0253] In some embodiments, the pharmaceutical composition may optionally contain one or more other therapeutic agents having pharmaceutically active properties, such as another therapeutic agent or drug that can be used to treat inflammatory diseases and / or autoimmune diseases.

[0254] Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous or non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspensions / dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, and various other components or combinations thereof known in the art.

[0255] In some embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can be manufactured in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for preparing a liquid solution, suspension, or emulsion. Injectable formulations include: immediately injectable sterile and / or pyrogen-free solutions; sterile, dried, soluble products such as lyophilized powders containing subcutaneous tablets to be combined with a solvent immediately before use; immediately injectable sterile suspensions; sterile, dried, insoluble products to be combined with a solvent immediately before use; and sterile and / or pyrogen-free emulsions. The solutions may be aqueous or non-aqueous.

[0256] In some embodiments, the unit dose of the parenteral formulation is packaged in an ampoule, vial, or syringe with a needle. All formulations intended for parenteral administration must be sterile and pyrogen-free, as is known and practiced in the art.

[0257] In some embodiments, formulations suitable for parenteral administration (e.g., injection) include aqueous or nonaqueous, isotonic, pyrogen-free, and sterile liquids (e.g., solutions, suspensions) in which the active ingredient is provided by dissolution, suspension, or other means (e.g., liposomes or other microparticles). These liquids may further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, antimicrobial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the subject's blood (or other relevant body fluids). Examples of excipients include, for example, water, alcohol, polyols, glycerol, and vegetable oils. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or Ringer's lactate injection.

[0258] In some embodiments, sterile lyophilized powders are prepared by dissolving the antigen-binding molecules disclosed herein in a suitable solvent. The solvent may contain excipients or other pharmacological components that improve the stability of the powder or the reconstituted solution prepared from the powder. Possible excipients include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable reagents. The solvent may contain buffers such as citrate, sodium phosphate, potassium phosphate, or other buffers known to those skilled in the art. In one embodiment, the buffer has a nearly neutral pH. This solution is then sterile filtered under standard conditions known to those skilled in the art and subsequently lyophilized to obtain the desired formulation. In one embodiment, the obtained solution is dispensed into vials and lyophilized. Each vial may contain a single dose or multiple doses of the antigen-binding molecules or compositions described herein. To facilitate accurate sample extraction and accurate dosing, it is permissible to overfill the vials by a small amount (e.g., about 10%) beyond the amount required for the single dose or multiple doses. Freeze-dried powder can be stored under appropriate conditions, such as from approximately 4°C to room temperature.

[0259] A formulation for parenteral administration can be obtained by reconstituting the lyophilized powder with sterile water for injection. In one embodiment, sterile and / or pyrogen-free water, or other suitable liquid carrier, is added to the lyophilized powder for reconstitution. The exact amount depends on the chosen treatment and can be determined empirically. Purpose

[0260] The TL1A-binding molecules, IL-23p19-binding molecules, multispecific antigen-binding molecules, bispecific antigen-binding molecules, fusion proteins, conjugates, and / or compositions according to the present invention have a variety of applications in vitro and in vivo. For example, by contacting cultured cells with therapeutically effective amounts of these molecules in vitro or ex vivo, or by administering them to human subjects in vivo, it is possible to treat, prevent, or alleviate inflammatory diseases in subjects, inhibit or block the specific binding of TL1A to DR3 and / or the specific binding of IL-23p19 to the IL-23R and IL-12Rβ1 complex, or inhibit the activation of downstream signaling pathways mediated by TL1A and / or IL-23p19.

[0261] In another aspect, the present invention also provides the use of TL1A-binding molecules, IL-23p19-binding molecules, multispecific antigen-binding molecules, bispecific antigen-binding molecules, fusion proteins, conjugates, and / or compositions according to the present invention in the manufacture of drugs for treating, preventing, and / or alleviating inflammatory diseases and / or autoimmune diseases.

[0262] In some embodiments, the use includes, for example, concomitant administration with other therapeutic agents or drugs for treating inflammatory diseases and / or autoimmune diseases. When administered concomitantly with other therapeutic agents or drugs, they may be administered in any order or simultaneously. When administered sequentially, appropriate intervals may be maintained to maximize the therapeutic effect.

[0263] Preferred subjects include patients suffering from inflammatory diseases and / or autoimmune diseases. The method is particularly suitable for treating, preventing, and / or alleviating inflammatory diseases and / or autoimmune diseases in human patients by inhibiting the activation of downstream signaling pathways mediated by TL1A and / or IL-23p19, by blocking the specific binding of TL1A to DR3 and / or the specific binding of IL-23p19 to the complex of IL-23R and IL-12Rβ1. In a specific embodiment, the method is suitable for treating, preventing, and / or alleviating inflammatory diseases and / or autoimmune diseases in vivo, such as asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, or psoriasis. In particular, the method is particularly suitable for Crohn's disease or ulcerative colitis.

[0264] TL1A-binding molecules, IL-23p19-binding molecules, multispecific antigen-binding molecules, bispecific antigen-binding molecules, fusion proteins, conjugates, and / or compositions can be administered to a subject by any suitable route of administration. Those skilled in the art will understand that the route of administration and / or method of administration will vary depending on the desired outcome. The suitable routes of administration preferably include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes such as injection or infusion. As used herein, the term “parenteral administration” refers to methods of administration other than enteral and topical administration, which are usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intra-articular, intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, sub-articular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0265] Those skilled in the art will understand that the appropriate dose may vary from patient to patient. Determining the optimal dose usually requires balancing the level of therapeutic effect with the risks or side effects. In some embodiments, the antigen-binding molecules described herein may be administered in therapeutically effective doses ranging from about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the dose regimen may be adjusted to obtain the optimal desired response (e.g., therapeutic response). For example, it may be administered as a single dose or in divided doses over time. If multiple doses are administered, they may be given at appropriate intervals to maximize the therapeutic effect. In some embodiments, the dose may be changed during treatment in response to the subject's response. Sequence List

[0266] This application includes a sequence listing containing numerous amino acid sequences. Table A below provides an overview of the amino acid sequences included therein.

[0267] [Table A(1)] [Table A(2)] [Table A(3)] [Table A(4)] [Table A(5)] [Table A(6)] [Table A(7)] [Table A(8)] [Table A(9)] [Table A(10)] [Table A(11)] [Table A(12)] [Table A(13)] [Table A(14)] [Table A(15)] Examples

[0268] The present invention, as outlined herein, can be more readily understood by referring to the following examples. Those skilled in the art can appropriately modify process parameters in implementation by referring to the contents of this specification. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be within the scope of the protection of the present invention. The following examples are provided for illustrative purposes only and are not intended to limit the present invention. Furthermore, these examples are not intended to indicate that the following experiments are all or only experiments in which they were performed.

[0269] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Example 1: Screening of anti-TL1A antibodies

[0270] TL1A antigen (TL1A-his-avi, manufactured by Beijing Baipusais Co., Ltd., TLA-H52Q1, trimer form) was loaded onto streptavidin-conjugated magnetic beads (Thermo Scientific, 88817), and a full-human Fab phage display library was panned. The panning process and results are shown in Table 1. After three panning cycles, the phages eluted from the TL1A antigen group were significantly enriched compared to the control group, with an enrichment ratio of 27.1 times.

[0271] [Table 1] Example 2: Binding screening of crude extracts of periplasmic lumen anti-TL1A-Fab fragments based on a single clone.

[0272] The concentrated phage elution library obtained by the third panning in Example 1 was used to infect TG1 bacteria in the logarithmic growth phase, and after dilution, was spread onto plates. Then, single clones were picked and inoculated into 96-well deep-well bacterial culture plates to which 1 mL / well of 2×YT medium (containing 1‰ amp) had been added, and incubated overnight at 28°C.

[0273] The following day, the culture plate was centrifuged at 4200 rpm at 4°C for 30 minutes, and the cells were resuspended in 0.3 mL of PBST. Subsequently, three freeze-thaw cycles from -80°C to 37°C were repeated to release the Fab fragment from the periplasmic lumen. After centrifuging again at 4200 rpm at 4°C for 30 minutes, 200 μL of the supernatant was aspirated and transferred to a 96-well plate to prepare a crude extract of single-clonal periplasmic lumen anti-TL1A-Fab fragment.

[0274] Next, the supernatant of the crude extract was subjected to ELISA-bound detection. Wells coated with TL1A-His protein (TLA-H5243, trimer form) were used as measurement wells, and wells coated with unrelated target proteins were used as control wells. As the secondary antibody, HRP (horseradish peroxidase)-conjugated anti-HA tag antibody (100028-MM10-H, Beijing Yiqiao Shenzhou Co., Ltd.) was used. The readings of the measurement wells (OD) were recorded. 450nm Bacterial suspensions corresponding to clones with a value of 1.0 or greater and a control well reading of 0.1 or less were selected, and plasmid extraction and gene sequencing were performed. After converting the sequenced sequences to amino acid sequences, sequence alignment was performed, and duplicate sequences and similar sequences with differences of 2 amino acids or less in the CDR (CDR region defined using the AbM method) were removed, ultimately yielding 30 unique sequences. Example 3: Detection of the blocking function of periplasmic cavity crude extract based on anti-TL1A-Fab fragment

[0275] Human DR3 / TNFRSF25 (His-tagged, Baipusais, TN5-H52H3) was coated onto ELISA well plates at 300 ng / well overnight at 4°C. The following day, the ELISA well plates were blocked at room temperature for 2 hours with 1% BSA (bovine serum albumin). The crude extract was mixed with 0.8 μg / mL TL1A-his-avi in ​​equivolute proportions. The crude extract was serially diluted 1:2 from the stock solution to establish two concentration points: stock solution (1×) and 1:2 (0.5×). The crude extract was incubated with TL1A-his-avi at room temperature for 30 minutes, then transferred to the DR3-coated and blocked well plates and incubated at room temperature for 1 hour. After incubation, the plates were washed, and streptavidin-HRP (Baipusais, STN-NH913) was added at a ratio of 1:3,000 for color development. PRA-023 (sequence referenced from patent US2022 / 0259320A1, initial concentration 100 nM) was used as a positive control, and crude extracts of Fab fragments targeting unrelated targets were used as a negative control. Absorbance values ​​at a wavelength of 450 nm were measured using a microplate reader (Biotek, Synergy H1MF).

[0276] As shown in Figure 1, out of 30 clones with unique sequences, eight clones (numbers 4, 5, 8, 9, 11, 12, 24, and 30) showed a certain level of blocking effect against TL1A-DR3. Example 4: Measurement of anti-TL1A antibody expression and in vitro activity

[0277] Fab fragment sequences corresponding to clones 4, 5, 8, 9, 11, 12, 24, and 30 (their heavy chain variable domain sequences are shown in SEQ ID NOs. 7, 8; SEQ ID NOs. 14, 15; SEQ ID NOs. 20, 21; SEQ ID NOs. 24, 25; SEQ ID NOs. 30, 31; SEQ ID NOs. 34, 35; SEQ ID NOs. 38, 39; SEQ ID NOs. 42, 43, respectively) were ligated to the N-terminus of human IgG1 Fc domains (including L234A / L235A mutations) and transiently transfected into HEK293 cells for expression and purification. The resulting molecules were named HYB1801 through HYB1808, respectively.

[0278] Next, the inhibitory effect of the expressed anti-TL1A antibody on the TL1A-mediated caspase 3 / 7 apoptosis signaling pathway was detected using TF-1 cells. Anti-TL1A antibody samples were serially diluted using RPMI 1640 medium (containing 10% FBS) without GM-CSF to prepare cycloheximide (CHX) at a concentration of 80 μg / mL and TL1A-His (TLA-H5243, trimer form) at a concentration of 40 ng / mL. Subsequently, 30 μL each of the antibody dilution, cycloheximide solution, and TL1A solution were added to a sterile 96-well plate and incubated in a 37°C incubator for 1 hour. Simultaneously, TF-1 cells in the logarithmic growth phase were seeded at a seeding rate of 30,000 cells / 50 μL / well in a 96-well white-walled clear-bottom cell culture plate. The incubated antibody:CHX:TL1A mixture was added to a cell culture plate at 50 μL / well and incubated in an incubator for 6 hours. After incubation, 100 μL / well of Caspase-Glo® 3 / 7 reagent (Promega, G8091) was added, and luminescence detection was performed according to the reagent instructions.

[0279] As shown in Figure 2, HYB1803 and HYB1805 exhibited similar blocking effects to the reference molecule Duvakitug (sequence referenced in patent US2016 / 0333104A1), superior to another reference molecule 1D1 (sequence referenced in patent US9683998B2), and significantly superior to PRA-023. Therefore, HYB1803 and HYB1805 were selected for further study. In previous transient HEK293 transfection and expression purification, the yields and SEC main peak purity of HYB1803 and HYB1805 were 165 mg / L, 99.49%, and 421 mg / L, 100%, respectively, after one-step purification using protein A. Example 5: Measurement of the blocking activity of an anti-TL1A antibody against TL1A-DR3

[0280] ELISA detection of blockade of anti-TL1A antibodies against TL1A-DR3 binding was performed according to the method described in Example 3, using 1D1, PRA-023, and Duvakitug as positive controls.

[0281] As shown in Figure 3, HYB1803 and HYB1805 exhibited similar blocking effects to the reference molecule Duvakitug, and were significantly superior to the other two reference molecules, 1D1 and PRA-023. Example 6: Study of the binding activity of anti-TL1A antibodies against cynomolgus monkey TL1A.

[0282] Cynomolgus monkey TL1A protein (TLA-C5241, manufactured by Baipusais) was coated into ELISA well plates at 200 ng / well, and then serially diluted anti-TL1A antibody was added and incubated. Starting with an initial anti-TL1A antibody concentration of 0.2 μg / mL, four concentration points (0.2, 0.05, 0.0125, and 0.003125 μg / mL) were established, and serial dilutions were performed in a 1:4 ratio. Chromogenic development was performed using an HRP-conjugated anti-human Fc secondary antibody. As shown in Figure 4, HYB1803 and HYB1805 showed strong binding affinity to cynomolgus monkey TL1A, similar to the reference molecule Duvakitug. Example 7: Measurement of the blocking activity of an anti-TL1A antibody against TL1A-DcR3

[0283] DcR3 and DR3 are two receptors for TL1A, both belonging to the TNF receptor superfamily. However, unlike DR3, DcR3 exists in a soluble form in vivo, lacks a transmembrane domain (Yu, KY. et al. (1999) J. Biochem. Chem. 274:13733), does not transmit signals into cells, and acts as a decoy receptor (Wroblewski VJ. et al. (2003) Biochem. Pharmacol. 65:657), playing a negative regulatory role in the TL1A-DR3 signaling pathway. Therefore, the development of therapeutic agents that selectively block TL1A-DR3 is expected to demonstrate superior activity and clinical development potential.

[0284] To detect the blocking effect of anti-TL1A antibody against TL1A-DcR3, DcR3-Fc (Beijing Yiqiao Shenzhou Co., Ltd., 10224-H02B) was coated onto ELISA well plates at 200 ng / well. Serially diluted anti-TL1A antibody was mixed in equivolume with TL1A-His-avi at a concentration of 40 ng / mL and incubated at room temperature for 30 minutes. The mixture was then added to the well plates and incubated with shaking at room temperature for 1 hour. After incubation, the plates were washed and colored using streptavidin-HRP.

[0285] As shown in Figure 5, HYB1803, HYB1805, and the reference molecule Duvakitug did not show complete blocking ability against TL1A-DcR3 even at a concentration of 100 μg / mL, while the reference molecule PRA-023 showed normal blocking ability against TL1A-DcR3, and its EC 50 The value was approximately 0.2 μg / mL, indicating that reference molecule 1D1 exhibits the strongest blocking ability against TL1A-DcR3. Example 8: Affinity (K D ) measurement

[0286] The affinity of HYB1803 and HYB1805 for TL1A protein (TLA-H5243, trimer form) was measured using a Biacore 8K instrument. Test antibodies were captured on a Protein A tip (Cytiva, 29127556) at a flow rate of 10 μL / min, followed by the passage of antigen proteins at different concentrations at a flow rate of 30 μL / min. HBS-EP was used as the running buffer. + The antigen-antibody binding time was 120 seconds, the dissociation time was 450 seconds, and the regeneration buffer was 10 mM pH 1.5 Gly-HCl. After the measurement was completed, the antibody-antigen binding rate constant K was fitted according to the 1:1 binding model using the instrument's built-in software. on , dissociation rate constant K off , and the equilibrium dissociation constant K D The value was calculated. The results are shown in Table 2.

[0287] [Table 2A] Example 9: Detection based on TL1A / CHO cells and DR3 reporter gene cells

[0288] In epithelial cells, membrane-bound TL1A can bind to its functional receptor via intercellular contact and, upon activation, induce enhanced secretion of downstream inflammatory factors, including interferon. Anti-TL1A antibodies designed for the treatment of inflammatory and / or immune disorders need to be validated to block both forms of endogenous TL1A in order to achieve optimal therapeutic efficacy.

[0289] To detect the blocking effect of anti-TL1A antibodies against membrane-bound TL1A, this example used CHO cells that stably express human TL1A on their membrane surface and reporter cells that stably express human DR3.

[0290] The detection method was as follows: 20,000 huTL1A / CHO cells and 40,000 DR3 reporter cells per well, along with serially diluted test antibodies, were added to each well of a white-walled, clear-bottom cell culture plate at a concentration of 100 μL / well. RPMI 1640 medium containing 10% FBS was used as the culture medium, and the plates were incubated in a 37°C incubator for 6 hours. After incubation, 100 μL of fluorescence detection reagent (Cobioer, CBPH0001) was added to each well, and fluorescence detection was performed.

[0291] As shown in Figure 6, HYB1805 and Duvakitug exhibited superior blocking effects against fluorescence signals, followed by HYB1803. Both were superior to 1D1 and significantly superior to PRA-023. Example 10: Preparation and screening of single-domain antibodies targeting IL-23p19 (IL-23A) Alpaca immunity

[0292] Human IL-23A-huFc protein (purchased from Beijing Yiqiao Shenzhou Co., Ltd., 13062-H02H) and IL23A&IL12B-His protein (heterodimer, purchased from Beijing Baipusais Co., Ltd., ILB-H52W5) were used to immunize two alpacas by subcutaneous injection. The immunization dose was 0.5 mg / alpaca / injection. To preferentially enrich the alpacas with antibodies targeting IL23A, IL-23A-huFc was used in the first two immunizations, and IL23A&IL12B was used in the subsequent immunizations from the third to the eighth. A complete adjuvant was used in the first immunization, followed by an incomplete adjuvant, with a total of eight immunizations performed at two-week intervals. Measurement of serum immunosuppression

[0293] The study began with a second immunization, and 5 mL of blood was collected one week after each immunization to measure serum immunotrichosis. Specifically, 200 ng each of the IL23A and IL12B-His antigen proteins used in the previous step were coated into each well of an ELISA well plate. After blocking and washing, serum samples were added in serial dilutions starting from a 1:2,000 ratio. After incubation, binding, and washing, the samples were color-developed using a secondary antibody against the anti-camel single-domain antibody (GenScript, A01861). Building a Phage Library

[0294] After each alpaca was immunized eight times, 10 mL of blood was collected and combined to construct a phage display library of single-domain antibodies. First, lymphocytes were isolated using lymphocyte isolate (Solarbio, P8610), treated and lysed with RNA extraction reagent (TaKaRa, 9109), impurities were removed with chloroform, RNA was precipitated with isopropanol, the precipitate was washed with 75% ethanol, dried at room temperature, and then dissolved in sterile water for injection. Next, the RNA was reverse transcribed using a reverse transcription kit (Thermo Scientific, K1622) to synthesize cDNA. Then, the DNA encoding VHH was amplified by nested PCR, digested with Sfi I enzyme, and ligated with a display vector similarly digested with Sfi I enzyme via T4 DNA ligase. The ligation product was purified, electroporated into TG1 competent cells, and infected with M13KO7 helper phage to prepare the phage display library. The effective library size of the final constructed libraries (HYC7-8 and HYBC8-8) is 4 × 10⁶ each. 9 and 4.8×10 9 It was CFU. Panning of bacteriophage libraries

[0295] Antigens (IL23A & IL12B-His-Avi, manufactured by Beijing Baipusais Co., Ltd., ILB-H82W6, heterodimer form) were loaded onto streptavidin-conjugated magnetic beads (Thermo Scientific, 88817), and panning was performed on HYC7-8 and HYBC8-8 phage libraries. The panning process and results are shown in Table 2. After one panning, the phages eluted from the IL23A & IL12B antigen group were significantly enriched compared to the control group, with enrichment ratios of 200-fold and 405.4-fold, respectively.

[0296] [Table 2B] Phage-based single-clonal screening

[0297] Using the concentrated phage library eluted in the previous step, TG1 bacteria in the logarithmic growth phase were infected, diluted, plated, and cultured. Then, single clones were picked and inoculated into 2×YT medium containing Carb and M13KO7 helper phages, and cultured overnight. The following day, the culture plates were centrifuged at 4200 rpm, and the supernatant was used for phage ELISA detection. Wells coated with IL23A & IL12B-His protein (heterodimer, Beijing Yiqiao Shenzhou Co., Ltd., CT012-H08H) were used as the measurement wells, and wells coated with unrelated target proteins were used as control wells. As the secondary antibody, an anti-M13 antibody conjugated with HRP (horseradish peroxidase) (Beijing Yiqiao Shenzhou Co., Ltd., 11973-MM05T-H) was used. (OD) 450nm Clone bacterial suspensions with a reading of 1.0 or higher in the control well and a reading of 0.1 or lower in the control well were selected, and plasmid extraction and gene sequencing were performed. After converting the sequenced sequences to amino acid sequences, sequence alignment was performed to remove duplicate sequences and similar sequences with differences of 2 amino acids or less in the CDR (CDR region defined using the AbM method), ultimately obtaining 103 unique sequences. Example 11: Detection of the blocking function of periplasmic cavity crude extract based on IL-23p19 single-domain antibody

[0298] Plasmids containing a unique sequence were transformed into competent BL21 Rosetta (DE3) bacteria and inoculated into 2 mL of 2×YT medium containing a Carb antibiotic. OD 600 When the concentration reached 0.4, 3 mL of 2×YT medium containing IPTG and Carb was added to bring the final IPTG concentration to 0.1 mM, and the culture was incubated overnight at 28°C. The following day, the bacterial suspension was centrifuged at 4200 rpm at 4°C for 20 minutes, and the cells were resuspended in 0.5 mL of PBS. The cells were subjected to three freeze-thaw cycles from -80°C to 37°C to release single-domain antibodies in the periplasmic lumen. After centrifuging at 10,000 rpm at 4°C for 15 minutes, the supernatant was collected to prepare a crude extract (10×) of single-domain antibodies in the periplasmic lumen.

[0299] Next, the blocking of human IL-23R-Fc binding to IL23A & IL12B-His-Avi was detected by ELISA using the crude single-domain antibody extract described above. First, human IL-23R-Fc (ILR-H5254, manufactured by Baipusais Co., Ltd.) was coated onto well plates overnight at 4°C with 300 ng per well. The following day, the well plates were blocked at room temperature for 2 hours with 1% BSA (bovine serum albumin). The crude extract was mixed in equivolute with 0.2 μg / mL of IL23A & IL12B-His-Avi. The crude extract was serially diluted 1:2 from the stock solution to establish two concentration points (the final concentrations of the crude single-domain antibody extract were 5× and 2.5×, respectively). After incubation of the crude extract with IL23A & IL12B-His-Avi at room temperature for 30 minutes, it was transferred to well plates coated with IL23R-Fc and blocked, and incubated at room temperature for 1 hour. After incubation, the plates were washed, and streptavidin-HRP (Shanghai Bioengineering Co., Ltd., D111054) was added at a ratio of 1:3,000 for color development. A risankizumab biosimilar was used as a positive control, and a crude extract of a single-domain antibody targeting an unrelated target was used as a negative control. Absorbance values ​​at a wavelength of 450 nm were measured using a microplate reader (Biotek, Synergy H1MF).

[0300] The results showed that 8 out of 103 clones exhibited a significant blocking effect on the binding of IL-23R to IL-23. Furthermore, when the binding ability of the crude extracts of these 103 clones to IL-23 coated on well plates was detected, it was found that most of them showed significant binding, while they did not bind to control wells coated with unrelated target proteins (data not shown). Example 12: Expression of IL-23p19 heavy chain antibody and identification of interspecies cross-reactivity (monkeys)

[0301] The VHH sequences corresponding to the eight clones obtained above are assigned to the human IgG1 Fc domain (C 220 A / L 234 A / L 235 The A mutation-containing molecule (sequence shown in SEQ ID NO: 100) was ligated to the N-terminus and transiently transfected into HEK293 cells for expression and purification. The resulting molecules were named HYB1901, HYB1902, HYB1903, HYB1904, HYB1905, HYB1906, HYB1907, and HYB1908, respectively (their respective variable domain sequences are shown in SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, and 74). Next, the binding ability of these molecules to monkey IL23A & mouse IL12B-His protein (ILB-CM52W8, manufactured by Baipusais) coated on well plates was measured by ELISA, with risankizumab biosimilars used as positive controls.

[0302] As shown in Figure 7, HYB1901, HYB1903, HYB1905, HYB1906, and HYB1907 all effectively bind to monkey IL23A and mouse IL12B proteins. These five molecules—HYB1901, HYB1903, HYB1905, HYB1906, and HYB1907—were selected for further study. Example 13: Measurement of the blocking activity of IL-23p19 heavy chain antibody against the binding of IL-23R to IL-23.

[0303] The blocking ability of IL-23p19 heavy chain antibodies against the binding of IL-23R-Fc and IL-23-Avi was detected according to the method described in Example 11. A risankizumab biosimilar was used as a positive control, and an isotype single-domain antibody targeting an unrelated target was used as a negative control.

[0304] As shown in Figure 8, each molecule has a clear blocking effect, and among them, HYB1903 has the best blocking effect, and its EC 50 The concentration was found to be 0.030 μg / mL, which was superior to the control molecule risankizumab's concentration of 0.126 μg / mL. Example 14: Inhibitory effect of IL-23p19 heavy chain antibody on IL-23-induced IL-17A release from mouse splenocytes.

[0305] Mouse splenocytes (Oribiotech, MSPL-002F) were added to a 96-well cell culture plate at a rate of 50,000 cells / 100 μL / well and incubated at 37°C for 2 hours. During this time, serially diluted IL-23p19 heavy chain antibody samples and human IL-23-His (Beijing Baipusais Co., Ltd., ILB-H52W5) were mixed in a 1:1 volume ratio (60 μL + 60 μL) and incubated at 37°C for 1 hour. After cell incubation, 100 μL of the incubated mixture was added to the mouse splenocyte wells to achieve a final human IL-23-His concentration of 10 ng / mL. Subsequently, the cells were cultured in a 37°C / 5% CO2 incubator for 48 hours. After culturing, the mIL-17A secretion level in the supernatant culture medium was detected using an mIL-17A quantification kit (Biosharp, BSEM-041-96T).

[0306] As shown in Figure 9, HYB1903 exhibited the most superior blocking effect against IL-23-induced IL-17A release from mouse splenocytes, and its blocking EC 50 It was found to be 0.022 μg / mL. Example 15: Humanization and activity measurement of IL-23p19 heavy chain antibody

[0307] Structural simulations of the antibody HYB1903 were performed using SWISS-MODEL. Based on the obtained structure, humanization mutations in the camel-derived amino acid site within the framework region were evaluated. Human germline gene V 3-23 *01 and J1*01 were used as target sequences for humanization mutations. Amino acid sites located on the surface of the antibody structure and not adjacent to the CDR were preferentially mutated, while mutations in sites located inside the antibody structure (buried) and sites adjacent to the CDR were postponed.

[0308] Based on the above principles, multiple humanized antibody sequences from HYB1903-hz1 to HYB1903-hz5 (variable domain sequences are shown in SEQ ID NOs. 75-79) were designed and then transiently transfected into HEK293 cells for expression. Expression and purification results showed that the SEC main peak purity of HYB1903-hz5 was less than 90%, while the purity of other molecules was over 97%. Next, the cellular-level blocking effects of HYB1903-hz1 to HYB1903-hz4 were detected using the method described in Example 14.

[0309] As shown in Figure 10, all of HYB1903-hz1 to HYB1903-hz4 demonstrated excellent blocking effects against IL-23-induced IL-17A release from mouse splenocytes, and their blocking effect was found to be close to that of the positive control risankizumab. Example 16: Construction of bispecific antibodies targeting TL1A and IL-23p19

[0310] For the TL1A target site, the conventional four-chain antibody molecule HYB1805, screened from a complete human phage display library, was used, and for the IL-23p19 target site, the single-domain antibody (sdAb) HYB1903-hz3, prepared by immunizing and humanizing alpacas, was used. HYB1903-hz3 was ligated to the C-terminus of the HYB1805 heavy chain via (GGGGS)3 and (GGGGS)4 linkers, respectively. Furthermore, M was added to the Fc domain. 252 Y / S 254 T / T256 E / L 234 A / L 235 A / K 447 The del gene was introduced. The constructed molecules were named HYB1805-2 and HYB1805-3, respectively (heavy chain sequences are shown in SEQ ID NOs. 115 and 116, and light chain sequences in SEQ ID NOs. 92). These molecules were transiently transfected into HEK293 cells, expressed, and purified. Both molecules showed good yield and SEC main peak purity. Example 17: Inhibitory effect of a bispecific antibody on IL-23-induced IL-17A release from mouse splenocytes.

[0311] Mouse splenocytes (Oribiotech, MSPL-002F) were added to a 96-well cell culture plate at a rate of 50,000 cells / 100 μL / well and incubated at 37°C for 2 hours. During this time, serially diluted bispecific antibody samples and human IL-23-His (Beijing Baipusais Co., Ltd., ILB-H52W5) were mixed in a 1:1 volume ratio (60 μL + 60 μL) and incubated at 37°C for 1 hour. After cell incubation, 100 μL of the incubated mixture was added to the mouse splenocyte wells to achieve a final human IL-23-His concentration of 10 ng / mL. The plate was incubated at 37°C / 5% CO2 for 48 hours. After incubation, the mIL-17A secretion level in the supernatant culture medium was detected using an mIL-17A quantification kit (Biosharp, BSEM-041-96T).

[0312] As shown in Figure 11, both bispecific antibody molecules demonstrated excellent blocking effects against IL-23-induced IL-17A release from mouse splenocytes. HYB1805-2, with its shorter linker length, was selected for further validation. Example 18: Inhibitory effect of a bispecific antibody on the TL1A-mediated caspase 3 / 7 apoptosis signaling pathway

[0313] The inhibitory effect of HYB1805-2 on the caspase 3 / 7 apoptotic signaling pathway mediated by TL1A was detected using TF-1 cells. Bispecific antibody samples were serially diluted using RPMI 1640 medium (containing 10% FBS) without GM-CSF to prepare cycloheximide (CHX) at a concentration of 80 μg / mL and TL1A-His (TLA-H5243, trimer form) at a concentration of 40 ng / mL. Next, 30 μL each of the antibody dilution, cycloheximide solution, and TL1A solution were added to a sterile 96-well plate and incubated in a 37°C incubator for 1 hour. Simultaneously, TF-1 cells in the logarithmic growth phase were seeded at a seeding rate of 30,000 cells / 50 μL / well in a 96-well white-walled clear-bottom cell culture plate. After incubation, 50 μL of antibody:CHX:TL1A mixture was added to a cell culture plate at 50 μL / well and incubated for 6 hours. After incubation, 100 μL / well of Caspase-Glo® 3 / 7 reagent (Promega, G8091) was added, and luminescence detection was performed according to the reagent instructions.

[0314] As shown in Figure 12, HYB1805-2 exhibits superior in vitro blocking effects against TL1A, and its blocking effect is superior to that of the control drug Duvakitug and significantly superior to that of the control drug PRA-023.

[0315] Although the present invention has been described illustratively according to its preferred embodiments, it is not limited to those embodiments described above. For those skilled in the art, the present invention is subject to various modifications and changes. The selection and application of specific technical solutions can be appropriately adjusted and modified according to actual requirements. Therefore, several simple substitutions are possible by those skilled in the art without departing from the concept and principles of the present invention, and all of these fall within the scope of protection of the present invention.

Claims

1. A tumor necrosis factor-like protein 1A (TL1A) binding molecule comprising at least one antigen-binding unit targeting TL1A, wherein the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

6. 2) The VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 11, and the VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

13. 3) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

19. 4) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

23. 5) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

29. 6) The VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 32, and the VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

33. 7) The VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36, and the VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 37, or 8) The TL1A binding molecule wherein VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 40, and VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

41.

2. The TL1A binding molecule according to claim 1, wherein the VH comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by sequence numbers 7, 14, 20, 24, 30, 34, 38, or 42.

3. The TL1A binding molecule according to claim 1, wherein the VL comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NOs: 8, 15, 21, 25, 31, 35, 39, or 43.

4. Here, 1) The VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO:

8. 2) The VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO:

15. 3) The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO:

21. 4) The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO:

25. 5) The VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO:

31. 6) The VH comprises the amino acid sequence shown in SEQ ID NO: 34, and the VL comprises the amino acid sequence shown in SEQ ID NO:

35. 7) The VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO: 39, or 8) The TL1A binding molecule according to claim 1, wherein VH comprises the amino acid sequence shown in SEQ ID NO: 42, and VL comprises the amino acid sequence shown in SEQ ID NO:

43.

5. The TL1A binding molecule according to any one of claims 1 to 4, wherein the VH and / or VL further comprises substitution, addition and / or deletion of one or more amino acid residues and maintains specific binding to TL1A, wherein the substitution, addition and / or deletion of one or more amino acid residues is present in the VH and / or VL sequence but not in either CDR sequence.

6. The TL1A binding molecule according to any one of claims 1 to 4, wherein the TL1A binding molecule is an anti-TL1A antibody or its antigen-binding fragment.

7. The TL1A-binding molecule according to any one of claims 1 to 4, wherein the TL1A-binding molecule is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody, or an antigen-binding fragment thereof.

8. The TL1A binding molecule is F(ab), F(ab'), F(ab') ２ A TL1A-binding molecule according to any one of claims 1 to 4, which is scFab, Fd, Fv, dsFv, dAb, double-chain antibody (diabody), or scFv.

9. The TL1A binding molecule according to any one of claims 1 to 4, further comprising an immunoglobulin heavy chain constant domain.

10. The TL1A binding molecule according to claim 9, wherein the immunoglobulin heavy chain constant domain is a human immunoglobulin heavy chain constant domain, preferably a heavy chain constant domain derived from human IgG, and more preferably a heavy chain constant domain derived from human IgG1 or IgG4.

11. The immunoglobulin heavy chain constant domain further comprises one or more amino acid mutations, and the one or more amino acid mutations are L ２３４ A, L ２３５ A, M ２５２ Y, S ２５４ T, T ２５６ E and K ４４７ A TL1A-binding molecule according to claim 9, selected from del.

12. The TL1A binding molecule according to claim 9, wherein the immunoglobulin heavy chain constant domain sequence includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence that is at least 90%.

13. The TL1A binding molecule according to any one of claims 1 to 4, further comprising an immunoglobulin light chain constant domain.

14. The TL1A binding molecule according to claim 13, wherein the immunoglobulin light chain constant domain is derived from a human λ light chain constant domain or a κ light chain constant domain.

15. The TL1A binding molecule according to claim 13, wherein the immunoglobulin light chain constant domain sequence comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:

82.

16. The TL1A binding molecule according to any one of claims 1 to 4, wherein the TL1A binding molecule comprises a plurality of antigen-binding units that target the TL1A.

17. The TL1A-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain, the heavy chain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by TL1A.

18. The TL1A-binding molecule according to any one of claims 1 to 4, wherein the antigen-binding unit targeting TL1A comprises an immunoglobulin light chain, the light chain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by TL1A.

19. The antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain and an immunoglobulin light chain, where, 1) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 83, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:

84. 2) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 85, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:

86. 3) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 87, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:

88. 4) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 89, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:

90. 5) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 91, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:

92. 6) The immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 93, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:

94. 7) The immunoglobulin heavy chain contains the amino acid sequence shown in SEQ ID NO: 95, and the immunoglobulin light chain contains the amino acid sequence shown in SEQ ID NO: 96, or 8) The TL1A binding molecule according to any one of claims 1 to 4, wherein the immunoglobulin heavy chain comprises the amino acid sequence shown in SEQ ID NO: 97, and the immunoglobulin light chain comprises the amino acid sequence shown in SEQ ID NO:

98.

20. An interleukin-23p19 subunit (IL-23p19) binding molecule comprising at least one immunoglobulin monovariable domain that specifically binds to IL-23p19, wherein the immunoglobulin monovariable domain that specifically binds to IL-23p19 is 1) CDR1 of the amino acid sequence shown in SEQ ID NO: 44, CDR2 of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 of the amino acid sequence shown in SEQ ID NO: 46 2) CDR1 of the amino acid sequence shown in SEQ ID NO: 48, CDR2 of the amino acid sequence shown in SEQ ID NO: 49, and CDR3 of the amino acid sequence shown in SEQ ID NO: 50 3) CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54 4) CDR1 of the amino acid sequence shown in SEQ ID NO: 56, CDR2 of the amino acid sequence shown in SEQ ID NO: 57, and CDR3 of the amino acid sequence shown in SEQ ID NO: 58 5) CDR1 of the amino acid sequence shown in SEQ ID NO: 60, CDR2 of the amino acid sequence shown in SEQ ID NO: 61, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 6) CDR1 of the amino acid sequence shown in SEQ ID NO: 64, CDR2 of the amino acid sequence shown in SEQ ID NO: 65, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 7) CDR1 of the amino acid sequence shown in SEQ ID NO: 67, CDR2 of the amino acid sequence shown in SEQ ID NO: 68, and CDR3 of the amino acid sequence shown in SEQ ID NO: 69, or 8) The IL-23p19 binding molecule comprising CDR1 of the amino acid sequence shown in SEQ ID NO: 71, CDR2 of the amino acid sequence shown in SEQ ID NO: 72, and CDR3 of the amino acid sequence shown in SEQ ID NO:

73.

21. The IL-23p19 binding molecule according to claim 20, wherein the immunoglobulin monovariate domain that specifically binds to IL-23p19 comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 99%, and maintains specific binding to IL-23p19 with respect to the amino acid sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78, or 79, and has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78, or 79.

22. The IL-23p19 binding molecule according to claim 20 or 21, wherein the immunoglobulin monovariable domain further comprises substitution, addition and / or deletion of one or more amino acid residues and maintains specific binding to IL-23p19, and the substitution, addition and / or deletion of one or more amino acid residues is present in the immunoglobulin monovariable domain sequence but not in any CDR sequence.

23. The IL-23p19 binding molecule according to claim 20 or 21, wherein the immunoglobulin single variable domain is VHH.

24. The IL-23p19 binding molecule according to claim 20 or 21, wherein the immunoglobulin single variable domain is VHH derived from a camelid, and the camelid is preferably an alpaca or a llama.

25. The IL-23p19 binding molecule according to claim 20 or 21, wherein the immunoglobulin single variable domain is humanized VHH.

26. The IL-23p19 binding molecule according to claim 20 or 21, wherein the IL-23p19 binding molecule is an anti-IL-23p19 antibody or its antigen-binding fragment.

27. The IL-23p19 conjugating molecule according to claim 20 or 21, wherein the IL-23p19 conjugating molecule is a heavy chain antibody, a heavy chain single-domain antibody, a chimeric antibody, or a humanized antibody.

28. The IL-23p19 binding molecule according to claim 20 or 21, further comprising an immunoglobulin Fc domain.

29. The IL-23p19 binding molecule according to claim 28, wherein the immunoglobulin Fc domain is a human immunoglobulin Fc domain, preferably a human IgG-derived Fc domain, and more preferably a human IgG1 or IgG4-derived Fc domain.

30. The immunoglobulin Fc domain further comprises one or more amino acid mutations, and the one or more amino acid mutations are selected from C ２２０ A, L ２３４ A, L ２３５ A, M ２５２ Y, S ２５４ T, T ２５６ E and K ４４７ The IL-23p19 binding molecule according to claim 28, selected from del.

31. The IL-23p19 binding molecule according to claim 28, wherein the immunoglobulin Fc domain is an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence represented by sequence number 99, 100, 118, or 119.

32. The IL-23p19 binding molecule according to claim 28, wherein the immunoglobulin Fc domain and the immunoglobulin single variable domain are optionally linked via an immunoglobulin hinge region or a linker, preferably the linker being (GS)n, (GGS)n, (GGGS)n or (GGGGGS)n, where n is 1, 2, 3, 4 or 5.

33. The IL-23p19 binding molecule according to claim 20 or 21, wherein the IL-23p19 binding molecule comprises a plurality of immunoglobulin monovariable domains that specifically bind to the IL-23p19.

34. The IL-23p19 binding molecule according to claim 33, wherein the immunoglobulin single variable domains that specifically bind to the plurality of IL-23p19 are optionally linked via a linker, preferably the linker being (GS)n, (GGS)n, (GGGS)n, or (GGGGGS)n, where n is selected from 1, 2, 3, 4, or 5.

35. An IL-23p19 binding molecule according to claim 20 or 21, comprising an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by any one of the amino acid sequences represented by any one of the amino acid sequences 101 to 113, and that maintains specific binding to IL-23p19.

36. A multispecific antigen-binding molecule comprising a TL1A-binding molecule according to any one of claims 1 to 19 and / or an IL-23p19-binding molecule according to any one of claims 20 to 35, and one or more other antigen-binding functional regions, wherein the one or more other antigen-binding functional regions bind to an antigen different from the TL1A-binding molecule and / or the IL-23p19-binding molecule, or to a different epitope of the same antigen.

37. A bispecific antigen-binding molecule comprising a TL1A-binding molecule according to any one of claims 1 to 19 and an IL-23p19-binding molecule according to any one of claims 20 to 35.

38. A bispecific antigen-binding molecule comprising a first antigen-binding functional region and a second antigen-binding functional region, wherein the first antigen-binding functional region comprises at least one antigen-binding unit targeting TL1A, and the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 3, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

6. 2) The VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 11, and the VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

13. 3) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

19. 4) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

23. 5) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

29. 6) The VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 32, and the VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

33. 7) The VH includes HCDR1 of the amino acid sequence shown in SEQ ID NO: 9, HCDR2 of the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36, and the VL includes LCDR1 of the amino acid sequence shown in SEQ ID NO: 12, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 37, or 8) The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 40, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

41. Furthermore, the second antigen-binding functional region includes at least one immunoglobulin monovariate domain that specifically binds to IL-23p19, and the immunoglobulin monovariate domain that specifically binds to IL-23p19 is 1) CDR1 of the amino acid sequence shown in SEQ ID NO: 44, CDR2 of the amino acid sequence shown in SEQ ID NO: 45, and CDR3 of the amino acid sequence shown in SEQ ID NO: 46 2) CDR1 of the amino acid sequence shown in SEQ ID NO: 48, CDR2 of the amino acid sequence shown in SEQ ID NO: 49, and CDR3 of the amino acid sequence shown in SEQ ID NO: 50 3) CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO: 54 4) CDR1 of the amino acid sequence shown in SEQ ID NO: 56, CDR2 of the amino acid sequence shown in SEQ ID NO: 57, and CDR3 of the amino acid sequence shown in SEQ ID NO: 58 5) CDR1 of the amino acid sequence shown in SEQ ID NO: 60, CDR2 of the amino acid sequence shown in SEQ ID NO: 61, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 6) CDR1 of the amino acid sequence shown in SEQ ID NO: 64, CDR2 of the amino acid sequence shown in SEQ ID NO: 65, and CDR3 of the amino acid sequence shown in SEQ ID NO: 62 7) CDR1 of the amino acid sequence shown in SEQ ID NO: 67, CDR2 of the amino acid sequence shown in SEQ ID NO: 68, and CDR3 of the amino acid sequence shown in SEQ ID NO: 69, or 8) The bispecific antigen-binding molecule comprising CDR1 of the amino acid sequence shown in SEQ ID NO: 71, CDR2 of the amino acid sequence shown in SEQ ID NO: 72, and CDR3 of the amino acid sequence shown in SEQ ID NO:

73.

39. The bispecific antigen-binding molecule comprises a first antigen-binding functional region and a second antigen-binding functional region, wherein the first antigen-binding functional region comprises at least one antigen-binding unit targeting TL1A, and the antigen-binding unit targeting TL1A comprises an immunoglobulin heavy chain variable domain (VH) and an immunoglobulin light chain variable domain (VL), where, 1) The VH comprises the amino acid sequence shown in SEQ ID NO: 7, and the VL comprises the amino acid sequence shown in SEQ ID NO:

8. 2) The VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO:

15. 3) The VH comprises the amino acid sequence shown in SEQ ID NO: 20, and the VL comprises the amino acid sequence shown in SEQ ID NO:

21. 4) The VH comprises the amino acid sequence shown in SEQ ID NO: 24, and the VL comprises the amino acid sequence shown in SEQ ID NO:

25. 5) The VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO:

31. 6) The VH comprises the amino acid sequence shown in SEQ ID NO: 34, and the VL comprises the amino acid sequence shown in SEQ ID NO:

35. 7) The VH comprises the amino acid sequence shown in SEQ ID NO: 38, and the VL comprises the amino acid sequence shown in SEQ ID NO:

39. 8) The VH includes the amino acid sequence shown in SEQ ID NO: 42, and the VL includes the amino acid sequence shown in SEQ ID NO: 43, or includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the above amino acid sequence and maintains specific binding with TL1A. Furthermore, the second antigen-binding functional region includes at least one immunoglobulin monovariate domain that specifically binds to IL-23p19, and the immunoglobulin monovariate domain that specifically binds to IL-23p19 is at least 80% of the amino acid sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78 or 79, or at least 80% of the amino acid sequence shown in SEQ ID NOs: 47, 51, 55, 59, 63, 66, 70, 74, 75, 76, 77, 78 or 79. The bispecific antigen-binding molecule according to claim 38, comprising an amino acid sequence having at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity and maintaining specific binding to IL-23p19.

40. The bispecific antigen-binding molecule according to claim 38, wherein VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

29.

41. The bispecific antigen-binding molecule according to claim 38, wherein VH includes the amino acid sequence shown in SEQ ID NO: 30, and VL includes the amino acid sequence shown in SEQ ID NO: 31, or includes an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 30 or 31, and maintains specific binding to TL1A.

42. The bispecific antigen-binding molecule according to claim 38, wherein the immunoglobulin single variable domain comprises CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO:

54.

43. The bispecific antigen-binding molecule according to claim 38, wherein the immunoglobulin monovariable domain comprises the amino acid sequence shown in SEQ ID NO: 77, or comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 77, and maintains specific binding to IL-23p19.

44. The VH comprises HCDR1 of the amino acid sequence shown in SEQ ID NO: 26, HCDR2 of the amino acid sequence shown in SEQ ID NO: 27, and HCDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the VL comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 17, LCDR2 of the amino acid sequence shown in SEQ ID NO: 18, and LCDR3 of the amino acid sequence shown in SEQ ID NO:

29. Furthermore, the bispecific antigen-binding molecule according to claim 38, wherein the immunoglobulin single variable domain comprises CDR1 of the amino acid sequence shown in SEQ ID NO: 52, CDR2 of the amino acid sequence shown in SEQ ID NO: 53, and CDR3 of the amino acid sequence shown in SEQ ID NO:

54.

45. The VH comprises the amino acid sequence shown in SEQ ID NO: 30, and the VL comprises the amino acid sequence shown in SEQ ID NO: 31, or comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 30 or 31, and that maintains specific binding with TL1A. Furthermore, the monovariable immunoglobulin domain comprises the amino acid sequence shown in SEQ ID NO: 77, or comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 77, and that maintains specific binding to IL-23p19, according to claim 38.

46. The bispecific antigen-binding molecule according to any one of claims 37 to 45, wherein the VH, VL and / or immunoglobulin monovariable domain further comprises substitution, addition and / or deletion of one or more amino acid residues and maintains specific binding to TL1A and / or IL-23p19, and the substitution, addition and / or deletion of one or more amino acid residues is present in the VH, VL and / or immunoglobulin monovariable domain sequence but not in any of the CDR sequences.

47. The bispecific antigen-binding molecule according to claim 46, wherein the immunoglobulin monovariate domain further comprises one or more amino acid mutations that reduce immunogenicity.

48. The bispecific antigen-binding molecule according to any one of claims 37 to 45, wherein the immunoglobulin monovariable domain is VHH.

49. The bispecific antigen-binding molecule according to claim 48, wherein the immunoglobulin monovariate domain is VHH derived from a camelid, and the camelid is preferably an alpaca or a llama.

50. The bispecific antigen-binding molecule according to claim 48, wherein the immunoglobulin monovariate domain is humanized VHH.

51. The antigen-binding units targeting TL1A are F(ab), F(ab'), and F(ab'). ２ A bispecific antigen-binding molecule according to any one of claims 37 to 45, wherein the bispecific antigen-binding molecule is scFab, Fd, Fv, dsFv, dAb, double-chain antibody (diabody), or scFv.

52. The bispecific antigen-binding molecule according to claim 51, wherein the antigen-binding unit targeting TL1A is F(ab), and the VH is linked to the immunoglobulin CH1 domain, and the VL is linked to the constant domain of the immunoglobulin light chain.

53. The bispecific antigen-binding molecule according to claim 52, wherein the immunoglobulin CH1 domain is a human immunoglobulin CH1 domain, preferably a human IgG-derived CH1 domain, more preferably a human IgG1 or IgG4-derived CH1 domain, and / or the immunoglobulin light chain constant domain is derived from a human λ light chain constant domain or a κ light chain constant domain.

54. The immunoglobulin CH1 domain sequence includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in SEQ ID NO: 117, and / or the immunoglobulin CH1 domain sequence includes an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 890%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at The bispecific antigen-binding molecule according to claim 52, wherein the brin light chain constant domain sequence comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:

82.

55. A bispecific antigen-binding molecule according to any one of claims 37 to 45, further comprising an immunoglobulin Fc domain.

56. The bispecific antigen-binding molecule according to claim 55, wherein the immunoglobulin Fc domain is a human immunoglobulin Fc domain, preferably a human IgG-derived Fc domain, and more preferably a human IgG1 or IgG4-derived Fc domain.

57. The immunoglobulin Fc domain further comprises one or more amino acid mutations, and the one or more amino acid mutations are L ２３４ A, L ２３５ A, M ２５２ Y, S ２５４ T, T ２５６ E and K ４４７ A bispecific antigen-binding molecule according to claim 55, selected from del.

58. The bispecific antigen-binding molecule according to claim 55, wherein the immunoglobulin Fc domain is an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence represented by sequence number 99, 100, 118, or 119.

59. The bispecific antigen-binding molecule according to any one of claims 37 to 45, wherein the first antigen-binding functional region comprises a plurality of antigen-binding units targeting TL1A, and / or the second antigen-binding functional region comprises a plurality of immunoglobulin monovariable domains that specifically bind to IL-23p19.

60. The bispecific antigen-binding molecule according to any one of claims 37 to 45, wherein the first antigen-binding functional region and the second antigen-binding functional region are each independently one, two, or two or more.

61. The bispecific antigen-binding molecule according to claim 59 or 60, wherein the plurality of antigen-binding units, the plurality of immunoglobulin monovariable domains, and / or the plurality of antigen-binding functional regions are optionally linked via linkers, preferably the linkers being (GS)n, (GGS)n, (GGGS)n, or (GGGGGS)n, where n is selected from 1, 2, 3, 4, or 5.

62. The aforementioned bispecific antigen-binding molecule is From the N-terminus to the C-terminus, the immunoglobulin heavy chain comprises the VH, immunoglobulin CH1 domain, Fc domain, an optional linker, and the immunoglobulin single variable domain, A bispecific antigen-binding molecule according to any one of claims 37 to 45, comprising an immunoglobulin light chain including the VL and the constant region of the immunoglobulin light chain from the N-terminus to the C-terminus.

63. The bispecific antigen-binding molecule according to claim 62, wherein the linker is (GS)n, (GGS)n, (GGGS)n, or (GGGGGS)n, where n is selected from 1, 2, 3, 4, or 5.

64. The immunoglobulin heavy chain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence represented by SEQ ID NO: 115 or 116, and The bispecific antigen-binding molecule according to any one of claims 37 to 45, wherein the immunoglobulin light chain comprises an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 92, or an amino acid sequence that is identical to the amino acid sequence shown in SEQ ID NO: 92 by at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

65. A fusion protein comprising a TL1A binding molecule according to any one of claims 1 to 19, an IL-23p19 binding molecule according to any one of claims 20 to 35, a multispecific antigen binding molecule according to claim 36, and / or a bispecific antigen binding molecule according to any one of claims 37 to 64, and a polypeptide or protein expressed in fusion with it.

66. A conjugate comprising a TL1A binding molecule according to any one of claims 1 to 19, an IL-23p19 binding molecule according to any one of claims 20 to 35, a multispecific antigen binding molecule according to claim 36, a bispecific antigen binding molecule according to any one of claims 37 to 64, and / or a fusion protein according to claim 65, and a conjugate conjugated thereto.

67. A nucleic acid molecule comprising a TL1A binding molecule according to any one of claims 1 to 19, an IL-23p19 binding molecule according to any one of claims 20 to 35, a multispecific antigen binding molecule according to claim 36, a bispecific antigen binding molecule according to any one of claims 37 to 64, and / or a nucleotide sequence encoding a fusion protein according to claim 65.

68. An expression vector comprising a nucleic acid molecule according to claim 67, operably linked to an expression regulatory element.

69. A host cell comprising the nucleic acid molecule described in claim 67 and / or the expression vector described in claim 68.

70. A method for producing a TL1A binding molecule according to any one of claims 1 to 19, an IL-23p19 binding molecule according to any one of claims 20 to 35, a multispecific antigen binding molecule according to claim 36, a bispecific antigen binding molecule according to any one of claims 37 to 64, and / or a fusion protein according to claim 65, wherein the method is: The method comprising culturing the host cells described in claim 69 under conditions suitable for the expression of the vector described in claim 68, and optionally isolating and / or purifying the TL1A binding molecule, the IL-23p19 binding molecule, the multispecific antigen binding molecule, the bispecific antigen binding molecule, and / or the fusion protein from the host cells or the host cell culture.

71. A composition comprising a TL1A binding molecule according to any one of claims 1 to 19, an IL-23p19 binding molecule according to any one of claims 20 to 35, a multispecific antigen binding molecule according to claim 36, a bispecific antigen binding molecule according to any one of claims 37 to 64, a fusion protein according to claim 65, and / or a conjugate according to claim 66.

72. The composition according to claim 71, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier and / or excipient, and optionally one or more therapeutic agents having other pharmaceutically active properties.

73. Use of a TL1A binding molecule according to any one of claims 1 to 19, an IL-23p19 binding molecule according to any one of claims 20 to 35, a multispecific antigen binding molecule according to claim 36, a bispecific antigen binding molecule according to any one of claims 37 to 64, a fusion protein according to claim 65, a conjugate according to claim 66, and / or a composition according to claim 71 in the manufacture of a drug for treating, preventing or alleviating inflammatory diseases and / or autoimmune diseases.

74. The use according to claim 73, wherein the inflammatory disease and / or autoimmune disease is selected from asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, or psoriasis.

75. The use according to claim 74, wherein the inflammatory bowel disease is selected from Crohn's disease or ulcerative colitis.

76. The use according to claim 73, wherein the drug further comprises one or more other therapeutic agents for treating inflammatory diseases and / or autoimmune diseases.

77. A method for treating, preventing or alleviating an inflammatory disease and / or autoimmune disease, comprising administering a therapeutically effective amount of a TL1A-binding molecule according to any one of claims 1 to 19, an IL-23p19-binding molecule according to any one of claims 20 to 35, a multispecific antigen-binding molecule according to claim 36, a bispecific antigen-binding molecule according to any one of claims 37 to 64, a fusion protein according to claim 65, a conjugate according to claim 66, and / or a composition according to claim 71 to a subject in need thereof.

78. The method according to claim 77, wherein the inflammatory disease and / or autoimmune disease is selected from asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, or psoriasis.

79. The method according to claim 78, wherein the inflammatory bowel disease is selected from Crohn's disease or ulcerative colitis.

80. The method according to claim 77, further comprising using it in combination with one or more other therapeutic agents for treating inflammatory diseases and / or autoimmune diseases.

81. Use of a TL1A-binding molecule according to any one of claims 1 to 19, an IL-23p19-binding molecule according to any one of claims 20 to 35, a multispecific antigen-binding molecule according to claim 36, a bispecific antigen-binding molecule according to any one of claims 37 to 64, a fusion protein according to claim 65, a conjugate according to claim 66, and / or the composition according to claim 71 in in vivo or in vitro inhibition or blockade of TL1A and / or IL-23p19 signaling.

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