Antibodies targeting IL23 and their bispecific antibodies

Abstract

This invention relates to antibodies targeting IL23 and bispecific antibodies constructed therefrom. More specifically, it provides an antibody that specifically binds to IL23 or its antigen-binding moiety and its uses. In some aspects, the invention describes a method for blocking the biological activity of the IL23-IL23R&IL12Rβ1 complex signaling pathway. In some aspects, the antibodies and methods described herein are used to treat autoimmune diseases or diseases associated with the induction of the IL23-IL23R&IL12Rβ1 complex pathway. In some aspects, the invention describes bispecific antibodies targeting IL23 and other autoimmune targets TL1A, α4β7, TNF-α, and PD-1.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-human interleukin-23 (IL23) monoclonal antibody or its antigen-binding fragment, a pharmaceutical composition containing the antibody, and its pharmaceutical uses. More specifically, this invention relates to a monoclonal antibody that specifically binds to the human IL23p19 subunit, which can efficiently block the binding of IL23 to its receptor IL23R, thereby inhibiting IL23-mediated Th17 cell differentiation and downstream inflammatory signaling pathways. The antibody of this invention, together with a TL1A antibody, forms a bispecific antibody, simultaneously inhibiting the IL23 / IL23R signaling pathway and the TL1A / TNFRSF25 (DR3) signaling pathway, thus doubly blocking Th17 cell differentiation, activation, and downstream inflammatory cascade reactions, producing significant synergistic anti-inflammatory and immunomodulatory effects. The antibodies and bispecific antibodies described in this invention can be used to prevent and / or treat diseases associated with IL23 overexpression or abnormal activation of the Th17 / IL23 / TL1A signaling pathway, including but not limited to plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, hidradenitis suppurativa, generalized pustular psoriasis, giant cell arteritis, non-infectious uveitis, Behçet's disease, and other autoimmune and inflammatory diseases. Background Technology

[0002] Autoimmune diseases are a group of chronic inflammatory diseases caused by the immune system abnormally attacking its own tissues. They affect a large number of people worldwide and mainly include ankylosing spondylitis (AS), hidradenitis suppurativa (HS), plaque psoriasis (PsO), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis (UC), generalized pustular psoriasis (GPP), giant cell arteritis (GCA), systemic lupus erythematosus (SLE, especially the cutaneous form), non-infectious uveitis (NIU), and Behçet's disease. Diseases such as dysplasia of the skin (BD), especially those with uveitis or intestinal involvement, Sjögren's syndrome (SjS), pyodermagangrenosum (PG), relapsing polychondritis (RP), VEXAS syndrome, adult-onset Still's disease (AOSD), rheumatoid arthritis (RA), multiple sclerosis (MS, especially the classic RRMS type), primary biliary cholangitis (PBC), sarcoidosis, and asthmatic COPD are among the many diseases that severely impact patients' quality of life and impose a huge medical burden.

[0003] Interleukin-23 (IL23) is a member of the IL12 family and a heterodimeric cytokine composed of two subunits, IL23 (p19) and IL12b (p40). It is primarily secreted by activated dendritic cells, macrophages, and monocytes. IL23 activates the JAK2-TYK2-STAT3 signaling pathway by binding to a receptor complex composed of IL12Rβ1 and IL23R, promoting Th17 cell differentiation, survival, and proliferation. It also induces Th17 cells to secrete pro-inflammatory factors such as IL17A, IL17F, IL22, IL26, and TNF-α, thus playing a central role in the maintenance of chronic inflammation and tissue destruction in various autoimmune diseases. Genetic studies have confirmed that IL23R and IL12B gene polymorphisms are significantly associated with susceptibility to various diseases, including psoriasis, PsA, AS, and IBD.

[0004] Animal model studies have further confirmed the crucial role of the IL23 / Th17 axis in disease development and progression. For example, in DSS-induced colitis, TNBS colitis, IL10 knockout spontaneous colitis, collagen-induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE), imiquimod-induced psoriatic dermatitis, and pilonidal sinus inflammation models, knocking out IL23a or using anti-IL23a antibodies can significantly reduce inflammation scores, histopathological damage, Th17 cell infiltration, and pro-inflammatory factor levels.

[0005] Existing biologics targeting the IL23 or IL12 pathway mainly include anti-p40 monoclonal antibodies (such as ustekinumab and briakinumab), which have been approved for the treatment of psoriasis, PsA, CD, and UC. However, the p40 subunit is shared by both IL12 and IL23, and blocking p40 will also inhibit the IL12 / Th1 axis, potentially increasing the risk of infection and malignancy. Monoclonal antibodies targeting the IL23-specific subunit IL23p19 (such as guselkumab, tildrakizumab, risankizumab, and mirikizumab) block only IL23 signaling while preserving IL12 / IFN-γ for anti-infection and anti-tumor immunity. They have shown higher complete skin clearance rates (PASI 100), endoscopic remission rates, and better safety in multiple phase III clinical trials, making them a preferred treatment option for psoriasis, PsA, AS, IBD, and other diseases.

[0006] Nevertheless, in clinical practice, approximately 30%-50% of patients still exhibit insufficient response or secondary loss of response to existing anti-IL23a antibodies, and some indications (such as pilonidal sinusitis, non-infectious uveitis, Behçet's disease, and juvenile idiopathic arthritis) lack approved specific anti-IL23a treatments. Furthermore, existing antibodies still have room for improvement in terms of tissue penetration, half-life, immunogenicity, and dosing frequency.

[0007] Therefore, developing novel, high-affinity, long-acting, and low-immunogenic anti-IL23a monoclonal antibodies that can more efficiently neutralize free IL23 and block its binding to IL23R is of great significance for meeting unmet clinical needs of existing treatments and providing safe and effective precision treatment options for more patients with autoimmune diseases. By specifically blocking the interaction between IL23 and the IL23R&IL12Rβ1 complex, the antibody of this invention can effectively inhibit the inflammatory response and fibrotic process mediated by the Th17 / IL23 axis in autoimmune diseases, providing a new treatment option for patients who are unresponsive to or intolerant of existing treatments. Summary of the Invention

[0008] In a first aspect, the present invention provides an antibody or antigen-binding moiety thereof that specifically binds to IL23 or a functional fragment thereof, characterized in that the antibody or antigen-binding moiety thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). Wherein, the VH includes: I. Containing a heavy chain complementarity-determining region 1 (HCDR1) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 82, wherein SEQ ID NO: 82: NYWX0G; Wherein, X0 is selected from I or M; II. Containing a heavy chain complementarity-determining region 2 (HCDR2) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 83, wherein SEQ ID NO: 83: IIX1PSGSX2TWYAQKFQG; Wherein, X1 is selected from N or S; X2 is selected from T or S; III. Contains a heavy chain complementarity-determining region 3 (HCDR3) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, and The VL includes: Ⅰ) Contains a light chain complementarity-determining region 1 (LCDR1) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; II) Contains a light chain complementarity-determining region 2 (LCDR2) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. III) Contains a light chain complementarity-determining region 3 (LCDR3) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 84, wherein SEQ ID NO: 84: X3SX4TX5X6X7X8X9X 10 X 11 ; Wherein, X3 is selected from A or S; X4 is selected from W or Y; X5 is selected from D or P; X6 is selected from T or S; The X7 is selected from P, S, or L; The X8 is selected from N, V, or S; X9 is selected from M, V, or L; The X 10 Selected from I, F, or V; The X 11 Choose from V or G.

[0009] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL). Wherein, the VH includes: i. A heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 12; or a heavy chain complementarity-determining region 1 (HCDR1) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 12. Alternatively, it may contain a heavy chain complementarity-determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO: 13; or a heavy chain complementarity-determining region 1 (HCDR1) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 13. ii. A heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 14; or a heavy chain complementarity-determining region 2 (HCDR2) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 14. Alternatively, it may contain a heavy chain complementarity-determining region 2 (HCDR2) as shown in SEQ ID NO: 15; or a heavy chain complementarity-determining region 2 (HCDR2) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 15. Alternatively, it may contain a heavy chain complementarity-determining region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 16; or a heavy chain complementarity-determining region 2 (HCDR2) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 16. and iii. A heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence shown in SEQ ID NO: 17; or a heavy chain complementarity-determining region 3 (HCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 17; and The VL includes: i) A light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 18; or a light chain complementarity-determining region 1 (LCDR1) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 18. ii) A light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 19; or a light chain complementarity-determining region 2 (LCDR2) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 19. iii) A light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 20; or a light chain complementarity-determining region 3 (LCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 20. Alternatively, it may contain a light chain complementarity-determining region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 21; or a light chain complementarity-determining region 3 (LCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 21. Alternatively, it may contain a light chain complementarity-determining region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 22; or a light chain complementarity-determining region 3 (LCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 22. Alternatively, it may contain a light chain complementarity-determining region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 23; or a light chain complementarity-determining region 3 (LCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 23. Alternatively, it may contain a light chain complementarity-determining region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 24; or a light chain complementarity-determining region 3 (LCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 24. Preferably, the amino acid difference is an amino acid substitution; more preferably, the amino acid difference is a conserved amino acid substitution.

[0010] In some embodiments, the antibody or its antigen-binding portion comprises: (a) Includes a heavy chain backbone comprising the amino acid sequence shown in SEQ ID NO: 25, wherein the complementary region of the heavy chain is: Including the heavy chain complementarity determination region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO: 12, the heavy chain complementarity determination region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 14, and the heavy chain complementarity determination region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 17. Alternatively, it may include the heavy chain complementarity determination region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO: 13, the heavy chain complementarity determination region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 14, and the heavy chain complementarity determination region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 17. Alternatively, it may include the heavy chain complementarity determination region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO: 12, the heavy chain complementarity determination region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain complementarity determination region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 17. Alternatively, it may include the heavy chain complementarity determination region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO: 13, the heavy chain complementarity determination region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain complementarity determination region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 17. Alternatively, it may include the heavy chain complementarity determination region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO: 12, the heavy chain complementarity determination region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain complementarity determination region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 17. Alternatively, it may include the heavy chain complementarity-determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO: 13, the heavy chain complementarity-determining region 2 (HCDR2) of the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain complementarity-determining region 3 (HCDR3) of the amino acid sequence shown in SEQ ID NO: 17; and (b) Includes a light chain backbone comprising the amino acid sequence shown in SEQ ID NO: 26, wherein the light chain complementary region: Including the light chain complementarity-determining region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity-determining region 2 (LCDR2) of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity-determining region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 20. Alternatively, it may include the light chain complementarity determination region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity determination region 2 (LCDR2) of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity determination region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 21. Alternatively, it may include the light chain complementarity determination region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity determination region 2 (LCDR2) of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity determination region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 22. Alternatively, it may include the light chain complementarity determination region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity determination region 2 (LCDR2) of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity determination region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 23. Alternatively, it may include light chain complementarity determination region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO: 18, light chain complementarity determination region 2 (LCDR2) of the amino acid sequence shown in SEQ ID NO: 19, and light chain complementarity determination region 3 (LCDR3) of the amino acid sequence shown in SEQ ID NO: 24.

[0011] In some implementations, the VH includes: The heavy chain variable region backbone region 1 (H-FR1) contains at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in any of SEQ ID NO: 87-89. The heavy chain variable region backbone region 2 (H-FR2) contains at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 90 or 100. Includes a heavy chain variable region backbone region 3 (H-FR3) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 91-94; and / or The heavy chain variable region backbone region 4 (H-FR4) contains at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 95.

[0012] In some implementations, the VL includes: It includes a light chain variable region backbone region 1 (L-FR1) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 96. It includes a light chain variable region backbone region 2 (L-FR2) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 97. Contains a light chain variable region backbone region 3 (L-FR3) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 98; and / or It contains a light chain variable region backbone region 4 (L-FR4) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 99.

[0013] In some embodiments, the VH has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-6; and / or The VL has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequences selected from SEQ ID NO: 7-11.

[0014] In some implementations, the VH and VL are selected from combinations of the following: VH containing the amino acid sequence shown in SEQ ID NO: 1; and VL containing the amino acid sequence shown in SEQ ID NO: 7; or VH, comprising the amino acid sequence shown in SEQ ID NO: 1; and VL, comprising the amino acid sequence shown in SEQ ID NO: 8; or VH, comprising the amino acid sequence shown in SEQ ID NO: 1; and VL, comprising the amino acid sequence shown in SEQ ID NO: 9; or VH comprising the amino acid sequence shown in SEQ ID NO: 1; and VL comprising the amino acid sequence shown in SEQ ID NO: 10; or VH comprising the amino acid sequence shown in SEQ ID NO: 1; and VL comprising the amino acid sequence shown in SEQ ID NO: 11; or VH, comprising the amino acid sequence shown in SEQ ID NO: 2; and VL, comprising the amino acid sequence shown in SEQ ID NO: 7; or VH, comprising the amino acid sequence shown in SEQ ID NO: 2; and VL, comprising the amino acid sequence shown in SEQ ID NO: 8; or VH containing the amino acid sequence shown in SEQ ID NO: 2; and VL containing the amino acid sequence shown in SEQ ID NO: 9; or VH comprising the amino acid sequence shown in SEQ ID NO: 2; and VL comprising the amino acid sequence shown in SEQ ID NO: 10; or VH comprising the amino acid sequence shown in SEQ ID NO: 2; and VL comprising the amino acid sequence shown in SEQ ID NO: 11; or VH containing the amino acid sequence shown in SEQ ID NO: 3; and VL containing the amino acid sequence shown in SEQ ID NO: 7; or VH, comprising the amino acid sequence shown in SEQ ID NO: 3; and VL, comprising the amino acid sequence shown in SEQ ID NO: 8; or VH containing the amino acid sequence shown in SEQ ID NO: 3; and VL containing the amino acid sequence shown in SEQ ID NO: 9; or VH containing the amino acid sequence shown in SEQ ID NO: 3; and VL containing the amino acid sequence shown in SEQ ID NO: 10; or VH containing the amino acid sequence shown in SEQ ID NO: 3; and VL containing the amino acid sequence shown in SEQ ID NO: 11; or VH, comprising the amino acid sequence shown in SEQ ID NO: 4; and VL, comprising the amino acid sequence shown in SEQ ID NO: 7; or VH comprising the amino acid sequence shown in SEQ ID NO: 4; and VL comprising the amino acid sequence shown in SEQ ID NO: 8; or VH, comprising the amino acid sequence shown in SEQ ID NO: 4; and VL, comprising the amino acid sequence shown in SEQ ID NO: 9; or VH comprising the amino acid sequence shown in SEQ ID NO: 4; and VL comprising the amino acid sequence shown in SEQ ID NO: 10; or VH containing the amino acid sequence shown in SEQ ID NO: 4; and VL containing the amino acid sequence shown in SEQ ID NO: 11; or VH, comprising the amino acid sequence shown in SEQ ID NO: 5; and VL, comprising the amino acid sequence shown in SEQ ID NO: 7; or VH comprising the amino acid sequence shown in SEQ ID NO: 5; and VL comprising the amino acid sequence shown in SEQ ID NO: 8; or VH, comprising the amino acid sequence shown in SEQ ID NO: 5; and VL, comprising the amino acid sequence shown in SEQ ID NO: 9; or VH comprising the amino acid sequence shown in SEQ ID NO: 5; and VL comprising the amino acid sequence shown in SEQ ID NO: 10; or VH comprising the amino acid sequence shown in SEQ ID NO: 5; and VL comprising the amino acid sequence shown in SEQ ID NO: 11; or VH comprising the amino acid sequence shown in SEQ ID NO: 6; and VL comprising the amino acid sequence shown in SEQ ID NO: 7; or VH comprising the amino acid sequence shown in SEQ ID NO: 6; and VL comprising the amino acid sequence shown in SEQ ID NO: 8; or VH comprising the amino acid sequence shown in SEQ ID NO: 6; and VL comprising the amino acid sequence shown in SEQ ID NO: 9; or VH comprising the amino acid sequence shown in SEQ ID NO: 6; and VL comprising the amino acid sequence shown in SEQ ID NO: 10; or VH contains the amino acid sequence shown in SEQ ID NO: 6; and VL contains the amino acid sequence shown in SEQ ID NO: 11.

[0015] In some embodiments, the antibody or its antigen-binding portion is an antibody or its antigen-binding portion of a human synthetic library, preferably a fully human antibody or its antigen-binding portion.

[0016] In some embodiments, the antibody or its antigen-binding portion further comprises an immunoglobulin Fc region or a functional variant thereof, preferably a human immunoglobulin Fc region or a functional variant thereof, more preferably the Fc region of human IgG1, IgG2, IgG3 or IgG4 or a functional variant thereof. Optionally, functional variants of the Fc region may include point mutations or modifications to reduce or weaken ADCC activity, ADCP activity, CDC activity, or prolong antibody PK.

[0017] In some embodiments, the antibody or its antigen-binding portion further includes a constant region, preferably: I. The constant region connected to the light chain is SEQ ID NO: 43 or SEQ ID NO: 44; and the constant region connected to the heavy chain is selected from SEQ ID NO: 45-73; or II. The constant region connected to the light chain is SEQ ID NO: 43; and the constant region connected to the heavy chain is SEQ ID NO: 66. More preferably, the variable region is directly connected to the constant region or connected through a connector.

[0018] In some implementations, (a) The antibody is a monoclonal antibody; and / or (b) The antigen-binding portion is selected from Fab, Fab', F(ab')2, Fv, scFv, nanobody, Fd and Fd'.

[0019] In a second aspect, the present invention provides a fusion protein comprising the antibody or its antigen-binding portion described in the first aspect.

[0020] Thirdly, the present invention provides a multispecific antibody comprising: (a) the antibody or its antigen-binding portion as described in the first aspect, or the fusion protein as described in the second aspect; and (b) At least one other antigen-binding region, wherein the at least one other antigen-binding region binds to a different antigen or a different epitope of the same antigen to the antibody or its antigen-binding portion thereof; Preferably, the multispecific antibody is a bispecific antibody composed of the antibody described in the first aspect or its antigen-binding portion and an antibody targeting other targets of autoimmune diseases, wherein the other targets of autoimmune diseases are preferably antibodies targeting the following targets: TL1A, α4β7, TNF-α and PD-1; More preferably, the multispecific antibody is a bispecific antibody constructed by combining the antibody described in the first aspect or its antigen-binding portion with the scFv sequence of an antibody selected from Tulisokibart, Duvakitug, Afimkibart, Vedolizumab, Risenzizumab, Gusnilimab, Rosnilimab, Adalimumab, and infliximab, preferably wherein the scFv sequence is linked to the C-terminus (e.g., the C-terminus of the light chain or the C-terminus of the heavy chain) of the antibody described in the first aspect or its antigen-binding portion via a linker. Preferably, the multispecific antibody is a bispecific antibody constructed from the antibody or its antigen-binding portion described in the first aspect and a heavy chain sequence (preferably containing a heavy chain variable region selected from SEQ ID NO: 35, 37, 39, 41) and a light chain sequence (preferably containing a light chain variable region selected from SEQ ID NO: 36, 38, 40, 42) targeting TL1A, especially wherein the heavy chain sequence or light chain sequence of the antibody targeting TL1A is connected to the C-terminus (e.g., the C-terminus of the light chain or the C-terminus of the heavy chain) of the antibody or its antigen-binding portion described in the first aspect via a adapter, or the antibody or its antigen-binding portion described in the first aspect is connected to the C-terminus (e.g., the C-terminus of the light chain or the C-terminus of the heavy chain) of the antibody heavy chain sequence or the light chain sequence targeting TL1A via a adapter. Preferably, the multispecific antibody is an IgG-scFv structure constructed from the antibody described in the first aspect or its antigen-binding portion, the structure having biological activity in vitro and in vivo, such as having synergistic inhibitory activity; Preferably, the multispecific antibody is a bispecific antibody comprising the antibody described in the first aspect or its antigen-binding portion, for the treatment or prevention of inflammatory bowel disease (IBD), particularly ulcerative colitis (UC) and Crohn's disease (CD), as well as other autoimmune diseases or fibrosis-related diseases.

[0021] In some embodiments, the scFv in the multispecific or bispecific antibody of the present invention is a scFv further stabilized by internal disulfide bonds. For example, the antibody or antigen-binding portion thereof described in the first aspect further comprises amino acid substitutions (e.g., in the backbone region) to stabilize by internal disulfide bonds. In some embodiments, the heavy chain variable region of the antibody or antigen-binding portion thereof described in the first aspect comprises the amino acid sequence shown in SEQ ID NO: 106, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity with the sequence shown in SEQ ID NO: 106. In some embodiments, the light chain variable region of the antibody or antigen-binding portion thereof described in the first aspect comprises the amino acid sequence shown in SEQ ID NO: 107, or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 97% sequence identity with the sequence shown in SEQ ID NO: 107.

[0022] Fourthly, the present invention provides an isolated nucleic acid molecule that encodes the antibody or its antigen-binding portion as described in the first aspect, the fusion protein as described in the second aspect, or the multispecific antibody as described in the third aspect.

[0023] Fifthly, the present invention provides an expression vector comprising the nucleic acid molecule described in the fourth aspect, wherein the nucleic acid molecule is optionally operatively linked to an expression regulatory element.

[0024] In a sixth aspect, the present invention provides a recombinant cell comprising the nucleic acid molecule described in the fourth aspect or the expression vector described in the fifth aspect, wherein the recombinant cell is selected from mammalian cells or insect cells.

[0025] In a seventh aspect, the present invention provides a pharmaceutical composition comprising at least one component selected from the following: an antibody or antigen-binding portion thereof as described in the first aspect, a fusion protein as described in the second aspect, a multispecific antibody as described in the third aspect, a nucleic acid molecule as described in the fourth aspect, an expression vector as described in the fifth aspect, a recombinant cell as described in the sixth aspect; and optionally, at least one pharmaceutically acceptable carrier.

[0026] Eighthly, the present invention provides the use of the antibody or its antigen-binding portion according to the first aspect, the fusion protein according to the second aspect, the multispecific antibody according to the third aspect, the nucleic acid molecule according to the fourth aspect, the expression vector according to the fifth aspect, or the recombinant cell according to the sixth aspect in the preparation of a medicament for treating or preventing IL23-related diseases, wherein the IL23-related diseases are selected from autoimmune diseases, including but not limited to ankylosing spondylitis (AS), hidradenitis suppurativa (HS), plaque psoriasis (PsO), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis (UC), generalized pustular psoriasis (GPP), giant cell arteritis (GCA), and systemic lupus erythematosus. Erythematosus (SLE, especially cutaneous type), non-infectious uveitis (NIU), Behçet's disease (BD, especially with uveitis or intestinal involvement), Sjögren's syndrome (SjS), pyodermagangrenosum (PG), relapsing polychondritis (RP), VEXAS syndrome, adult-onset Still's disease (AOSD), rheumatoid arthritis (RA), multiple sclerosis (MS, especially classic RRMS), primary biliary cholangitis (PBC), sarcoidosis, and asthmatic COPD.

[0027] In a ninth aspect, the present invention provides a method for treating or preventing IL23-related diseases, comprising administering to a subject in need a therapeutically effective amount of a substance selected from: the antibody or its antigen-binding portion described in the first aspect, the fusion protein described in the second aspect, the multispecific antibody described in the third aspect, or the pharmaceutical composition described in the seventh aspect, wherein: (a) The subject is a mammal, preferably a human; (b) The IL23-related diseases are selected from autoimmune diseases, preferably from ankylosing spondylitis (AS), hidradenitis suppurativa (HS), plaque psoriasis (PsO), psoriatic arthritis (PsA), Crohn's disease (CD), ulcerative colitis (UC), generalized pustular psoriasis (GPP), giant cell arteritis (GCA), systemic lupus erythematosus (SLE, especially cutaneous type), non-infectious uveitis (NIU), Behçet's disease (BD, especially with uveitis or intestinal involvement), and Sjogren's syndrome (SjS).

[0028] In some embodiments, the method includes administering to the subject a combination of an antibody or antigen-binding portion thereof as described in the first aspect, a fusion protein as described in the second aspect, a multispecific antibody as described in the third aspect, or a pharmaceutical composition as described in the seventh aspect, and at least one other therapeutic agent.

[0029] In some embodiments, the other therapeutic agents may be any therapeutic agent for autoimmune diseases, including but not limited to corticosteroids such as prednisone, methylprednisolone, betamethasone, dexamethasone, or budesonide; nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, naproxen, diclofenac, celecoxib, or etoricoxib; conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) and immunosuppressants such as methotrexate, leflunomide, sulfasalazine, hydroxychloroquine, azathioprine, cyclosporine, ellamod, cyclophosphamide, mycophenolate mofetil, or tacrolimus; and biological disease-modifying antirheumatic drugs (bDMARDs) including tumor necrosis factor (TNF-α) inhibitors, integrin inhibitors, interleukin inhibitors, etc. Attached Figure Description

[0030] Figure 1 The binding affinity data of the candidate antibody to IL23 were analyzed by BLI assay. The binding activity of the candidate molecule of this invention is comparable to that of levothyroxine, and 4 times stronger than that of gusejinumab. Figure 2 The candidate antibody activates IL23 receptor-overexpressing reporter cells, and the inhibitory-induced activity of the candidate molecule of this invention is superior to that of levothyroxine or gusejinumab. Figure 3 The candidate antibody inhibits the release of IL17 induced by IL23-induced PBMC activation. The inhibitory activity of the candidate molecule of this invention is slightly higher than that of levothyroxine or gusejinumab. Figure 4 Bispecific antibodies activate reporter cells overexpressing the TL1A-DR3 receptor; the inhibitory activity of the bispecific antibody molecule of this invention against TL1A-induced activation is comparable to that of the candidate molecule. Figure 5 The bispecific antibody activates IL23 receptor-overexpressing reporter cells, and the inhibitory activity of the bispecific antibody molecule of the present invention against IL23-induced activating cells is comparable to that of the candidate molecule. Figure 6 The bispecific antibody inhibits the release of IL17 induced by IL23+TL1A-induced PBMC activation. The inhibitory activity of the bispecific antibody molecule of the present invention is higher than that of anti-TL1A antibody or anti-IL23 antibody, and slightly higher than that of the combination antibody. Detailed Implementation

[0031] To further understand the present invention, preferred embodiments will be described below with reference to examples. These descriptions are merely illustrative of the features and advantages of the present invention and are not intended to limit the scope of protection of the present invention.

[0032] Unless otherwise defined, all techniques, symbols, and other scientific terms used in this invention are intended to have the meaning commonly understood by those skilled in the art. In some cases, the invention defines terms with a generally understood meaning for clarity and / or at reference, and the inclusion of such definitions in the invention should not necessarily be construed as representing a difference from the common understanding in the art. Those skilled in the art will generally fully understand the techniques and procedures described or referenced in this invention and will generally employ conventional methods, such as Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 4th edition (2012), Cold Spring Harbor Laboratory Press, NY. Unless otherwise stated, procedures involving the use of commercially available kits and reagents are generally performed according to the manufacturer's defined protocols and conditions.

[0033] The terms "comprising," "including," or "having" mean that any of the listed elements must be included, and other elements may optionally be included. The term "consisting of" means that all unlisted elements are excluded. The terms "comprising," "including," or "having" cover "consisting of" or "substantially consisting of."

[0034] The term “about” or “approximately”, when applied to one or more values ​​of interest, refers to a value that is close to or within an acceptable error range of the stated reference value, the acceptable error range being determined by those skilled in the art and depending in part on how the value is measured or determined, such as limitations of the measurement system. In one instance, the term “about” refers to any value within a variation range of up to ±10% or up to ±5% of the value modified by the term “about,” including both integer and fractional components. Alternatively, according to practice in the art, “about” can mean within 3 or more standard deviations. Or, for example, for biological systems or processes, the term “about” can mean within an order of magnitude of the value, within 5 times in some embodiments, and within 2 times in others. As described herein, when used to refer to values, the terms “about,” “approximately,” and “comparable to” refer to values ​​similar to a reference value in the context of the reference value. Generally, those skilled in the art will understand the relative degree of variation covered by the contexts “about,” “approximately,” and “comparable to.” For example, in some embodiments, the terms “about,” “approximately,” and “comparable to” may include a range of values ​​of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0035] The term "amino acid" refers to twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala;A), arginine (Arg;R), asparagine (Asn;N), aspartic acid (Asp;D), cysteine ​​(Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V).

[0036] The term "conservative amino acid substitution" refers to the replacement of an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acid it replaces. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid substitutions are well known in the art and can be selected based on the properties of the 20 major amino acids defined in Table A below. In cases where the amino acids have similar polarity, this can also be determined with reference to the table of hydrophilicity of amino acid side chains in Table B.

[0037] Table A - Chemical Properties of Amino Acids

[0038] Table B - Hydrophilicity Measurement

[0039] As is well known, conserved substitutions between amino acids with similar properties usually do not affect the activity of peptide sequences. Conserved substitutions are shown in Table C.

[0040] Table C Conservative Amino Acid Substitution

[0041] The term "antibody" is used herein in its broadest sense to include certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies include, among others, complete antibodies (e.g., complete immunoglobulins), antibody fragments, and multispecific antibodies. Antibodies according to the invention can be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4).

[0042] The term "human antibody" refers to an antibody having an amino acid sequence that corresponds to antibodies produced by humans or human cells, or an antibody derived from a non-human source using a human antibody lineage or a human antibody coding sequence (e.g., obtained from or redesigned from a human source). Human antibodies do not specifically include humanized antibodies.

[0043] "Fab" (also known as an antigen-binding fragment) comprises a constant domain of the light chain (CL) and a first constant domain (CH1) of the heavy chain, as well as variable domains on the VL and VH of both the light and heavy chains. The variable domains include complementarity-determining regions (CDRs, also known as hypervariable regions) involved in antigen binding. The term "Fab'" differs from Fab in that several residues are added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region.

[0044] “F(ab')2” contains two Fab' fragments linked by disulfide bonds near the hinge region. F(ab')2 can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab')2 can dissociate, for example, by treatment with β-mercaptoethanol.

[0045] The “Fv” fragment consists of a non-covalently linked dimer of a heavy chain variable domain and a light chain variable domain.

[0046] A “single-chain Fv” or “sFv” or “scFv” comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun, *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994). A “disulfide-bonded scFv” refers to an scFv further stabilized by internal disulfide bonds. An “IgG-scFv” refers to a multispecific molecular structure comprising an IgG-type antibody and an scFv fused to the C-terminus or N-terminus of the IgG heavy or light chain via a linker. Preferably, the IgG-type antibody and the scFv target different targets (antigens or epitopes). Preferably, the scFv in the IgG-scFv structure is a disulfide bond scFv.

[0047] The “scFv-Fc” fragment includes an scFv linked to an Fc domain. For example, the Fc domain may be linked to the C-terminus of the scFv. Depending on the orientation of the variable domains (i.e., VH-VL or VL-VH) in the scFv, the Fc domain may follow VH or VL. Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain includes an IgG4 Fc domain.

[0048] The term "single-domain antibody" or "sdAb" refers to a molecule in which one of the variable domains of an antibody binds specifically to an antigen in the absence of other variable domains. Single-domain antibodies and their fragments are described in Arabici Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245. Single-domain antibodies are also known as sdAbs or nanobodies. SdAbs are very stable and readily expressed as fusion couplers with the Fc chain of the antibody (Harmsen MM, De Haard HJ (2007). "Properties, production, and applications of camelid single-domain antibody fragments". Appl. Microbiol Biotechnol. 77(1):13-22).

[0049] The term "Fd" refers to the portion obtained from the heavy chain when IgG antibodies are hydrolyzed with papain, containing the VH and CH1 domains. "Fd'" refers to the fragment produced when antibodies are hydrolyzed with pepsin; it is similar to the Fd fragment but has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain.

[0050] The term "multispecific antibody" refers to an antibody that contains two or more different antigen-binding domains, which typically bind specifically to two or more different epitopes.

[0051] A "monospecific antibody" is an antibody that contains one or more binding sites that specifically bind to a single epitope. An example of a monospecific antibody is the naturally occurring IgG molecule, which, although bivalent (i.e., having two antigen-binding domains), recognizes the same epitope in each of the two domains. Binding specificity can exist at any suitable valence.

[0052] The term "affinity" refers to the strength of the total non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope). As used in this invention, unless otherwise stated, "affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).

[0053] Term "K" off (sec) -1 ( ) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also called K. off value.

[0054] Term "K" on (M) -l ×sec -1 (K) refers to the binding rate constant of a specific antibody-antigen interaction. This value is also called K. on value.

[0055] Term "K" D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. K D =K off / K on In some implementations, the antibody affinity is expressed as K... D To describe the interaction between this antibody and its antigen. For clarity, as described in the art, the smaller K... D A value of K indicates a higher affinity interaction, while a larger K value indicates a lower affinity interaction. D The value indicates a lower affinity interaction.

[0056] The terms "antagonist," "inhibitor," "neutralizer," or "blocker," when used to refer to an antigen-binding site or a molecule containing an antigen-binding site, mean that the binding of the antigen-binding site or molecule to its target results in the inhibition of at least some biological activities of the target. In this invention, "synergistic inhibitory activity" means that the inhibitory effect of the bispecific antibody is greater than that of either monoclonal antibody alone, or greater than that of the equimolar combination group.

[0057] "Fc mutation or modification," or "Fc functional variant," refers to the substitution of amino acids in the CH2 and CH3 regions of an antibody to alter its binding to the Fc receptor or to change its function after binding to the antigen. Mutation sites include human IgG1, S298A / E333A / K334A, S298A / E333A / K334A / K326A (Lu Y, Vernes JM, Chiang N et al., J Immunol Methods., 2011 Feb 28; 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L (Stavenhagen JB, Gorlatov S, Tuaillon N et al., Cancer Res., September 15, 2007; 67(18):8882-90; Nordstrom JL, Gorlatov S, Zhang W et al., Breast Cancer Res., November 30, 2011; 13(6):R123); F243L (Stewart R, Thom G, Levens M et al., Protein Eng Des Sel., September 2011; 24(9):671-8.), S298A / E333A / K334A (Shields RL, Namenuk AK, Hong K et al., J Biol Chem., March 2, 2001; 276(9):6591-604); S239D / I332E / A330L, S239D / I332E (Lazar GA, Dang W, Karki S et al., Proc NatlAcad Sci USA., March 14, 2006; 103(11):4005-10); S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S et al., Mol Immunol.September 2008; 45(15):3926-33), and 239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A33 0L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and other mutations listed in WO2011 / 120134 and WO2011 / 120135. In some embodiments, the Fc region includes one or more amino acid substitutions, wherein, compared to an Fc without one or more substitutions, one or more substitutions result in an increased antibody half-life, and a decrease or increase in one or more of ADCC activity, ADCP activity, or CDC activity. ADCC refers to antibody-dependent cell-mediated cytotoxicity; ADCP refers to antibody-dependent cell phagocytosis; and CDC refers to complement-dependent cytotoxicity. In some embodiments, compared to antibodies containing a wild-type Fc region, one or more amino acid substitutions result in prolonged antibody PK (pharmacokinetics), meaning the antibody is more stable and has a longer duration of action in vivo, preferably with a prolonged half-life. In some embodiments, one or more amino acid substitutions result in an increased antibody half-life at pH 6.0 compared to antibodies containing a wild-type Fc region. In some embodiments, the half-life of isolated antibodies containing an Fc region with one or more amino acid substitutions in humans is approximately 80 to 110 days.

[0058] Human antibody Fc mutations include one or more amino acid substitutions selected from the following groups: S228P (SP); M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E380A, M428L, N434A, N434S, N297A, D265A, L234A, L235A, and N434W.In some embodiments, the one or more amino acid substitutions are selected from the following amino acid substitutions: M428L / N434S (LS); M252Y / S254T / T256E (YTE); T250Q / M428L; T307A / E380A / N434A; T256D / T307Q (DQ); T256D / T307W (DW); M252Y / T256D (YD); T307Q / Q311V / A378V (QVV); T256D / H285D / T307R / Q311V / A378V (DVV). DRVV); L309D / Q311H / N434S; S228P / L235E (SPLE); L234A / L235A (LA), M428L / N434A L234A / G237A (LALA), L234A / L235A / G237A, L234A / L235A / P329G, N297A, D265A / YTE, LALA / YTE, LAGA / YTE, LALAGA / YTE, LALAPG / YTE, N297A / LS; D265A / LS; LALA / LS; SP / YTE;SPLE / YTE;SP / LS;SPLE / LS, SP / DHS;SPLE / DHS;N297A / LA;D265A / LA, LALA / LA, LAGA / LA, LALA / LA, LALA / LA, LALA PG / LA, N297A / N434A; D265A / N434A; LALA / N434A, LAGA / N434A, LALAGA / N434A, LALAPG / N434A, N297A / N434W, D265A / N434W, LALA / N434W, LAGA / N434W, LALAGA / N434W, LALA PG / N434W, N297A / DQ, D265A / DQ, LALA / DQ, LAGA / DQ, LAL AGA / DQ, LALAPG / DQ, N297A / DW, D265A / DW, LALA / DW, LAGA / DW, LALAGA / DW, LALAPG / DW N297A / YD, D265A / YD, LALA / YD, LAGA / YD, LALAGA / YD, LALAPG / YD, T307Q / Q311V / A378V (QVV), N297A / QVV, D265A / QVV, LALA / QVV, L AGA / QVV, LALAGA / QVV, LALAPG / QVV, DDRVV, N297A / DDRVV, D265A / DDRVV, LALA / DDRVV, LAGA / DDRVV, LALAGA / DDRVV and LALAPG / DDRVV.In some embodiments, the one or more amino acid substitutions are selected from: LALA / YTE, LAGA / YTE, LALA / LS, YTE, and LS. In some embodiments, the one or more amino acid substitutions comprise or consist of LALA / YTE. In some embodiments, the one or more amino acid substitutions comprise or consist of LAGA / YTE. In some embodiments, the one or more amino acid substitutions comprise or consist of LALA / LS. In some embodiments, the one or more amino acid substitutions comprise or consist of YTE. In some embodiments, the one or more amino acid substitutions comprise or consist of LS, and these mutations are incorporated herein by reference.

[0059] The term "linker" refers to an amino acid sequence that connects two polypeptide domains. In some embodiments, variable regions in an antibody or its antigen-binding fragment are linked to constant regions via linkers; for example, a light chain variable region (preferably at its C-terminus) is linked to a light chain constant region via a linker, and a heavy chain variable region (preferably at its C-terminus) is linked to a heavy chain constant region via a linker. In some embodiments, individual antigen-binding domains in a multispecific antibody are linked via linkers. In some embodiments, the linker is a P2A linker or a GS linker, such as (G4S)n, where n is an integer from 1 to 20, preferably an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Preferably, the adapter comprises an amino acid sequence shown in any one of SEQ ID NO: 101-105, or an amino acid sequence having at least 80%, 85%, 90%, 95% or 97% sequence identity with the sequence shown in any one of SEQ ID NO: 101-105.

[0060] An antibody's "antigen-binding fragment" comprises a portion of the complete antibody that remains capable of binding to the antigen. In some embodiments, papain digestion of the antibody yields two identical antigen-binding fragments, called the "Fab" fragment, and a residual "Fc" fragment, the names reflecting its ease of crystallization. The Fab fragment consists of the entire light chain variable region domain (Vc) of the heavy chain. H ) and the first constant structural domain of a heavy chain (C H1 The Fab fragment is composed of two disulfide-linked Fab fragments. Each Fab fragment is monovalent in its antigen binding, meaning it has a single antigen-binding site. In some embodiments, treating the antibody with pepsin produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments in that they are located at C... H1Several residues were added to the carboxyl terminus of the domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH stands for Fab', where the cysteine ​​residues of the constant domain have free thiol groups. The F(ab')2 antibody fragments were initially generated as Fab' fragment pairs with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0061] The term "antigen binding site" refers to any molecule or any part of a molecule capable of binding to an antigen. In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable region ("V") of the heavy chain ("H") and the light chain ("L"). Three highly divergent segments within the V region of the heavy and light chains are called "hypervariable regions," situated between more conserved flanking segments called "frame regions" or "FRs." Thus, the term "FR" refers to the naturally occurring amino acid sequence between and near the hypervariable regions of an immunoglobulin. In human antibodies, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged opposite each other in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of antigen binding, and the three hypervariable regions of the heavy and light chains are called "complementarity-determining regions" or "CDRs." In some animals, such as camels and cartilaginous fish, the antigen binding site may be formed by a single antibody chain, providing a "single-domain antibody." Antigen binding sites may also be present in antibody mimics.

[0062] CDRs can be determined by the methods described in Kabat et al., J. biol. chem. 252, 6609-6616 (1977), Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. mol. biol. 196: 901-917 (1987), and MacCallum et al., J. mol. biol. 262: 732-745 (1996). CDRs determined according to these definitions typically include overlapping or subsets of amino acid residues when compared with each other. In some embodiments, the term "CDR" is the CDR defined by MacCallum et al., J. mol. biol. 262: 732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann, and Dübel ed., pp. 422-439, Chapter 31, Springer-Verlag, Berlin (2001). In some embodiments, the term "CDR" is the CDR defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In some embodiments, the heavy chain CDR and light chain CDR of the antibody are defined using different conventions. For example, in some embodiments, the heavy chain CDR is defined according to MacCallum's definition, and the light chain CDR is defined according to Kabat's definition. HCDR1, HCDR2, and HCDR3 represent heavy chain CDRs, while LCDR1, LCDR2, and LCDR3 represent light chain CDRs.

[0063] The “binding affinity” of the molecules disclosed herein (e.g., antigen-binding sites) for a given target (e.g., IL23, IL23R & IL12Rβ1 complex) can be determined by a variety of methods known to those skilled in the art. Such methods include, but are not limited to, fluorescence titration, ELISA (enzyme-linked immunosorbent assay), calorimetry (e.g., isothermal titration calorimetry (ITC)), and surface plasmon resonance (SPR).

[0064] The term "bispecific" refers to a molecule capable of specifically binding to at least two different targets. Typically, a bispecific molecule comprises two antigen-binding sites, each specific to a different target. In some embodiments, a bispecific molecule can bind to two targets simultaneously.

[0065] The term "detectable affinity" refers to affinity measured by an affinity constant (usually expressed as K). D or EC 50 The ability of a given target to bind to a target (measured) typically has an affinity constant of about 10. -5 M or smaller (lower K) D or EC 50 The value reflects a better binding ability.

[0066] The term "epitope" refers to a specific antigen-binding site (composed of six complementarity-determining regions of the antibody) that interacts with a variable region of an antibody molecule. A single antigen can have multiple epitopes. Epitopes can be conformational or linear. Conformational epitopes consist of spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes consist of adjacent amino acid residues in a polypeptide chain.

[0067] The “Fc” fragment comprises the carboxyl-terminal portion of two heavy chains linked together by disulfide bonds. The effector function of the antibody is determined by the sequence of the Fc region, which is also recognized by Fc receptors in certain cell types. Regarding antigens (e.g., the IL23, IL23R & IL12Rβ1 complex), the term “fraction” refers to an N-terminal and / or C-terminal truncation or protein domain of the antigen. In some embodiments, the antigen fragment retains the ability of the full-length antigen to be recognized and / or bound by the antigen-binding fragment of this disclosure.

[0068] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced and their progeny. Host cells include “transformers” (or “transformed cells”) and “transfectants” (or “transfected cells”), each comprising primary transformed or transfected cells and their derived progeny. The nucleic acid content of these progeny cells may not be entirely identical to that of the parent cells and may contain mutations. “Recombinant host cell” or “host cell” refers to a cell containing exogenous polynucleotides, regardless of the method used for insertion (e.g., direct uptake, transduction, f-conjugation, or other methods known in the art for producing recombinant host cells).

[0069] The term "monoclonal antibody" refers to an antibody derived from a population of essentially homogeneous antibodies; that is, the individual antibodies constituting the population are identical, except for the possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific (targeting a single antigenic site). Furthermore, each monoclonal antibody targets a single determinant on an antigen, unlike the various antibodies that typically contain targets against different determinants (epitopes). The modifier "monoclonal" indicates that the antibody is derived from a population of essentially homogeneous antibodies and should not be construed as requiring production by any particular method. For example, monoclonal antibodies can be prepared using hybridoma methods or recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries.

[0070] The term "reference level" generally refers to a level considered "normal" for comparative purposes, such as a properly controlled level. For example, in the context of enhancing antibody effector function, "reference level" could refer to the effector function level of an antibody containing the wild-type constant region (Fc). As another example, in the context of target binding, "reference level" could refer to the binding affinity level of the target's natural ligand (e.g., by K...). D or EC 50 (Value definition). The reference level can be determined simultaneously or predetermined, for example, it can be known or inferred from past observations.

[0071] The terms "subject" and "patient" refer to an organism that will be treated by the methods and compositions described in this invention. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cattle, pigs, canines, felines, etc.), and more preferably include humans.

[0072] The terms "therapeutic effective amount" and "effective amount" are used interchangeably, referring to an effective amount that achieves the desired therapeutic effect at the necessary dose and over a given period of time. Therapeutic effective amounts can vary depending on a variety of factors, such as the type of disease (e.g., cancer), disease state, individual age, sex, and / or weight, and the ability of the antibody (or its pharmaceutical composition) to elicit the desired response in the individual. An effective amount can also be the amount at which any toxic or adverse effects of the antibody or its pharmaceutical composition are offset by the beneficial therapeutic effect.

[0073] "Treating" a disease (e.g., the disease described in this invention, such as an autoimmune disease) is a method of achieving a beneficial or desired outcome (e.g., clinical outcome). Beneficial or desired outcomes may include, but are not limited to, alleviating or improving one or more symptoms or conditions, reducing the severity of a disease, disorder, or condition, stabilizing (i.e., not worsening) the state of a disease, disorder, or condition, preventing the spread of a disease, disorder, or condition, delaying or slowing the progression of a disease, condition, or symptom, improving or palliating a disease, condition, or symptom, and partial or complete remission, whether detectable or undetectable. Examples of autoimmune diseases for which this invention provides a more favorable treatment include, but are not limited to: psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, non-radioactive axial spondyloarthritis, hidradenitis suppurativa, uveitis, multiple sclerosis, systemic lupus erythematosus, cutaneous lupus erythematosus, Sjögren's syndrome, inflammatory bowel disease-associated arthritis, bowel disease-associated spondyloarthritis, giant cell arteritis, high-intensity arteritis, granulomatous polyangiitis, ANCA-associated vasculitis, Behçet's disease, dermatomyositis, polymyositis, antisynthetic enzyme syndrome, relapsing polychondritis, SAPHO syndrome, adult-onset Still's disease, and autoimmune diseases. Encephalitis, neuropsychiatric lupus, adverse reactions related to immune checkpoint inhibitors, intestinal fibrosis, Crohn's disease with fibrosis, atopic dermatitis, alopecia, chronic spontaneous urticaria, sarcoidosis, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, non-alcoholic steatohepatitis, idiopathic pulmonary fibrosis, connective tissue disease-associated interstitial lung disease, chronic graft-versus-host disease, systemic sclerosis, IgE-related diseases, eosinophilic granulomatous polyangiitis, pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, chronic relapsing multiple osteomyelitis, Sweet's syndrome, neutrophilia, and pyoderma gangrenosum.

[0074] The term "epitope" is an epitope that interacts with a specific antigen-binding site (composed of six complementarity-determining regions of the antibody) in a variable region of an antibody molecule. A single antigen may have multiple epitopes. Epitopes can be conformational or linear. Conformational epitopes consist of spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes consist of adjacent amino acid residues in a polypeptide chain. In one aspect, this application provides antigen-binding sites capable of binding to a given antigen (e.g., an antigen on a cell involved in an immune disorder). In some embodiments, the antigen-binding site is capable of binding to an epitope within the antigen. In some embodiments, the antigen-binding site is capable of binding to an antigen on a cell involved in an autoimmune, allergic, or inflammatory disease. In some embodiments, the cell is an immune cell, and the antigen is IL23.

[0075] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention are described clearly and completely below with reference to the accompanying drawings. The embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments described in the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The technical solutions of the present invention can be implemented in other specific forms without departing from their basic characteristics. Without conflict, any embodiment of the present invention can be combined with the technical features of one or more other embodiments to form new embodiments. The scope of protection of the present invention includes other embodiments formed by such combinations. All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety. If the use or terminology in a publication, patent, or patent application incorporated by reference conflicts with the use or terminology in this specification, the use and terminology in this specification shall prevail. Chapter headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter. Unless otherwise expressly defined, all technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. For the purposes of this specification, the following definitions apply, and where appropriate, singular terms include plural forms, and plural terms include singular forms.

[0076] Example 1: Screening of a fully human synthetic library

[0077] Using the fully human antibody backbone from the therapeutic antibody (TheraSabDab), a fully synthetic phage display library (VH / VLκ, VH / VLλ, library size >10e10) was constructed using a reported method (Bostrom, J., Fuh, G., 2009). The library phages were incubated with biotinylated human IL23 antigen protein (ACRO Biosystems, ILB-52W5) and IL12 antigen protein (ACRO Biosystems, IL-H4210) as negative filters. After enrichment with SA-Bead (Thermo, 88816), non-specifically bound phages were washed with PBST (containing 0.1% Tween-20) to remove them. The bound phages were eluted with 0.2M glycine-HCl (pH 2.2) and then infected with *E. coli* for amplification. Three rounds of affinity panning were performed to select 200 phage clones. Human IL23 protein (1 μg / mL) or IL12 protein (1 μg / mL) was coated onto 96-well plates. Phage clones enriched with IL23 and flow-through with IL12 were added and incubated. Binding signals were detected using HRP-labeled anti-M13 antibody, and IL23-positive and IL12-negative clones were selected. In IL23-coated 96-well plates, the supernatant of selected phage clones and human IL23R-Avi protein (ACRO Biosystems, ILR-H82F3) were added. Anti-IL23R positive signals were detected using SA antibody, and clones inhibiting IL23 / IL23R complex binding were selected. Phage clones were sequenced to obtain WT antibody sequences.

[0078] Using the variable region sequence of the WT antibody as a template, the CDR amino acids were randomly mutated to other amino acids (the mutant pAk plasmid was provided by Genewiz) to construct a Fab phage mutant library (Holger Yhie, 2009). The library phages were incubated with biotinylated human IL23 antigen protein (ACRO Biosystems, ILB-52W5) at protein concentrations of 2.5 nM, 0.25 nM, and 0.025 nM. The protein was enriched by SA-Bead (Thermo, 88816), and 100 clones were selected. Positive clones were detected by IL23 protein ELISA. The top 20 clones were sequenced, and repetitive sequences were removed. The amino acid sequences of the light and heavy chain variable regions of 17 representative clones are listed in Table 1, the CDR sequences in Table 2, the backbone structures in Table 3, the sequences of the positive control antibodies used in the screening in Table 4, and the extracellular sequences of the antigens used in the screening in Table 5-2.

[0079] Table 1 Representative clones

[0080] Table 2 Antibody CDR Sequences

[0081] Table 3 Antibody backbone sequence

[0082] Table 4 Positive control variable region sequences

[0083] Table 5-1 Antibody Constant Region

[0084] Table 5-2 Antigen Amino Acid Sequences

[0085] Example 2: Antibody preparation and purification

[0086] The variable regions of antibodies VL and VH in Table 1 were synthesized using DNA. The positive reference antibody in Table 4 was derived from the sequence disclosed in the patent. The human homologous control antibody (Biointron, B117901) was used. The variable regions were ligated to the constant regions of antibodies in Table 5-1 via PCR to form the coding sequences for antibodies HC and LC (where the heavy chain IgG1, IgG1-LALA / LS, IgG1-LALA / YTE, hIgG1-LS, and IgG1-LAGA / LS, hIgG1-LALAPG / LS constant regions serve as weakened ADCC mutations; for the light chain, each clone in Table 1 uses light chain λ, and the control antibody uses light chain κ; for each antibody of this invention, unless otherwise specified, the variable region is directly ligated to the constant region sequence at the C-terminus). These sequences were then subcloned into a pDNA 3.0 plasmid, and the HC and LC gene sequences in the expression vector were confirmed by DNA sequencing. Approximately 5.5 x 10⁻⁶ cells were used in shake flasks. 6 / mL of CHO cells (Baiying Biotech CRO transient expression) were used as host cells. A mixture of 1 mg / L DNA (HC plasmid + LC plasmid) and 7 mg / L PEI was added to OptiMEM. TMTransfection was initiated by adding 5.5 E6 / ml CHO cells to culture medium (Invitrogen, 31985070) and gently mixing. Cells were cultured in a shaker at 110 rpm, 37°C, and 8% CO2 for 9 days. Protein purification was performed using an AKTA purification system (GE Lifesciences) via affinity chromatography and ion exchange chromatography. Conditioned medium expressing IL23 antibody was harvested by centrifugation at 4000 rpm for 50 min and filtered through a 0.22 μm filter. The harvested supernatant was loaded onto a MabSelect™ SuRe™ (GE HealthCARE) column. After washing the column with buffer A (PBS, pH 7.4), the protein was eluted with buffer B (1M glycine, pH 2.7) and immediately neutralized with 1 / 10 volume of buffer D (1M sodium citrate, pH 6.0). The affinity-purified antibody buffer was then exchanged for PBS buffer. The purified samples were tested by SEC-HPLC and SDS-PAGE, and the purity was greater than 90% in both tests.

[0087] Example 3: Determination of antibody affinity by BLI

[0088] The binding kinetics of the prepared antibody to human IL23 (ACRO Biosystems, ILB-52W5) protein were detected using biomembrane interference (BLI) (Sartorius, ForteBio Red96). The antibody was immobilized onto an AHC2.0 biosensor (Cat. No. 18-5142). The antigen protein IL23 was diluted to four concentrations (100 nM, 50 nM, 25 nM, and 12.5 nM) with 0.02% PBST20, and injected. Kinetic analysis was performed using a 1:1 binding model, and the binding rate (Kb) was calculated. on ) and dissociation rate (K off ), equilibrium dissociation constant (K D ) is K off / K on The ratios and antibody affinities are shown in Table 6. The variable region sequences of the antibodies are shown in Table 1, the light chain constant region is light chain λ, the heavy chain constant region is IgG1-LALA / LS, and the constant region sequences are shown in Table 5-1.

[0089] Table 6. Affinity of mutant clones

[0090] Based on affinity values, candidate molecules (WP03-1, clone 1) and positive control antibodies were selected. IL23 was diluted to four concentrations (100 nM, 50 nM, 25 nM, and 12.5 nM) for affinity testing. Antibody affinities are shown in Table 7. Figure 1 As shown. The variable region sequence of clone 1 is shown in Table 1, and the variable region sequence of the positive control antibody is shown in Table 4. The light chain constant region of clone 1 uses λ while the positive control uses κ. The heavy chain constant region is IgG1-LALA / LS. The constant region sequence is shown in Table 5-1.

[0091] Table 7. Multi-site BLI affinity of candidate molecules

[0092] Example 4: ELISA detection of IL23 binding in humans / cynomolgus monkeys / mice

[0093] The binding of antibody candidate molecules to human / cynomolgus monkey / mouse IL23 protein was detected using ELISA. Human IL23 (ACRO Biosystems, ILB-H52W5), cynomolgus monkey IL23 protein (ACRO Biosystems, ILB-C52W3), or mouse IL23 protein (ILB-M52W7) was diluted with PBS to 1 μg / mL, and 100 µl was added to each well of the ELISA plate. The plate was incubated overnight at 2–8°C, followed by blocking with 200 µl of blocking buffer (0.05% Tween 20 + 2% BSA + PBS) at room temperature for 1 hour. The diluted antibody was then added to each well at 100 µl. The plate was incubated with gentle shaking at room temperature for 1 hour. The washing step was repeated three times. The enzyme-labeled secondary antibody (goat anti-human IgG-HRP, ABCAM, AB204901) was diluted 1:10000 with blocking buffer. Add 100 µL of diluted secondary antibody to each well, wash, and then add 100 µL of TMB substrate solution (Solepro, PR1200) to each well. Incubate the plate at room temperature in the dark for 10 minutes. Add 50 µL of sulfuric acid stop solution to each well. Within 30 minutes of adding the stop solution, measure the absorbance at 450 nm using a microplate reader (ENVISION, PerkinElmer). The results are shown in Table 8. The candidate molecule can bind to human, cynomolgus monkey, and mouse IL23 protein.

[0094] Table 8 Antibody binding to recombinant IL23 protein

[0095] Example 5: Inhibition of inhibitory signaling in reporter cells of the IL23-IL23R&IL12Rβ1 complex by antibody

[0096] Free 20 ng / ml human IL23 (ACRO Biosystems, ILB-52W5) diluted with DMEM and 293-IL23R & IL12Rβ1-Luc cells (1E5 cells / well, using HEK-293-stats-luc (BPS, #79800-P) as the cell line, co-electropographed pDNA3.1-IL23R (SEQ ID NO:80) and pDNA3.1-IL12Rβ1 (SEQ ID NO:81) plasmids (synthesized by Genewiz), and double-positive cell pools obtained by FACs sorting) were seeded into wells containing the test antibody at a starting concentration of 100 nM, diluted 1:4 in 7 gradients, and incubated for 6 h. The luciferase substrate (Bright-Glo™, Luciferase Substrate, Cat. No. DD1204-03, Vazyme) was added to the corresponding wells. The chemiluminescence signal was detected using a microplate reader (Meigu, i3). The emission reading is expressed as RLU (Relative Light Unit), and IC is calculated. 50 The results are shown in Table 9 and Figure 2 .

[0097] Table 9

[0098] Example 6: Antibody inhibits the release of cytokines from primary PBMCs

[0099] T cells were pre-stimulated with anti-CD3 / CD28 magnetic beads and PBMCs (1:10), and the beads were removed overnight. PBMCs (Schbio, PBMNC100C, 1E5 cells / well) were mixed with 10 ng / ml human IL23 protein (ACRO Biosystems, ILB-52W5) and seeded in wells containing 100 nM of the anti-IL23 antibody to be tested. After incubation for 24 h, the cell supernatant was collected, and the IL17 concentration was detected by ELISA. The IL17 release ratio was calculated (negative control: anti-CD3 / CD28 magnetic beads; positive control: anti-CD3 / CD28 magnetic beads + 10 ng / ml IL23). The results are as follows: Figure 3 .

[0100] Example 7: Antibody Competitive Epitope Detection

[0101] Using biomembrane interference (BLI) (Sartorius, ForteBio Red96), the competitive binding of prepared antibodies to human IL23 antigen was investigated. Octet HIS1K (Fotebio, 18-5077) was used to immobilize 10ug / ml human IL23 (ACRO Biosystems, ILB-52W5) for 60s. Binding to antibody 1 (candidate antibody WP03-1, 100nM) took 200s, followed by binding to antibody 2 (100nM) for 100s (with levothyroxine, ustekinumab, and gusejinumab as positive controls). Octet epitope mapping analysis showed that the candidate antibodies exhibited 100% inhibition with gusejinumab, indicating competition for the same epitope; 30% inhibition with levothyroxine, indicating partial competition; and no inhibition with ustekinumab, indicating different epitopes.

[0102] Example 8: Preparation of Bispecific Antibodies

[0103] Candidate anti-IL23 antibodies and anti-TL1A antibodies were synthesized using DNA (Table 10), wherein the anti-TL1A antibody used was an antibody developed internally by the company (Table 11, X in this embodiment). 12 Let S and X be the values ​​of S and X. 13 Let P, X 14 Let V, X 18 For example, using PCR, the variable region was linked to the antibody constant region in Table 5-1 to form the coding sequences for antibodies HC and LC (heavy chain IgG1, IgG1-LALA / LS, light chain κ). The C-terminus of the heavy chain or the C-terminus of the light chain was linked to the scFv sequence of the second antibody via 2X(G4S), 3X(G4S), or 4X(G4S) (SEQ ID NO: 101-103). Subsequently, it was subcloned into the pDNA3.0 plasmid, and the HC and LC gene sequences in the expression vector were confirmed by DNA sequencing. The sequences of the constructed bispecific antibodies are shown in Table 12. Approximately 5.5 x 10⁻⁶ ppm of the antibody in the shake flask was used. 6 / mL of CHO cells (Baiying Biotech CRO transient expression) were used as host cells. A mixture of 1 mg / L DNA (HC plasmid + LC plasmid) and 7 mg / L PEI was added to OptiMEM. TMThe culture medium (Invitrogen, 31985070) was added to 5.5 E6 / ml CHO cells and gently mixed to begin transfection. Cells were cultured in an incubator shaker at 110 rpm, 37°C, and 8% CO2 for 9 days. Protein purification was performed using an AKTA purification system (GE Lifesciences) via affinity chromatography and ion exchange chromatography. Conditioned medium expressing IL23 antibody was harvested by centrifugation at 4000 rpm for 50 min and filtered through a 0.22 μm filter. The harvested supernatant was loaded onto a MabSelect™ SuRe™ (GE HealthCARE) column. After washing the column with buffer A (PBS, pH 7.4), the protein was eluted with buffer B (1M glycine, pH 2.7) and immediately neutralized with 1 / 10 volume of buffer D (1M sodium citrate, pH 6.0). The affinity-purified antibody buffer was exchanged for PBS buffer. The purified samples were tested by SEC-HPLC and SDS-PAGE, and the purity was greater than 90% in both tests.

[0104] Table 10

[0105] Table 11

[0106] Table 12

[0107] Example 9: Bispecific antibody inhibits the inhibitory signaling of the TL1A-DR3 complex reporter cells

[0108] Free 10 ng / ml human TL1A (ACRO Biosystems, TLA-H5243) and Jurkat-DR3 complex-Luc cells (Genomeditech, GM-C30290, 1E5 cells / well) were diluted with DMEM and seeded in seven 1:4 dilutions of the test antibody at a starting concentration of 100 nM. After incubation for 6 h, the luciferase substrate (Bright-Glo™, Luciferase Substrate, Cat. No. DD1204-03, Vazyme) was added to the corresponding wells. Chemiluminescence signals were detected using a microplate reader (Meigu, i3). Luminescence readings were expressed as RLU (Relative Light Units), and IC50 was calculated. 50 The results are shown in Table 13 and as follows. Figure 4The variable region of the anti-TL1A antibody corresponds to the sequence in Table 11, the constant region of the light chain is light chain κ, and the constant region of the heavy chain is IgG1-LALA / LS. The constant region sequence is shown in Table 5-1. The bispecific antibody (BisAb) targeting TL1A and IL23 corresponds to the sequence in Table 12.

[0109] Table 13

[0110] Example 10: Bispecific antibody inhibits the inhibitory signaling of the IL23-IL23R&IL12Rβ1 complex reporter cells.

[0111] Free 10 ng / ml human IL23 (ACRO Biosystems, ILB-52W5) and Jurkat-IL23R&IL12Rβ1 complex-Luc cells (1E5 cells / well) were diluted with DMEM and seeded in seven 1:4 dilutions of the test antibody at a starting concentration of 100 nM. After incubation for 6 h, the luciferase substrate (Bright-Glo™, Luciferase Substrate, Cat. No. DD1204-03, Vazyme) was added to the corresponding wells. Chemiluminescence signals were detected using a microplate reader (Meigu, i3). Luminescence readings were expressed as RLU (Relative Light Units), and IC50 was calculated. 50 The results are shown in Table 14 and as follows. Figure 5 Among them, the anti-IL23 antibody is WP03-1.

[0112] Table 14

[0113] Example 11: Antibody inhibits the release of cytokines from primary PBMCs

[0114] T cells were pre-stimulated with anti-CD3 / CD28 magnetic beads and PBMCs (1:10), and the beads were removed overnight. PBMCs (Schbio, PBMNC100C, 1E5 cells / well) were mixed with 10 ng / ml IL23 (ACRO Biosystems, ILB-52W5) and TL1A (ACROBiosystems, TLA-H5243) protein, and seeded into wells containing 100 nM of the antibody to be tested. After incubation for 24 h, the cell supernatant was collected, and the IL17 concentration was detected by ELISA. The IL17 release ratio was calculated (negative control: anti-CD3 / CD28 magnetic beads; positive control: anti-CD3 / CD28 magnetic beads + 10 ng / ml IL23 + 10 ng / ml TL1A). The results are shown below. Figure 6 .

[0115] This embodiment demonstrates that the anti-IL23 antibody within the scope of this invention blocks or inhibits the binding of the IL23-IL23R&IL12Rβ1 complex by binding to free IL23. The anti-IL23 antibody within the scope of this invention targets the IL23a subunit and exhibits more significant inhibitory activity against downstream signals of the IL23R&IL12Rβ1 complex. The anti-IL23 antibody within the scope of this invention weakens ADCC and CDC at the Fc level, reduces DC interference, and simultaneously prolongs the pharmacokinetic half-life due to LS mutation. When the anti-IL23 antibody within the scope of this invention is combined with an anti-TL1A antibody to form a bispecific antibody, the synergistic activity is higher than the combined inhibitory activity of the two antibodies. Having described at least several aspects and embodiments of the invention, it is understood that various equivalent substitutions, changes, modifications, and improvements will be readily apparent to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to conform to the spirit and scope of the invention. Therefore, the above description is by way of example only, and the following claims provide a detailed description of the invention.

[0116] All references cited in this invention, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as each reference individually and expressly indicated by reference and fully listed herein. The foregoing description is intended only to illustrate the technical solutions of this invention and does not limit its scope of protection. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solutions of this invention do not depart from the essence and scope of this invention.

Claims

1. An antibody or its antigen-binding moiety that specifically binds to IL23, characterized in that, The antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes: Ⅰ. A heavy chain complementarity-determining region 1 containing at least 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 82, wherein SEQ ID NO: 82 is NYWX0G; Wherein, X0 is selected from I or M; II. A heavy chain complementarity-determining region 2 containing at least 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 83, wherein SEQ ID NO: 83 is IIX1PSGSX2TWYAQKFQG; Wherein, X1 is selected from N or S; X2 is selected from T or S; III. A heavy chain complementarity-determining region 3 containing at least 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and The light chain variable region includes: I) Contains a light chain complementarity-determining region 1 having at least 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; II) Contains a light chain complementarity-determining region 2 having at least 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; III) Contains a light chain complementarity-determining region 3 having at least 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 84, wherein SEQ ID NO: 84 is X3SX4TX5X6X7X8X9X 10 X 11 ; Wherein, X3 is selected from A or S; X4 is selected from W or Y; X5 is selected from D or P; X6 is selected from T or S; The X7 is selected from P, S, or L; The X8 is selected from N, V, or S; X9 is selected from M, V, or L; The X 10 Selected from I, F, or V; The X 11 Choose from V or G.

2. The antibody or its antigen-binding portion according to claim 1, characterized in that, The antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes: i. A heavy chain complementarity-determining region 1 comprising the amino acid sequence shown in SEQ ID NO: 12; or a heavy chain complementarity-determining region 1 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 12; Alternatively, it may contain a heavy chain complementarity determination region 1 comprising the amino acid sequence shown in SEQ ID NO: 13; or a heavy chain complementarity determination region 1 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO:

13. ii. A heavy chain complementarity determination region 2 comprising the amino acid sequence shown in SEQ ID NO: 14; or a heavy chain complementarity determination region 2 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 14; Alternatively, it may contain a heavy chain complementarity determination region 2 comprising the amino acid sequence shown in SEQ ID NO: 15; or a heavy chain complementarity determination region 2 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO:

15. Alternatively, the heavy chain complementarity determination region 2 may contain the amino acid sequence shown in SEQ ID NO: 16; or the heavy chain complementarity determination region 2 may have one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO:

16. and iii. A heavy chain complementarity-determining region 3 comprising the amino acid sequence shown in SEQ ID NO: 17; or a heavy chain complementarity-determining region 3 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 17; and The light chain variable region includes: i) A light chain complementarity-determining region 1 containing the amino acid sequence shown in SEQ ID NO: 18; or a light chain complementarity-determining region 1 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 18; ii) A light chain complementarity determination region 2 comprising the amino acid sequence shown in SEQ ID NO: 19; or a light chain complementarity determination region 2 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 19; iii) A light chain complementarity determination region 3 comprising the amino acid sequence shown in SEQ ID NO: 20; or a light chain complementarity determination region 3 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 20; Alternatively, it may contain a light chain complementarity determination region 3 comprising the amino acid sequence shown in SEQ ID NO: 21; or a light chain complementarity determination region 3 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO:

21. Alternatively, it may contain a light chain complementarity determination region 3 comprising the amino acid sequence shown in SEQ ID NO: 22; or a light chain complementarity determination region 3 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO:

22. Alternatively, it may contain a light chain complementarity determination region 3 comprising the amino acid sequence shown in SEQ ID NO: 23; or a light chain complementarity determination region 3 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO:

23. Alternatively, it may contain a light chain complementarity determination region 3 comprising the amino acid sequence shown in SEQ ID NO: 24; or a light chain complementarity determination region 3 having one, two, or three amino acid differences compared to the amino acid sequence shown in SEQ ID NO:

24.

3. The antibody or its antigen-binding portion according to claim 1, characterized in that, The antibody or its antigen-binding portion includes: (a) The heavy chain backbone of the amino acid sequence shown in SEQ ID NO: 25, wherein the complementary region of the heavy chain is any combination of the following: Including the heavy chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 12, the heavy chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 14, and the heavy chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO: 17; Alternatively, it may include the heavy chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 13, the heavy chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 14, and the heavy chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

17. Alternatively, it may include the heavy chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 12, the heavy chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

17. Alternatively, it may include the heavy chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 13, the heavy chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

17. Alternatively, it may include the heavy chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 12, the heavy chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

17. Alternatively, it may include the heavy chain complementarity-determining region 1 of the amino acid sequence shown in SEQ ID NO: 13, the heavy chain complementarity-determining region 2 of the amino acid sequence shown in SEQ ID NO: 16, and the heavy chain complementarity-determining region 3 of the amino acid sequence shown in SEQ ID NO: 17; and (b) The light chain backbone of the amino acid sequence shown in SEQ ID NO: 26, wherein the complementary regions of the light chains are any combination of the following: Including the light chain complementarity-determining region 1 of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity-determining region 2 of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity-determining region 3 of the amino acid sequence shown in SEQ ID NO: 20; Alternatively, it may include the light chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

21. Alternatively, it may include the light chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

22. Alternatively, it may include the light chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

23. Alternatively, it may include the light chain complementarity determination region 1 of the amino acid sequence shown in SEQ ID NO: 18, the light chain complementarity determination region 2 of the amino acid sequence shown in SEQ ID NO: 19, and the light chain complementarity determination region 3 of the amino acid sequence shown in SEQ ID NO:

24.

4. The antibody or its antigen-binding portion according to claim 1, characterized in that, The heavy chain variable region has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences selected from SEQ ID NO: 1-6; and The light chain variable region has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences selected from SEQ ID NO: 7-11.

5. The antibody or its antigen-binding portion according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region are selected from the following combinations: The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 5; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9; or The heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6; and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10; or The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 6; and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

11.

6. The antibody or its antigen-binding portion according to claim 1, characterized in that, The antibody or its antigen-binding portion is an antibody or its antigen-binding portion of a human synthetic library.

7. The antibody or its antigen-binding portion according to claim 1, characterized in that, The antibody or its antigen-binding portion further includes the immunoglobulin Fc region.

8. The antibody or its antigen-binding portion according to claim 1, characterized in that, The antibody or its antigen-binding portion further comprises a constant region, wherein: I. The constant region connected to the light chain is SEQ ID NO: 43 or SEQ ID NO: 44; and the constant region connected to the heavy chain is selected from SEQ ID NO: 45-73; or II. The constant region connected to the light chain is SEQ ID NO: 43; and the constant region connected to the heavy chain is SEQ ID NO:

66.

9. The antibody or its antigen-binding portion according to claim 1, characterized in that: (a) The antibody is a monoclonal antibody; or (b) The antigen-binding portion is selected from Fab, Fab', F(ab')2, Fv, scFv, nanobody, Fd and Fd'.

10. A fusion protein comprising the antibody or its antigen-binding portion as described in any one of claims 1-9.

11. A multispecific antibody comprising: (a) the antibody or antigen-binding portion thereof of any one of claims 1-9, or the fusion protein of claim 10; and (b) At least one other antigen-binding region, wherein the at least one other antigen-binding region binds to a different antigen or a different epitope of the same antigen to the antibody or its antigen-binding portion.

12. An isolated nucleic acid molecule encoding an antibody or antigen-binding portion thereof as described in any one of claims 1-9, a fusion protein as described in claim 10, or a multispecific antibody as described in claim 11.

13. An expression vector comprising the nucleic acid molecule of claim 12.

14. A recombinant cell comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13, wherein the recombinant cell is selected from mammalian cells or insect cells.

15. A pharmaceutical composition comprising at least one component selected from the group consisting of: an antibody or antigen-binding portion thereof according to any one of claims 1-9, a fusion protein according to claim 10, a multispecific antibody according to claim 11, a nucleic acid molecule according to claim 12, an expression vector according to claim 13, a recombinant cell according to claim 14; and at least one pharmaceutically acceptable carrier.

16. Use of the antibody or antigen-binding portion thereof according to any one of claims 1-9, the fusion protein according to claim 10, the multispecific antibody according to claim 11, the nucleic acid molecule according to claim 12, the expression vector according to claim 13, or the recombinant cell according to claim 14 in the preparation of a medicament for the treatment or prevention of IL23-related diseases, wherein the IL23-related diseases are autoimmune diseases.

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