Anti-tl1a / il-23 bispecific antibodies and uses thereof

Abstract

The present application relates to the field of biological medicine, and in particular to an anti-TL1A / IL-23 bispecific antibody and use thereof. The anti-TL1A / IL-23 bispecific antibody comprises a first antigen binding domain D1; and a second antigen binding domain D2; the D1 is an antibody or antigen binding fragment specifically binding to an antigen IL-23p19, and the D2 is an antibody or antigen binding fragment specifically binding to an antigen TL1A. The anti-TL1A / IL-23 bispecific antibody of the present application simultaneously targets two target points of TL1A and IL-23, has a synergistic effect, has a good curative effect on inflammatory diseases or disorders, and has application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an anti-TL1A / IL-23 bispecific antibody and its uses. Background Technology

[0002] Inflammatory bowel disease (IBD) is a specific type of chronic inflammatory bowel disease, including Crohn's disease (CD) and ulcerative colitis (UC). The etiology and pathogenesis of IBD are not fully understood, but it is generally believed to be caused by the interaction of multiple factors, such as abnormal intestinal mucosal immune regulation, persistent intestinal infection, intestinal mucosal barrier defects, genetic factors, and environmental factors. Both intrinsic and adaptive immune responses of the intestinal mucosa are involved in the pathogenesis of IBD, and abnormal immune responses of the intestinal mucosa are an important cause of mucosal tissue damage.

[0003] TL1A (also known as tumor necrosis factor superfamily member 15 [TNFSF15]) is a member of the TNF ligand superfamily and, like other members, is a homotrimer. TL1A is a type II transmembrane protein that exists in both membrane-bound and soluble forms. Its receptors include DR3 and soluble decoy receptor 3 (DcR3). DR3, also known as APO3, LARD, or WSL1, is a member of the tumor necrosis factor receptor superfamily (TNFRSF). TL1A is a ligand of DR3; upon binding, it induces activation of nuclear factor-κB (NF-κB), promotes the transcription of inflammatory factors, and activates apoptotic proteases, thereby exerting pro-inflammatory and pro-apoptotic effects. DcR3 competitively binds to TL1A, thereby weakening the generation of T cell co-stimulatory signals and exerting anti-apoptotic and pro-inflammatory factor secretion effects.

[0004] Recent studies have found that TL1A can promote intestinal mucosal inflammation and fibrosis by affecting the activation and proliferation of T cells in the lamina propria of the intestinal epithelium, promoting the polarization and effector function of Th1 cells, and upregulating the expression of various inflammatory factors such as IFN-γ, TNF-α, IL-17, IL-22, IL-1β, IL-4, IL-13, IL-6, and TGF-β. Simultaneously, TL1A can also act on Th17 cells, increasing their secretion of IL-17, leading to intestinal mucosal immune dysregulation and triggering intestinal inflammation. Chronic inflammation is the primary factor in the development of intestinal fibrosis, and the inflammatory response can further activate intestinal fibroblasts, producing large amounts of extracellular matrix (such as collagen I and III), ultimately leading to the formation of intestinal fibrosis.

[0005] IL-23 is a pro-inflammatory cytokine produced by innate immune cells (such as dendritic cells and macrophages) and belongs to the IL-12 cytokine family. It consists of two subunits: p19 (an IL-23-specific subunit) and p40 (a subunit shared with IL-12). IL-23 activates the downstream JAK-STAT signaling pathway by binding to the receptor complexes IL-23R and IL-12Rβ1, thereby promoting the differentiation and proliferation of Th17 cells. Th17 cells are key regulators of inflammatory responses, secreting pro-inflammatory cytokines such as IL-17 and IL-22, and participating in the pathogenesis of various autoimmune and chronic inflammatory diseases. Studies have shown that IL-23 plays a central role in the pathogenesis of IBD. IL-23 promotes the activation and maintenance of Th17 cells, leading to the excessive production of pro-inflammatory cytokines such as IL-17 and IL-22 in the intestinal mucosa, thereby triggering intestinal inflammation and tissue damage. Furthermore, IL-23 also acts directly on innate immune cells in the lamina propria (such as macrophages and dendritic cells), further amplifying the inflammatory response. Therefore, IL-23 is considered an important target for IBD treatment.

[0006] Since TL1A and IL-23 do not completely overlap in the molecular mechanisms of IBD pathogenesis, bispecific antibodies targeting these two targets are expected to achieve positive efficacy in a broader IBD patient population.

[0007] Therefore, there is an urgent need in this field to develop bispecific antibodies that simultaneously target TL1A and IL-23. Summary of the Invention

[0008] The purpose of this invention is to provide a bispecific antibody that simultaneously targets TL1A and IL-23.

[0009] In a first aspect of the present invention, a bispecific antibody is provided, the bispecific antibody comprising:

[0010] The first antigen-binding domain D1; and

[0011] Second antigen-binding domain D2;

[0012] D1 is an antibody or antigen-binding fragment that specifically binds to antigen IL-23p19, and D2 is an antibody or antigen-binding fragment that specifically binds to antigen TL1A.

[0013] In another preferred embodiment, the antibody includes: nanobodies, chimeric antibodies, murine antibodies, and humanized antibodies.

[0014] In another preferred embodiment, the antigen-binding fragment comprises a Fab fragment, an F(ab')2 fragment, an Fv fragment, or a single-chain Fv (scFv).

[0015] In another preferred embodiment, D1 and / or D2 are scFv.

[0016] In another preferred embodiment, D1 and / or D2 are IgG antibodies. The IgG antibodies are IgG1, IgG2, IgG3, or IgG4 antibodies.

[0017] In another preferred embodiment, D1 is an anti-IL-23p19 scFv and D2 is an anti-TL1A IgG antibody, wherein the anti-IL-23p19 scFv is linked to the anti-TL1A IgG antibody via a linker.

[0018] In another preferred embodiment, D1 comprises one, two, three or more scFvs that are anti-IL-23p19.

[0019] In another preferred embodiment, D1 comprises two scFvs that resist IL-23p19.

[0020] In another preferred embodiment, D2 is an IgG antibody.

[0021] In another preferred embodiment, D1 is connected to either the N-terminal or the C-terminal of D2.

[0022] In another preferred embodiment, D1 and D2 are connected by a connector or directly connected.

[0023] In another preferred embodiment, the anti-IL-23p19 scFv is linked to the heavy chain variable region of the anti-TL1A IgG antibody.

[0024] In another preferred embodiment, the anti-IL-23p19 scFv is linked to the heavy chain constant region of the anti-TL1A IgG antibody.

[0025] In another preferred embodiment, the bispecific antibody is a homodimer or a heterodimer.

[0026] In another preferred embodiment, the bispecific antibody is a homodimer.

[0027] In another preferred embodiment, the bispecific antibody comprises a monomer having a structure of formula (I), (II), (III), or (IV) from its N-terminus to its C-terminus:

[0028]

[0029] in,

[0030] VL A This indicates the light chain variable region of the anti-IL-23p19 antibody;

[0031] VH A This represents the heavy chain variable region of the anti-IL-23p19 antibody;

[0032] VL B This represents the light chain variable region of the anti-TL1A antibody;

[0033] VH B Heavy chain variable region representing anti-TL1A antibody

[0034] CH1, CH2, and CH3 represent the heavy chain constant regions of the anti-TL1A antibody;

[0035] CL represents the light chain constant region of the anti-TL1A antibody;

[0036] L1 and L2 represent connectors;

[0037] "~" indicates a disulfide bond or a covalent bond;

[0038] "-" indicates a peptide bond.

[0039] In another preferred embodiment, the sequence of connector L1 is (G4S)n, preferably n is 1-5.

[0040] In another preferred embodiment, the sequence of connector L2 is (G4S)n, preferably n is 1-5.

[0041] The sequences of connectors L1 and L2 can be the same or different.

[0042] In another preferred embodiment, the light chain variable region VL of the anti-IL-23p19 antibody A It contains the amino acid sequence shown in SEQ ID NO:7.

[0043] In another preferred embodiment, the heavy chain variable region VH of the anti-IL-23p19 antibody A It contains the amino acid sequence shown in SEQ ID NO:8.

[0044] In another preferred embodiment, the amino acid sequences of the heavy chain complementarity-determining region of the anti-TL1A antibody are HCDR1: GYTMN (SEQ ID NO: 17), HCDR2: LINPYSGGTNYNQKFKG (SEQ ID NO: 18), and HCDR3: IYQRHDGIAY (SEQ ID NO: 19), and the amino acid sequences of the light chain complementarity-determining region are LCDR1: SASSSVNYMH (SEQ ID NO: 20), LCDR2: DTSKLAS (SEQ ID NO: 21), and LCDR3: QQWSSSPYT (SEQ ID NO: 22) (determined according to Kabat rules).

[0045] In another preferred embodiment, the heavy chain variable region sequence of the anti-TL1A antibody has the sequence shown in SEQ ID NO:9.

[0046] In another preferred embodiment, the light chain variable region sequence of the anti-TL1A antibody has the sequence shown in SEQ ID NO:10.

[0047] In another preferred embodiment, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 or human IgG4.

[0048] In another preferred embodiment, the light chain constant region is selected from human κ (Kappa) or human λ (Lambda).

[0049] In another preferred embodiment, the heavy chain of the bispecific antibody has a sequence as shown in any one of SEQ ID NOs:1-4, or a sequence having at least 80% identity with any one of SEQ ID NOs:1-4; and / or the light chain has a sequence as shown in SEQ ID NO:5, or a sequence having at least 80% identity with SEQ ID NO:5.

[0050] In another preferred embodiment, the bispecific antibody comprises the active fragment of the bispecific antibody, which retains 70-100% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) of the anti-IL-23p19 activity and 70-100% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) of the anti-TL1A activity.

[0051] In another preferred embodiment, the sequence of the bispecific antibody comprises the deletion, insertion, and / or substitution of one or more amino acid residues, and has at least 85% identity with the sequence shown in any one of SEQ ID NO: 1 to 5; wherein the identity may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0052] In another preferred embodiment, the substitution of the amino acid residue is a conserved amino acid substitution.

[0053] In another preferred embodiment, the deletion, insertion, and / or substitution of the amino acid residues are located in the frame region or constant region.

[0054] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO:1, and the light chain amino acid sequence is shown in SEQ ID NO:5.

[0055] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO:2, and the light chain amino acid sequence is shown in SEQ ID NO:5.

[0056] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO:3, and the light chain amino acid sequence is shown in SEQ ID NO:5.

[0057] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO:4, and the light chain amino acid sequence is shown in SEQ ID NO:5.

[0058] In a second aspect of the invention, a polynucleotide molecule is provided, said polynucleotide molecule encoding a bispecific antibody as described in the first aspect of the invention.

[0059] In another preferred embodiment, the sequence of the polynucleotide is shown in any one of SEQ ID NOs:11-15.

[0060] In a third aspect of the invention, an expression vector is provided, the expression vector containing a polynucleotide molecule as described in the second aspect of the invention.

[0061] In another preferred embodiment, the expression vector is a virus or plasmid, preferably a lentivirus, bacteriophage, or phage particle.

[0062] In another preferred embodiment, the expression vector is selected from the group consisting of pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, preferably pcDNA3.4.

[0063] In a fourth aspect of the invention, a host cell is provided, the host cell containing the expression vector as described in the third aspect of the invention.

[0064] In another preferred embodiment, the host cell is a eukaryotic cell, preferably a CHO cell or a 293F cell.

[0065] In a fifth aspect of the invention, a method for preparing a bispecific antibody as described in the first aspect of the invention is provided, the method comprising the following steps:

[0066] a) Under expression conditions, host cells as described in the fourth aspect of the present invention are cultured to express bispecific antibodies;

[0067] b) Isolate and purify the bispecific antibody described in step a).

[0068] In a sixth aspect of the invention, a pharmaceutical composition is provided comprising a bispecific antibody as described in the first aspect of the invention.

[0069] In a seventh aspect of the invention, the use of a bispecific antibody as described in the first aspect of the invention, or a pharmaceutical composition as described in the sixth aspect of the invention, in the preparation of a medicament for treating inflammatory diseases or conditions is provided.

[0070] In another preferred embodiment, the inflammatory disease or condition includes diseases or conditions of the gastrointestinal tract or skin.

[0071] In another preferred embodiment, the gastrointestinal disease or condition is inflammatory bowel disease. More preferably, it is Crohn's disease or ulcerative colitis.

[0072] In another preferred embodiment, the skin disease or condition is psoriasis.

[0073] In an eighth aspect of the invention, a method for treating an inflammatory disease or condition is provided, the method comprising administering to a subject in need a bispecific antibody as described in the first aspect of the invention or a pharmaceutical composition as described in the sixth aspect of the invention.

[0074] In another preferred embodiment, the method further includes administration in combination with other drugs for treating inflammatory diseases or conditions.

[0075] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0076] Figure 1A And 1B is a schematic diagram of the structure of the bispecific antibody of the present invention.

[0077] Figure 2 This is the HPLC-SEC detection chromatogram of the bispecific antibody of the present invention.

[0078] Figure 3 The bispecific antibody of the present invention was demonstrated to bind to antigen TL1A.

[0079] Figure 4 The bispecific antibody was shown to bind to antigen IL-23.

[0080] Figure 5 The activity of the bispecific antibody in simultaneously binding to TL1A and IL23 proteins was demonstrated.

[0081] Figure 6 The activity of the bispecific antibody in inhibiting the downstream NF-κB signaling pathway of TL1A / DR3 was demonstrated.

[0082] Figure 7 The bispecific antibody was shown to inhibit the activity of the downstream JAK / STAT signaling pathway of IL-23.

[0083] Figure 8 The bispecific antibody was shown to inhibit the secretion of IL-22 by PBMCs.

[0084] Figure 9 This demonstrates the ability of bispecific antibodies to inhibit TNFα secretion by PBMCs.

[0085] Figure 10 The changes in mouse body weight are shown.

[0086] Figure 11 The statistical results of the length of the colon and rectum in mice are shown.

[0087] Figure 12 The results show the statistical results of colorectal weight and density in mice. Detailed Implementation

[0088] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0089] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0090] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0091] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0092] The "immunoglobulin antibody IgG" described in this invention is a molecule of approximately 150 kDa, composed of four peptide chains. It contains two identical γ heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus exhibiting a tetrameric quaternary structure. The two heavy chains are interconnected by disulfide bonds and each is linked to a light chain. The resulting tetramer has two identical halves, forming a fork-like or Y-like shape, with each end of the fork containing an identical antigen-binding site. IgG antibodies can be classified into several subclasses (e.g., IgG1, 2, 3, 4) based on minor differences in the amino acid sequence of the constant region of the heavy chain.

[0093] In this invention, the term "bispecific antibody (or bispecific antibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes. Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules.

[0094] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment that has antigen-binding activity. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fv fragments; or (iv) single-chain Fv (scFv). As used herein, the term "antigen-binding fragment" also encompasses other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, bisomal antibodies, trisomal antibodies, tetrasomal antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0095] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells. The term "F(ab')2 fragment" refers to an F(ab')2 fragment antibody obtained by digesting an entire IgG antibody with pepsin, removing most of the Fc region while retaining some hinge regions intact. It has two antigen-binding F(ab) portions linked together by disulfide bonds.

[0096] In this invention, the term "scFv" or "single-chain variable region fragment scFv" refers to a single-chain antibody fragment (scFv), which is a fusion protein containing the variable regions of the immunoglobulin heavy chain (VH) and light chain (VL). The VH and VL are linked by a linker of 15 to 25 amino acids, and the fusion protein retains the same antigen specificity as the complete immunoglobulin.

[0097] In this invention, the term "Fv fragment" or "Fv antibody" refers to the smallest antibody fragment containing variable regions of the antibody heavy chain and light chain, but no constant regions, and having all antigen-binding sites. Generally, an Fv fragment also includes a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding.

[0098] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for both the light and heavy chain domains to fold into an exchangeable dual variable region immunoglobulin. Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in a linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0099] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0100] Bispecific antibodies

[0101] This invention provides a bispecific antibody that simultaneously and specifically binds to TL1A and IL-23, comprising an anti-TL1A antibody portion and an anti-IL-23p19 antibody portion. Specifically, it comprises an immunoglobulin antibody IgG and two identical scFv fragments, wherein each scFv fragment is tandemly linked to the immunoglobulin antibody IgG via a linker.

[0102] The "bispecific antibody" described in this invention refers to a bispecific antibody that has two different antigen-binding sites and can simultaneously bind to TL1A and IL-23. It comprises two scFvs and conjugated immunoglobulin antibody IgG. Each scFv is linked to each heavy chain of immunoglobulin antibody IgG via a peptide linker to form a heavy chain fusion protein of the bispecific antibody.

[0103] Typically, peptide linkers possess sufficient length and flexibility to ensure that the two linked proteins have adequate spatial freedom to perform their functions. Preferably, they are flexible peptide linkers (linkers), for example, 0-30 amino acids. More preferably, they are (G4S)n, where n is a positive integer from 1 to 5.

[0104] The bispecific antibody of the present invention can be a dimer, trimer, or multimer, preferably a homodimer or heterodimer. The antibody portion against TL1A or against IL-23p19 in the bispecific antibody of the present invention may include one or more antibodies or their antigen-binding fragments, preferably 1, 2, 3, 4, 5, or 6.

[0105] As a preferred embodiment, the bispecific antibody of the present invention comprises two scFv antibodies against IL-23p19 and an IgG antibody against TL1A.

[0106] As a preferred embodiment, the anti-IL-23p19 scFv is constructed using the Guselkumab antibody.

[0107] As a preferred embodiment, the light chain variable region of the anti-IL-23p19 scFv contains the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:8.

[0108] As a preferred embodiment, the light chain variable region and the heavy chain variable region in the scFv for resisting IL-23p19 are connected by a connector, and the sequence of the connector is GGGGSGGGGSGGGGSGGGGSGGGGS.

[0109] As a preferred embodiment, the amino acid sequences of the heavy chain complementarity-determining region of the TL1A antibody are HCDR1: GYTMN (SEQ ID NO: 17), HCDR2: LINPYSGGTNYNQKFKG (SEQ ID NO: 18), and HCDR3: IYQRHDGIAY (SEQ ID NO: 19), and the amino acid sequences of the light chain complementarity-determining region are LCDR1: SASSSVNYMH (SEQ ID NO: 20), LCDR2: DTSKLAS (SEQ ID NO: 21), and LCDR3: QQWSSSPYT (SEQ ID NO: 22) (determined according to Kabat rules).

[0110] As a preferred embodiment, the anti-TL1A IgG antibody comprises a heavy chain variable region as shown in SEQ ID NO:9 and a light chain variable region as shown in SEQ ID NO:10.

[0111] As a preferred embodiment, the two anti-IL-23p19 scFvs are respectively linked to the N-terminus or C-terminus of the anti-TL1A IgG antibody via a linker, and the linker sequence is GGGGSGGGGSGGGGS.

[0112] More preferably, the heavy chain of the bispecific antibody is as shown in any one of SEQ ID NOs:1-4, and the light chain is as shown in SEQ ID NO:5.

[0113] In this invention, the bispecific antibody also includes its conserved variants, which refer to antibodies formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the bispecific antibody of this invention. Amino acids of similar or analogous properties are known to those skilled in the art.

[0114] In constructing the bispecific antibody of the present invention, problems related to the chemical and physical stability of the bispecific antibody were also solved, such as expressing physically stable molecules, increasing heat and salt-dependent stability, reducing aggregation, increasing solubility at high concentrations, and maintaining affinity for the two antigens IL-23 and TL1A, respectively.

[0115] Nucleic acid encoding and expression vector

[0116] In another aspect, the present invention provides a polynucleotide molecule encoding the aforementioned bispecific antibody. The polynucleotide of the present invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0117] Preferably, the sequence of the polynucleotide is shown in any one of SEQ ID NOs:11-15.

[0118] The method for preparing the nucleotide molecule described in this invention is a conventional method in the art, and preferably includes the following preparation method: obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR, or obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody by artificial full-sequence synthesis.

[0119] Those skilled in the art will understand that the nucleotide sequence encoding the amino acid sequence of the aforementioned bispecific antibody can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases encoding the bispecific antibody gene within the range of maintaining antibody activity.

[0120] In another aspect, the present invention provides an expression vector containing the above-described nucleotide molecules.

[0121] The expression vector described herein is a conventional expression vector in the art, referring to an expression vector containing appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and / or sequences, and other appropriate sequences. The expression vector can be a virus or plasmid, such as a suitable lentivirus, bacteriophage, or phage particle. For more technical details, please refer to, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols for nucleic acid manipulation can be found in *Current Protocols in Molecular Biology*, 2nd edition, edited by Ausubel et al. The expression vector of this invention is preferably pDR1, pcDNA3.1(+), pcDNA3.4, pcDNA3.1 / ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF, or pCHO 1.0, more preferably pcDNA3.4.

[0122] The present invention also provides a host cell containing the above-described expression vector.

[0123] The host cells described in this invention are any conventional host cells in the art, as long as they can stably and spontaneously replicate the recombinant expression vector and effectively express the carried nucleotides. The host cells include prokaryotic expression cells and eukaryotic expression cells. Preferably, the host cells include: COS, CHO (Chinese hamster ovary), NSO, sf9, sf21, DH5α, BL21(DE3), TG1, or 293F cells. Transforming the aforementioned expression vector into the host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation, heat shock method, or electroporation method.

[0124] As a preferred embodiment, the host cell is a eukaryotic cell. CHO cells or 293F cells are preferred.

[0125] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0126] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0127] Another aspect of the present invention provides a method for preparing the above-mentioned bispecific antibody that specifically binds to TL1A and IL-23, the method comprising the following steps:

[0128] a) Under the expression conditions, the above-mentioned host cells were cultured to express a bispecific antibody that specifically binds to TL1A and IL-23;

[0129] b) Isolate and purify the bispecific antibody described in step a).

[0130] The host cell culture method and the antibody isolation and purification method described in this invention are conventional methods in the art. For specific operating procedures, please refer to the relevant cell culture technology manual and antibody isolation and purification technology manual. The preparation method of the anti-TL1A / IL-23 bispecific antibody disclosed in this invention includes: culturing the aforementioned host cells under expression conditions to express a bispecific antibody that specifically binds to TL1A and IL-23; and isolating and purifying the anti-TL1A / IL-23 bispecific antibody. Using the above method, the recombinant protein can be purified into a substantially homogeneous substance.

[0131] The anti-TL1A / IL-23 bispecific antibody disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the anti-TL1A / IL-23 bispecific antibody bound to the affinity column. The inventors of this invention conducted detection experiments on the obtained anti-TL1A / IL-23 bispecific antibody. The experimental results show that the anti-TL1A / IL-23 bispecific antibody can bind well to target cells and antigens, exhibiting high affinity.

[0132] Pharmaceutical Composition

[0133] Another aspect of the present invention provides a pharmaceutical composition comprising the bispecific antibody described above that specifically binds to TL1A and IL-23.

[0134] The bispecific antibodies provided by this invention can be combined with one or more pharmaceutically acceptable excipients to form pharmaceutical formulations, thereby achieving more stable therapeutic effects. These formulations ensure the conformational integrity of the amino acid core sequence of the bispecific antibodies of this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. Generally, for liquid formulations, they can be stored stably for at least one year at 2°C-8°C, and for lyophilized formulations, they remain stable for at least six months at 30°C. The bispecific antibody formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, and lyophilized formulations.

[0135] In this invention, the term "pharmaceutical composition" refers to the bispecific antibody of this invention that can be combined with one or more pharmaceutically acceptable excipients to form a pharmaceutical formulation composition to exert its therapeutic effect more stably. These formulations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).

[0136] In this invention, the term "excipient" refers to pharmaceutically acceptable inactive ingredients used to improve the stability, solubility, release characteristics, safety, or ease of use of a drug, such as carriers, buffers, surfactants, etc.

[0137] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described bispecific antibody (or conjugate thereof) of the present invention, along with pharmaceutically acceptable excipients. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. Furthermore, the bispecific antibody of the present invention can also be used in conjunction with other therapeutic agents.

[0138] When using a drug composition, a safe and effective amount of bispecific antibody is administered to mammals. The specific dosage should also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill range of a skilled physician.

[0139] application

[0140] In another aspect, the present invention provides the use of the above-mentioned bispecific antibody that specifically binds to TL1A and IL-23, or the above-mentioned pharmaceutical composition, in the preparation of a medicament for the treatment of inflammatory bowel disease.

[0141] When the bispecific antibodies and their compositions of the present invention are administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage should be determined with reference to animal experimental results and various other factors, and the total dosage should not exceed a certain range. For example, the intravenous injection dose is 1-1800 mg / day.

[0142] The bispecific antibody and its composition of the present invention can also be administered in combination with other drugs for treating inflammatory bowel disease to achieve a more effective treatment of inflammatory bowel disease.

[0143] The main advantages of this invention include

[0144] (1) The bispecific antibody provided by the present invention can bind to TL1A and IL23 proteins with high affinity, effectively inhibiting the activity of the downstream NF-κB signaling pathway of TL1A / DR3 and the downstream JAK / STAT signaling pathway of IL-23, and has a synergistic effect.

[0145] (2) The bispecific antibody provided by the present invention has a better therapeutic effect on inflammatory bowel disease than TL1A monoclonal antibody, Guselkumab monoclonal antibody and the combination of the two monoclonal antibodies.

[0146] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all percentages below are weight percentages or molar percentages.

[0147] Example 1: Construction and Expression of Bispecific Antibodies

[0148] (I) Construction of bispecific antibodies

[0149] This invention constructs an scfv from the variable region sequence of a gusselukumab antibody and links it to the N-terminus or C-terminus of the heavy chain of an anti-TL1A monoclonal antibody (our company's SSGJ-627) (sequence derived from patent CN202410160677.5) to construct an anti-TL1A / IL-23 bispecific antibody, the structure of which is as follows: Figure 1A As shown in Table 1B, four bispecific antibodies were constructed and named TI001, TI002, TI003 and TI004, respectively. Their specific sequences are shown in Table 1.

[0150] The heavy and light chain DNA fragments of the bispecific antibody were cloned into the pCDNA3.4 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells and / or 293F cells. After culturing 293F cells for 5-7 days, the culture medium was centrifuged at high speed, filtered through a microporous membrane under vacuum, and then loaded onto a HiTrap MabSelectSuRe column. The protein was eluted in one step with elution buffer containing 100 mM citrate (pH 3.5). The target sample was recovered and dialyzed against PBS at pH 7.4. The purified protein was detected by HPLC. The HPLC-SEC chromatograms of the bispecific antibody are shown below. Figure 2 As shown, the antibody molecules are in a uniform state, and the monomer purity after one purification reaches over 96%.

[0151] Table 1. Partial sequence information of the present invention

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Example 2: Detection of the binding activity of bispecific antibody to TL1A protein

[0158] Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding ability of bispecific antibodies to human-TL1A-his protein. The specific method is as follows: The antigen human-TL1A-his was diluted to 1 μg / mL with coating buffer, and 100 μL / well was added to the ELISA plate and incubated overnight at 4°C. The plate was washed three times with PBST, and 250 μL / well of 1% BSA blocking buffer was added. The plate was incubated at 37°C for 1 hour, and washed three times with PBST. The positive control antibody SSGJ-627 and the bispecific antibody to be tested were diluted to 100 nM with 1% BSA-PBS, forming eight concentration gradients (5-fold dilution). These were then added sequentially to the blocked ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. The plate was washed three times with PBST, and HRP-labeled goat anti-human Fc antibody (working concentration: 1:3000, purchased from Sinopharm, catalog number: SSA001) was added. The plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, pat dry any remaining droplets on absorbent paper. Add 100 μL of TMB to each well and incubate at room temperature (20±5℃) in the dark for 5 minutes. Stop the substrate reaction by adding 70 μL of 1M H2SO4 stop solution to each well. Read the OD values ​​at 450 nm using a microplate reader, and analyze the data using GraphPad Prism 10 to plot graphs and calculate EC50. 50 The result is as follows Figure 3 As shown in the figure. Experimental results indicate that the binding activity of the bispecific antibody to antigen TL1A is comparable to that of its parent monoclonal antibody SSGJ-627.

[0159] Example 3: Detection of the binding activity of bispecific antibody to IL-23 protein

[0160] Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding ability of bispecific antibodies to human-IL23-his protein. The specific method is as follows: The antigen human-IL23-his was diluted to 1 μg / mL with coating buffer, and 100 μL / well was added to the ELISA plate and incubated overnight at 4°C. The plate was washed three times with PBST, and 250 μL / well of 1% BSA blocking buffer was added. The plate was incubated at 37°C for 1 h, and washed three times with PBST. The positive control gusselukab and the bispecific antibody were diluted to 200 nM with 1% BSA-PBS, forming eight concentration gradients (4-fold dilution). These gradients were added sequentially to the blocked ELISA plate at 100 μL / well and incubated at 37°C for 1 h. The plate was washed three times with PBST, and HRP-labeled goat anti-human Fab antibody (working concentration: 1:3000, purchased from Merck, catalog number: A0293) was added. The plate was incubated at 37°C for 1 h. After washing the plate three times with PBST, pat dry any remaining droplets on absorbent paper. Add 100 μL of TMB to each well and incubate at room temperature (20±5℃) in the dark for 5 minutes. Stop the substrate reaction by adding 70 μL of 1M H2SO4 stop solution to each well. Read the OD values ​​at 450 nm using a microplate reader, and analyze the data using GraphPad Prism 10 to plot graphs and calculate EC50. 50 The result is as follows Figure 4 As shown in the figure. Experimental results indicate that the binding activity of TI001 and TI002 molecules to antigen IL-23 is superior to that of the parental monoclonal antibody Guselkumab.

[0161] Example 4: Detection of the activity of bispecific antibodies simultaneously binding to TL1A and IL23 proteins

[0162] Enzyme-linked immunosorbent assay (ELISA) was used to determine the bridging binding ability of bispecific antibodies to human-TL1A-his and human-IL23-his proteins. The specific method is as follows: The antigen human-TL1A-his was diluted to 1 μg / mL with coating buffer, and 100 μL / well was added to the ELISA plate and incubated overnight at 4°C. The plate was washed three times with PBST, and 250 μL / well of 1% BSA blocking buffer was added. The plate was incubated at 37°C for 1 h, and washed three times with PBST. The bispecific antibody, the positive control SSGJ627 monoclonal antibody, and Guselkumab were diluted to 100 nM with 1% BSA-PBS, forming 11 concentration gradients (4-fold dilution). These were then added sequentially to the blocked ELISA plate at 100 μL / well and incubated at 37°C for 1 h. The plate was washed three times with PBST, and 1 μg / mL of human IL23-his (biotin) protein was added. The plate was incubated at 37°C for 1 h. Wash the plate three times with PBST, add Streptavidin HRP (working concentration: 1:3000), and incubate at 37°C for 1 hour. After washing three times with PBST, blot dry any remaining droplets on absorbent paper, add 100 μL of TMB to each well, and incubate at room temperature (20±5°C) in the dark for 5 minutes. Stop the substrate reaction by adding 70 μL of 1M H2SO4 stop solution to each well. Read the OD value at 450 nm using a microplate reader, analyze the data using a GraphPad Prism 10, plot the graphs, and calculate the EC50. 50 The result is as follows Figure 5 As shown in the figure. Experimental results indicate that the bispecific antibody can bind to both TL1A and IL23 proteins with high affinity, while the parental monoclonal antibody cannot bind to both proteins simultaneously.

[0163] Example 5: Detection of the activity of bispecific antibody in inhibiting the downstream NF-κB signaling pathway of TL1A / DR3

[0164] In this experiment, TF-1-NF-κB-Luc (an engineered cell line of TF-1NF-κB-Luc constructed in the laboratory using lentivirus) was used as the target cell. TF-1-NF-κB-Luc cells were centrifuged and counted. Cells with adjusted density were then added to 3903 cell plates at a density of 1×10⁻⁶. 5Add 50 μL to each well. Dilute the positive control antibody SSGJ-627 and the bispecific antibody to 200 nM with 1640 medium (containing 10% FBS and 1% PS), and then dilute 2-fold to create 8 concentration gradients. Add 25 μL of each gradient to the 3903 cell plate. Dilute human-TL1A-his to 800 ng / mL with 1640 medium (containing 10% FBS and 1% PS), and add 25 μL to each well of the 3903 cell plate. The final concentration of human-TL1A-his is 200 ng / mL, and the initial and final concentration of TL1A antibody is 50 nM. After incubating the cell culture plates at 37°C for 24 hours, the plates were allowed to equilibrate to room temperature for about 15 minutes beforehand. 100 μL of Bio-Lite Luciferase (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: DD1201-02) was added to each well. After incubation at room temperature for 10 minutes, Luminescence (RLU) values ​​were read using a microplate reader. Data analysis was performed using a GraphPad Prism 10, and graphs were plotted and IC50 calculated. 50 The result is as follows Figure 6 As shown in the figure. Experimental results indicate that the inhibitory capacity of the bispecific antibodies TI002 and TI003 is comparable to that of their parent monoclonal antibody SSGJ-627.

[0165] Example 6: Detection of the activity of bispecific antibody in inhibiting the downstream JAK / STAT signaling pathway of IL-23

[0166] In this experiment, the H_IL-23Reporter 293 cell line (a Luciferase reporter gene cell line highly expressing the IL23 receptor, constructed by Jimon Biotechnology based on the JAK / STAT signaling pathway) was used as the target cell. H_IL-23Reporter 293 cells were centrifuged and counted. The cells, after being adjusted to the desired density, were added to the aforementioned 3903 cell plate at a density of 1×10⁻⁶. 5Add 50 μL to each well. Dilute the positive control antibody Guselkilumab and the bispecific antibody to 800 nM in DMEM medium (containing 10% FBS and 1% PS), and then dilute 6-fold to create 8 concentration gradients. Add 25 μL of each gradient to the 3903 cell plate. Dilute human-IL23-his to 50 ng / mL in DMEM medium (containing 10% FBS and 1% PS), and add 25 μL to each well of the 3903 cell plate. The final concentration of human-IL23-his is 12.5 ng / mL, and the initial and final concentration of the antibody is 200 nM. After incubating the cell culture plates at 37°C for 24 hours, the plates were allowed to equilibrate to room temperature for about 15 minutes beforehand. 100 μL of Bio-Lite Luciferase (purchased from Nanjing Novizan Biotechnology Co., Ltd., catalog number: DD1201-02) was added to each well. After incubation at room temperature for 10 minutes, Luminescence (RLU) values ​​were read using a microplate reader. Data analysis was performed using a GraphPad Prism 10, and graphs were plotted and IC50 calculated. 50 The result is as follows Figure 7 As shown in the figure. Experimental results indicate that the bispecific antibodies TI002 and TI003 exhibit superior inhibitory activity compared to the parent monoclonal antibody Guselkinumab.

[0167] Example 7: Detection of the activity of bispecific antibody in inhibiting IL-22 secretion by PBMCs

[0168] This embodiment uses ELISA to detect the IL22 content in cell supernatant and evaluate the ability of the antibody to inhibit IL22 secretion by PBMCs. A single-cell suspension of PBMCs was prepared in PRMI 1640 + 3% FBS medium, and the cell density was adjusted to 4 × 10⁶ cells / cell. 6 / ml, 50μL / well. Dilute IL23 to 4μg / ml, 25μL / well; dilute TL1A to 4μg / ml, 25μL / well; dilute the bispecific antibody to be tested and the positive controls Guselkumab and SSGJ-627 to 800nM, using a 4-fold serial dilution. That is, the final concentration of IL23 is 1μg / ml, the final concentration of TL1A is 1μg / ml, and the initial concentration of the antibody is 200nM. After incubating the cell plate in a 37°C incubator for 72h, remove the plate, centrifuge to collect the cell supernatant, detect the IL22 content in the supernatant, plot the graph, and calculate the IC50. 50 The result is as follows Figure 8 As shown in the figure. The experimental results indicate that the inhibitory effect of the bispecific antibody TI002 is significantly enhanced compared to the monoclonal antibodies SSGJ-627 and Guselkumab used alone, and is superior to the combination of SSGJ-627 and Guselkumab.

[0169] Example 8: Detection of the ability of bispecific antibodies to inhibit TNFα secretion by PBMCs

[0170] This embodiment uses ELISA to detect the TNFα content in cell supernatant and evaluate the ability of the antibody to inhibit TNFα secretion by PBMCs. A single-cell suspension of PBMCs was prepared in PRMI 1640 + 3% FBS medium, and the cell density was adjusted to 4 × 10⁶ cells / cell. 6 Dilute IL-2 to 80 U / ml, 25 μL / well; dilute IL-23 to 4 μg / ml, 25 μL / well; dilute TL1A to 8 μg / ml, 25 μL / well; dilute the bispecific antibody and positive controls Guselkumab and SSGJ-627 to 800 nM, using a 3-fold serial dilution. That is, the final concentration of IL-2 is 20 U / ml, the final concentration of IL-23 is 1 μg / ml, the final concentration of TL1A is 2 μg / ml, and the initial and final concentrations of the antibody are 200 nM. Incubate the cell plates at 37°C for 72 h. After incubation, remove the plates, centrifuge to collect the cell supernatant, and detect the TNF-α content in the supernatant. Plot the data and calculate the IC50. 50 The result is as follows Figure 9 As shown in the figure. Experimental results indicate that the bispecific antibody has a better inhibitory effect than gusselukab.

[0171] Example 9: Pharmacodynamic study of bispecific antibody against a TNBS-induced mouse enteritis model

[0172] TNBS is a small molecule hapten that, when administered via rectal perfusion, binds to host proteins, triggering an immune response that leads to transmural necrosis and widespread inflammatory cell infiltration in the animal colon, accompanied by weight loss and intestinal obstruction. It is a commonly used modeling agent for acute enteritis in animals simulating clinical Crohn's disease. We validated the efficacy of a bispecific antibody in treating acute enteritis in rats using this model and compared it with its parent monoclonal antibody.

[0173] Forty-eight female B-hTL1A / hIL23A / hIL12B mice, weighing 18-20g, were randomly divided into six groups (n=8): the TNBS model group and the test group received 0.06mL of 1.5% TNBS solution rectally at a site approximately 4cm from the anus, and were held upside down for 2 minutes after enema; the control group (normal group) received 0.06mL of 50% ethanol solution rectally in the same manner as the model group. Before enema, the mice were fasted and then anesthetized with an intraperitoneal injection of a mixture of sulfadiazine and sulfadiazine. The test group received intraperitoneal injections on Day-1, Day 1, Day 3, and Day 5, with Day 5 being the experimental endpoint. Specific groupings are shown in Table 2. During the experiment, the mice's various states (behavioral activity, mental state, feeding status, coat color, etc.) were observed, and mouse weight was recorded.

[0174] Table 2 Animal grouping and administration regimen

[0175]

[0176] Note: Mice in the control group and TNBS model group were injected intraperitoneally with the same amount of the test solvent for intervention; the dosage in the TI002 group was the same as the dosage in equimolar amounts.

[0177] At the experimental endpoint, animal samples were collected: Blood was drawn from the ophthalmic venous plexus of mice after anesthesia, and the mice were euthanized by cervical dislocation. The abdomen was disinfected with 75% alcohol, and the colon, rectum, and cecum of the mice were removed, fixed, photographed, and the length of the colon and rectum was recorded. The cecum was cut off, the colon and rectum were longitudinally dissected, and the contents of the colon and rectum were rinsed with PBS buffer and weighed. 1-2 cm of the distal colon and rectum was cut off, soaked in 4% paraformaldehyde solution for fixation, and then subjected to pathological examination. The samples were placed in sterile 1.5 ml EP tubes and immediately flash-frozen in liquid nitrogen and stored at -80°C. Serum preparation: Mouse blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3500g for 15 minutes at 4°C, and the supernatant was collected and stored at -80°C.

[0178] The experimental results showed that the mice in the normal group (Control group) were in good condition from the beginning to the end of the modeling process, with stable weight gain, shiny fur, agile reactions, and formed, non-loose stools. The mice in the modeling group had dull fur, began to show lethargy, and decreased activity. Compared with the TNBS model group, the mice in the SSGJ-627 group, Guselkumab group, SSGJ-627+Guselkumab combination group, and TI002 double antibody group all showed significant improvement in the above symptoms.

[0179] The body weight results of each group of mice are as follows: Figure 10 As shown, the body weight of mice in the normal control group (Control group) increased over time, while the body weight of mice in the TNBS model group gradually decreased after the onset of the model, which was statistically significant compared with the normal control group (P < 0.001). The administration of SSGJ-627, Guselkumab, SSGJ-627 + Guselkumab combination, and TI002 bispecific antibody significantly improved the degree of body weight loss in mice. Compared with the TNBS model group, the body weight of each treatment group was significantly different; the SSGJ-627 + Guselkumab combination and TI002 bispecific antibody groups were better than the individual monoclonal antibody administrations; the TI002 bispecific antibody group did not show a significant difference in body weight compared with the combination groups, but showed a significant upward trend.

[0180] At the end of the experiment, the mouse abdominal cavity was opened, the colon and rectum were dissected, photographed, and their length and weight were recorded. The statistical analysis of changes in mouse colon and rectum length is as follows: Figure 11 As shown, the weight and density of mouse colon and rectum are statistically analyzed. Figure 12As shown in the figure. In the normal control group (Control group), the colon and rectum of mice appeared normal, with a clean intestinal lumen and no signs of erosion, congestion, or ulceration. Formed granular feces were distributed within the intestinal lumen. In the TNBS model group, the intestinal lumen was filled with a large amount of dark red bloody stool, with necrotic tissue in some parts of the intestine. The stool was unformed, and the intestines showed edema, shortening, thickening, and weight gain, showing a significant difference compared to the normal control group (P<0.001). Compared with the model group, the SSGJ-627 group, Guselkumab group, SSGJ-627+Guselkumab combination group, and TI002 bispecific antibody group showed significantly improved congestion and edema in the colon and rectum, and significantly inhibited the shortening of colon and rectum and the increase in intestinal weight (P<0.0001). Furthermore, the inhibitory effect of the bispecific antibody TI002 was superior to that of SSGJ-627, Guselkumab, and the SSGJ-627+Guselkumab combination.

[0181] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A bispecific antibody, characterized in that, The bispecific antibody comprises: The first antigen-binding domain D1; and Second antigen-binding domain D2; D1 is an antibody or antigen-binding fragment that specifically binds to antigen IL-23p19, and D2 is an antibody or antigen-binding fragment that specifically binds to antigen TL1A.

2. The bispecific antibody as described in claim 1, characterized in that, D1 is an anti-IL-23p19 scFv, and D2 is an anti-TL1A IgG antibody, wherein the anti-IL-23p19 scFv is linked to the anti-TL1A IgG antibody via a linker.

3. The bispecific antibody as described in claim 1 or 2, characterized in that, The bispecific antibody comprises a monomer having a structure of formula (I), (II), (III), or (IV) from its N-terminus to its C-terminus: in, VL A This indicates the light chain variable region of the anti-IL-23p19 antibody; VH A This represents the heavy chain variable region of the anti-IL-23p19 antibody; VL B This represents the light chain variable region of the anti-TL1A antibody; VH B Heavy chain variable region representing anti-TL1A antibody CH1, CH2, and CH3 represent the heavy chain constant regions of the anti-TL1A antibody; CL represents the light chain constant region of the anti-TL1A antibody; L1 and L2 represent connectors; "~" represents a disulfide bond or a covalent bond; "-" indicates a peptide bond.

4. The bispecific antibody as described in claim 1, characterized in that, The heavy chain of the bispecific antibody has a sequence as shown in any one of SEQ ID NOs: 1 to 4, or a sequence having at least 80% identity with any one of SEQ ID NOs: 1-4; and / or the light chain has a sequence as shown in SEQ ID NO: 5, or a sequence having at least 80% identity with SEQ ID NO:

5.

5. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the bispecific antibody as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide molecule as described in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6.

8. A method for preparing a bispecific antibody as described in any one of claims 1 to 4, characterized in that, The preparation method includes the following steps: a) Under expression conditions, host cells as described in claim 7 are cultured to express bispecific antibodies; b) Isolate and purify the bispecific antibody described in step a).

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the bispecific antibody as described in any one of claims 1 to 4.

10. Use of the bispecific antibody as described in any one of claims 1 to 4, or the pharmaceutical composition as described in claim 9, in the preparation of a medicament for treating inflammatory diseases or conditions.

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