CN120058933B

CN120058933B - Anti-tumor necrosis factor-like ligand 1A antibodies and uses thereof

Info

Publication number: CN120058933B
Application number: CN202311632076.1A
Authority: CN
Inventors: 郝小勃; 刘志刚; 周晓巍; 万姝南; 胡俊杰; 刘玉兰
Original assignee: Chongqing Zhixiang Jintai Biopharmaceutical Co ltd; Genrix Shanghai Biopharmaceutical Co ltd; Beijing Wisdomab Biotechnology Co ltd
Current assignee: Beijing Wisdomab Biotechnology Co ltd; Chongqing Zhixiang Jintai Biopharmaceutical Co ltd; Genrix Shanghai Biopharmaceutical Co ltd
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2026-02-10
Anticipated expiration: 2043-11-30
Also published as: CN120058933A; WO2025112237A1

Abstract

本申请公开了结合肿瘤坏死因子样配体1A(TL1A)的抗体，编码所述抗体的核酸分子、包含所述核酸分子的载体、包含所述核酸分子或载体的宿主细胞、制备和纯化所述抗体的方法及所述抗体的应用。This application discloses an antibody that binds to tumor necrosis factor-like ligand 1A (TL1A), a nucleic acid molecule encoding the antibody, a vector containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the vector, a method for preparing and purifying the antibody, and the application of the antibody.

Description

Anti-tumor necrosis factor-like ligand 1A antibodies and uses thereof

Technical Field

The present application relates generally to the field of genetic engineering and antibody pharmaceuticals, and more particularly to antibodies that bind to tumor necrosis factor-like ligand 1A and uses thereof.

Background

Tumor necrosis factor-like ligand 1A (TL 1A/VEGI-251/TNFSF 15) is a member of the tumor necrosis factor family, with 251 amino acids in full length, which is a type II transmembrane glycoprotein. As a type II transmembrane protein, TL1A, like other members of the TNF family, forms a stable non-covalent homotrimeric structure consisting of a β -sandwich. TL1A is expressed in immune cells (e.g., monocytes, macrophages, dendritic cells, T lymphocytes, plasma cells, etc.) and non-immune cells (e.g., endothelial cells, synovial fibroblasts, etc.), and TL1A is initially present in membrane bound form and can be cleaved at amino acid 72 by variable cleavage or tumor necrosis factor α converting enzyme to function ¹ as soluble protein release. The corresponding functional receptor for TL1A is death receptor 3 (DR 3), and the nonfunctional receptor is decoy receptor 3 (DcR 3) ². TL1A acts to induce oligomerization of DR3 receptor by binding DR3, recruit adaptor protein-tumor necrosis factor receptor related Death domain protein (TRADD, TNFR-associated Death domain protein) through intracellular Death domain, regulate downstream pathways such as TRAF2, RIP1, PI3K, MAPK and NF-. Kappa.B to exert pro-inflammatory effects, or promote apoptosis through FADD, RIP3 and Caspase-8/-3/-7 pathways, and simultaneously NF-. Kappa.B pathway also activates c-IAP protein to thereby down regulate apoptosis ^1,3.

Abnormal expression of TL1A is significantly associated with autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease, psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, and the like ¹. The key role of TL1A and DR3 signaling pathways in autoimmune and inflammatory diseases suggests that inhibition of TL1A-DR3 interactions may be an effective therapeutic strategy for ameliorating autoimmune diseases and local inflammation of target organs. The currently developed monoclonal antibodies targeting TL1A, clinical indications include inflammatory bowel disease and systemic sclerosis-associated interstitial lung disease, etc., wherein both RVT3101 and PRA023 observe significant clinical remission rates and endoscopic improvement rates in clinical trials for the treatment of inflammatory bowel disease.

Inflammatory Bowel Disease (IBD) includes Crohn's Disease (CD) and Ulcerative Colitis (UC), and is a recurrent, inflammatory disease of the involved digestive tract characterized by inflammation of the intestinal tract and epithelial lesions. The etiology of IBD is not fully understood and may be related to many factors such as genetics, environment, intestinal microecology, intestinal immunity etc ⁴. Current IBD treatment drugs include aminosalicylic acid formulations, oral glucocorticoids, oral small molecule Janus kinase inhibitors, tumor necrosis factor inhibitors, integrin receptor antagonists, interleukin (IL) 12/23 antagonists, and the like ⁵. However, the above-described treatment regimen still does not elicit an adequate response in IBD patients, and there is an unmet clinical need for treatment of IBD.

Therefore, the development and use of novel anti-human TL1A antibodies is needed in the art, with important biological and medical implications.

Summary of The Invention

In a first aspect, the application provides an antibody that binds to tumor necrosis factor-like ligand 1A (TL 1A) comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein

The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5 and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6, or

The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 7, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 8, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 9, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 10, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 11 and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 12, or

The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 7, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 13, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 9, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 14, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 15 and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 16, or

The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 17, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 18, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 19, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 20, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 21 and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 22;

Wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 23, 24, 25 or 26.

In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO 27, 28, 29 or 30.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 23 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 27, or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 24, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 28, or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 25, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 29, or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 26, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 30.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity to SEQ ID NO. 23, 24, 25 or 26, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity to SEQ ID NO. 27, 28, 29 or 30.

In some embodiments of the first aspect, the antibody is a whole antibody, a Fab fragment, a F (ab') ₂ fragment, or a single chain Fv fragment (scFv).

In some embodiments of the first aspect, the antibody is a monoclonal antibody.

In some embodiments of the first aspect, the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype.

In some embodiments of the first aspect, the antibody further comprises a light chain constant region selected from the group consisting of kappa subtype or lambda subtype.

In some embodiments of the first aspect, the antibody binds TL1A of a primate and/or rodent, and/or

The antibodies inhibit binding of TL1A to death receptor 3 (DR 3), and/or

The antibodies do not inhibit binding of TL1A to decoy receptor 3 (DcR 3), and/or

The antibody inhibits caspase activation of TL1A stimulated cells, and/or

The antibodies inhibit NF- κB activation of TL1A stimulated cells, and/or

The antibodies inhibit the ability of TL1A to stimulate secretion of interferon gamma by PBMCs.

In a second aspect, the application provides a nucleic acid molecule encoding an antibody according to the first aspect.

In a third aspect, the application provides a pharmaceutical composition comprising an antibody according to the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.

In a fourth aspect, the application provides the use of an antibody according to the first aspect, or a pharmaceutical composition according to the third aspect, in the manufacture of a medicament for the prevention or treatment of a TL1A associated disease.

In a fifth aspect, the present application provides a method of preventing or treating a TL1A associated disease comprising administering an antibody of the first aspect, or a pharmaceutical composition of the third aspect, to a subject in need thereof.

Drawings

FIG. 1 shows the results of inhibition of caspase activation by TL1A stimulated TF-1 cells by anti-TL 1A murine monoclonal antibodies.

FIG. 2 shows the results of inhibition of NF-. Kappa.B activation by TL1A by anti-TL 1A murine monoclonal antibodies to 293T-hDR 3-NF-. Kappa.B-RE-luci cells.

FIG. 3 shows the results of inhibition of caspase activation by TL1A stimulated TF-1 cells by anti-TL 1A monoclonal antibody H23F1+L28E1.

FIG. 4 shows NF-. Kappa.B activation of anti-TL 1A monoclonal antibody H23F1+L28E1 by inhibiting TL1A stimulated 293T-hDR 3-NF-. Kappa.B-RE-luci cells.

FIG. 5 shows the ability of anti-TL 1A monoclonal antibody H23F1+L28E1 to inhibit the secretion of interferon gamma by TL1A stimulated PBMC.

FIG. 6 shows the results of ELISA assays for anti-human TL1A monoclonal antibodies blocking binding of human TL1A to human DR 3.

FIG. 7 shows the results of ELISA assays for anti-TL 1A monoclonal antibodies blocking the binding activity of human DcR3 and human TL 1A.

Figure 8 shows DNBS-induced stool score for acute ulcerative colitis in rats.

Fig. 9 shows the results of macroscopic colon injury scoring for DNBS-induced acute ulcerative colitis in rats.

DESCRIPTION OF THE SEQUENCES

SEQ ID NOS 1-3 show the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively, of the anti-TL 1A single chain antibody S4D 3.

SEQ ID NOS.4-6 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the anti-TL 1A single chain antibody S4D 3.

SEQ ID NO. 7 shows the amino acid sequences of HCDR1 of the anti-TL 1A single chain antibodies S9H9 and S7B 11.

SEQ ID NO. 8 shows the amino acid sequence of HCDR2 of the anti-TL 1A single-chain antibody S9H 9.

SEQ ID NO.9 shows the amino acid sequences of HCDR3 of the anti-TL 1A single-chain antibodies S9H9 and S7B 11.

SEQ ID NOS 10-12 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the anti-TL 1A single chain antibody S9H 9.

SEQ ID NO. 13 shows the amino acid sequence of HCDR2 of the anti-TL 1A single-chain antibody S7B 11.

SEQ ID NOS 14-16 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the anti-TL 1A single chain antibody S7B 11.

SEQ ID NOS.17-19 show the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively, of the fully human anti-TL 1A monoclonal antibody H23F1+L28E1.

SEQ ID NOS.20-22 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the fully human anti-TL 1A monoclonal antibody H23F1+L28E1.

SEQ ID NO. 23 shows the amino acid sequence of the heavy chain variable region of the anti-TL 1A single-chain antibody S4D 3.

SEQ ID NO. 24 shows the amino acid sequence of the heavy chain variable region of the anti-TL 1A single-chain antibody S9H 9.

SEQ ID NO. 25 shows the amino acid sequence of the heavy chain variable region of the anti-TL 1A single-chain antibody S7B 11.

SEQ ID NO. 26 shows the amino acid sequence of the heavy chain variable region of the fully human anti-TL 1A monoclonal antibody H23F1+L28E1.

SEQ ID NO. 27 shows the amino acid sequence of the light chain variable region of the anti-TL 1A single-chain antibody S4D 3.

SEQ ID NO. 28 shows the amino acid sequence of the light chain variable region of the anti-TL 1A single-chain antibody S9H 9.

SEQ ID NO. 29 shows the amino acid sequence of the light chain variable region of the anti-TL 1A single-chain antibody S7B 11.

SEQ ID NO. 30 shows the amino acid sequence of the light chain variable region of the fully human anti-TL 1A monoclonal antibody H23F1+L28E1.

SEQ ID NO. 31 shows the amino acid sequence of recombinant human TL1A (hTL 1A).

SEQ ID NO. 32 shows the amino acid sequence of recombinant cynomolgus monkey TL1A (mfTL A).

SEQ ID NO. 33 shows the amino acid sequence of recombinant mouse TL1A (mTL A).

SEQ ID NO. 34 shows the amino acid sequence of recombinant rat TL1A (rTL 1A).

SEQ ID NO. 35 shows the amino acid sequence of the Foldon domain of the T4 bacteriophage fibrin (Fib Foldon).

SEQ ID NO. 36 shows the amino acid sequence of the His tag.

SEQ ID NO. 37 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype heavy chain constant region (CH-IgG 1).

SEQ ID NO. 38 shows the amino acid sequence of the human IgG1 subtype heavy chain constant region mutant IgG1m3 (CH-IgG 1m 3).

SEQ ID NO. 39 shows the amino acid sequence of a human IgG1 subtype heavy chain constant region mutant IgG1m3-YTE (CH-IgG 1m 3-YTE).

SEQ ID NO. 40 shows the amino acid sequence of the human (homo sapiens) kappa subtype light chain constant region.

SEQ ID NO. 41 shows the amino acid sequence of the human (homo sapiens) lambda subtype light chain constant region.

SEQ ID NO. 42 shows the amino acid sequence of the recombinant protein His-fib-hTL 1A.

SEQ ID NO. 43 shows the amino acid sequence of the recombinant protein His-fib-mfTL A.

SEQ ID NO. 44 shows the amino acid sequence of the recombinant protein His-fib-mTL A.

SEQ ID NO. 45 shows the amino acid sequence of the recombinant protein His-fib-rTL 1A.

The nucleotide sequence of primer PmCGR is shown in SEQ ID NO. 46.

SEQ ID NO. 47 shows the nucleotide sequence of primer PmCKR.

SEQ ID NO. 48 shows the amino acid sequence of the single-chain antibody S4D 3.

SEQ ID NO. 49 shows the amino acid sequence of the single-chain antibody S9H 9.

SEQ ID NO. 50 shows the amino acid sequence of the single-chain antibody S7B 11.

SEQ ID NO. 51 shows the amino acid sequence of the heavy chain variable region of the anti-TL 1A monoclonal antibody RVT-3101.

SEQ ID NO. 52 shows the amino acid sequence of the light chain variable region of the anti-TL 1A monoclonal antibody RVT-3101.

SEQ ID NO. 53 shows the amino acid sequence of the heavy chain variable region of anti-TL 1A monoclonal antibody PRA 023.

SEQ ID NO. 54 shows the amino acid sequence of the light chain variable region of anti-TL 1A monoclonal antibody PRA 023.

SEQ ID NO. 55 shows the amino acid sequence of the heavy chain variable region of the negative control antibody DP 47.

SEQ ID NO. 56 shows the amino acid sequence of the light chain variable region of the negative control antibody DP 47.

Detailed Description

The inventors of the present application obtained novel antibodies that bind to tumor necrosis factor-like ligand 1A (TL 1A) by antibody engineering techniques. In various aspects of the application, novel anti-tumor necrosis factor-like ligand 1A (TL 1A) antibodies, nucleic acid molecules encoding said antibodies, vectors comprising said nucleic acid molecules, host cells comprising said nucleic acid molecules or vectors, methods for the preparation and purification of said antibodies and medical and biological uses of said antibodies are provided. According to the amino acid sequence of the variable region of the antibody provided by the application, a full-length antibody molecule can be constructed as a medicament for preventing or treating TL1A related diseases.

The practice of the present application employs, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

Definition of the definition

The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. As used herein, "antibody" includes not only intact (i.e., full length) antibodies, but also antigen binding fragments thereof (e.g., fab ', F (ab') ₂, fv), variants thereof, fusion proteins comprising an antibody moiety, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of immunoglobulin molecules comprising an antigen recognition site of a desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

Typically, an intact or full length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second and third constant regions (CH 1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). The full length antibody may be any kind of antibody, such as IgD, igE, igG, igA or IgM (or subclasses thereof as described above), but the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the heavy chain constant domain. Typically, immunoglobulins have five main classes IgA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

The term "antigen binding fragment or antigen binding portion" as used herein refers to a portion or region of an intact antibody molecule responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3.

It is well known to those skilled in the art that complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) are regions of the variable region that have the greatest influence on the affinity and specificity of an antibody. There are two common ways of defining CDR amino acid sequences for VH or VL, namely Chothia definition and kabat definition. See, e.g., Kabat,"Sequences of Proteins of Immunological Interest",National Institutes of Health,Bethesda,Md.(1991)⁷;A1-Lazikani et al, J.mol. Biol.273:927-948 (1997) ⁸, and Martin et al, proc. Natl. Acad. Sci. USA86:9268-9272 (1989) ⁹. For a given antibody variable region amino acid sequence, the CDR amino acid sequences in VH and VL amino acid sequences may be determined according to Chothia definition or Kabat definition. In an embodiment of the application, the CDR amino acid sequences are defined using Kabat.

For a given antibody variable region amino acid sequence, the mid CDR amino acid sequence of the variable region amino acid sequence can be analyzed in a number of ways, for example, as determined using on-line software Abysis (http:// www.abysis.org /).

Examples of antigen binding fragments include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain, (2) a F (ab ') ₂ fragment, which may be a divalent fragment having two Fab ' fragments linked by a disulfide bridge of the hinge region (i.e., a dimer of Fab '), (3) an Fv fragment having the VL and VH domains of a single arm of an antibody, (4) a single chain Fv (scFv), which may be a single polypeptide chain consisting of the VH and VL domains via a peptide linker, and (5) a (scFv) ₂, which may comprise two VH and two VL domains linked by a peptide linker, the two VL domains being combined with the two VH domains via a disulfide bridge.

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of antibodies that are substantially homogeneous, i.e., the individual antibodies that make up the population are identical except for the naturally occurring mutations that may be present in a small number of individuals.

Wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 23 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 27.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 24 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 28.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 25 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 29.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 26 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 30.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID No. 23, 24, 25 or 26.

In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID No. 27, 28, 29 or 30.

In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence set forth in SEQ ID NO. 23, 24, 25 or 26 by about 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and/or additions.

In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence set forth in SEQ ID NO 27, 28, 29 or 30 by about 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and/or additions.

In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 23, 24, 25 or 26 may also be truncated by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functions of the heavy chain variable region of the antibody.

In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20,25 or more amino acids may also be added at the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO. 23, 24, 25 or 26, the resulting amino acid sequence still retaining similar functions as the heavy chain variable region of the antibody.

In some embodiments of the first aspect, 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO. 23, 24, 25 or 26, provided that the altered amino acid sequence substantially retains similar function of the heavy chain variable region of the antibody.

In some embodiments of the first aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO 27, 28, 29 or 30 may also be truncated by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functions of the light chain variable region of the antibody.

In some embodiments of the first aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20,25 or more amino acids may also be added at the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO 27, 28, 29 or 30, the resulting amino acid sequence still retaining similar functions as the light chain variable region of the antibody.

In some embodiments of the first aspect, 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO. 27, 28, 29 or 30, provided that the altered amino acid sequence substantially retains similar function of the light chain variable region of the antibody.

In some embodiments of the first aspect, the antibody is a fully human antibody.

In some embodiments of the first aspect, the antibody is a monoclonal antibody.

In some embodiments of the first aspect, the heavy chain constant region is of the IgG1 subtype.

In some embodiments of the first aspect, the heavy chain constant region comprises an Fc segment sequence of an IgG1 subtype heavy chain constant region and the amino acid sequences at positions 234, 235, 331 of the Fc segment sequence are F, E, S, respectively, wherein the antibody constant region amino acid sequence is determined according to EU numbering.

In some embodiments of the first aspect, the heavy chain constant region comprises an Fc segment sequence of an IgG1 subtype heavy chain constant region and the amino acid sequences at positions 252, 254, 256 of the Fc segment sequence are Y, T and E, respectively, wherein the antibody constant region amino acid sequence is determined according to EU numbering.

In some embodiments of the first aspect, the heavy chain constant region comprises an Fc segment sequence of an IgG1 subtype heavy chain constant region, and the amino acid sequences at positions 234, 235, 331 of the Fc segment sequence are F, E, S, respectively, and the amino acid sequences at positions 252, 254, 256 of the Fc segment sequence are Y, T and E, respectively, wherein the antibody constant region amino acid sequence is determined according to EU numbering.

In some embodiments of the first aspect, the antibody binds TL1A of a primate. In some embodiments, the primate is a human, or a monkey (e.g., cynomolgus monkey).

In some embodiments of the first aspect, the antibody binds to TL1A of a rodent. In some embodiments, the rodent is a murine, such as a rat or mouse.

In some embodiments of the first aspect, the antibody binds recombinant human TL1A (SEQ ID NO: 31).

In some embodiments of the first aspect, the antibody binds to recombinant monkey (e.g., cynomolgus monkey) TL1A (SEQ ID NO: 32).

In some embodiments of the first aspect, the antibody binds recombinant mouse TL1A (SEQ ID NO: 33).

In some embodiments of the first aspect, the antibody binds recombinant rat TL1A (SEQ ID NO: 34).

In some embodiments of the first aspect, the antibody inhibits binding of TL1A to death receptor 3 (DR 3).

In some embodiments of the first aspect, the antibody does not inhibit binding of TL1A to decoy receptor 3 (DcR 3).

In some embodiments of the first aspect, the antibody inhibits caspase activation of TL1A stimulated cells (e.g., tumor cells, e.g., TF-1 cells).

In some embodiments of the first aspect, the antibody inhibits NF- κB activation of TL 1A-stimulated cells (e.g., 293T-hDR-NF- κB-RE-luci cells).

In some embodiments of the first aspect, the antibody inhibits the ability of TL1A to stimulate secretion of interferon gamma by PBMCs.

In some embodiments, the nucleic acid molecule is operably linked to regulatory sequences that can be recognized by a host cell transformed with the vector.

In some embodiments of the third aspect, the pharmaceutical composition is for use in preventing or treating a TL1A associated disease.

In some embodiments of the third aspect, the TL1A associated disease is rheumatoid arthritis, inflammatory bowel disease (e.g., crohn's Disease (CD) and Ulcerative Colitis (UC)), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, and systemic sclerosis associated interstitial lung disease.

In some embodiments of the third aspect, the pharmaceutical composition may further comprise one or more of lubricants such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying agents, suspending agents, preserving agents such as benzoic acid, sorbic acid and calcium propionate, sweetening and/or flavouring agents and the like.

In some embodiments of the third aspect, the pharmaceutical compositions of the present application may be formulated in the form of tablets, pills, powders, troches, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules, and the like.

In some embodiments of the third aspect, the pharmaceutical compositions of the present application may be delivered using any physiologically acceptable mode of administration including, but not limited to, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.

In some embodiments of the third aspect, the pharmaceutical composition for therapeutic use may be formulated for storage in the form of a lyophilized formulation or an aqueous solution by mixing an agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or the like.

In a fourth aspect, the application provides the use of an antibody according to the first aspect, a pharmaceutical composition according to the third aspect, in the manufacture of a medicament for the prevention or treatment of TL1A associated diseases.

In some embodiments of the fourth aspect, the TL1A associated disease is rheumatoid arthritis, inflammatory bowel disease (e.g., crohn's Disease (CD) and Ulcerative Colitis (UC)), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, and systemic sclerosis associated interstitial lung disease.

In some embodiments of the fifth aspect, the TL1A associated disease is rheumatoid arthritis, inflammatory bowel disease (e.g., crohn's Disease (CD) and Ulcerative Colitis (UC)), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, and systemic sclerosis associated interstitial lung disease.

In other aspects, the application also provides vectors comprising nucleic acid molecules encoding the antibodies of the application or light or heavy chains thereof, host cells comprising the nucleic acid molecules or the vectors, and methods of producing the antibodies. In some embodiments, the nucleic acid molecule is operably linked to a regulatory nucleotide sequence that can be recognized by a host cell transformed with the vector. In some embodiments, the method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid. In some embodiments, the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.

It should be understood that the foregoing detailed description is only for the purpose of making apparent to those skilled in the art the contents of the application, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will occur to those skilled in the art.

Examples

The following examples are given for the purpose of illustration only and are not intended to limit the scope of the application.

EXAMPLE 1 preparation of recombinant proteins

A number of different recombinant proteins are required for the preparation and identification of anti-TL 1A antibodies, including human TL1A (hTL 1A, SEQ ID NO: 31), cynomolgus monkey TL1A (mfTL A, SEQ ID NO: 32), mouse TL1A (mTL A, SEQ ID NO: 33) and rat TL1A (rTL 1A, SEQ ID NO: 34). The addition of the Foldon domain of T4 phage fibrin (Fib Foldon, SEQ ID NO: 35) at the N-terminus of these recombinant proteins helps to maintain the native trimeric conformation of TL1A and enhances stability. Meanwhile, his tag (His, SEQ ID NO: 36) is added to the N end of the recombinant proteins, which is favorable for purification and functional identification of the recombinant proteins. In the preparation of recombinant antibodies, the antibody heavy chain constant region may be of the human IgG1 subtype (CH-IgG 1, SEQ ID NO: 37) or various mutants of the defined human IgG1 subtype, such as IgG1m3 (CH-IgG 1m3, SEQ ID NO: 38) and IgG1m3-YTE (CH-IgG 1m3-YTE, SEQ ID NO: 39), and the light chain constant region may be of the human kappa subtype (CK, SEQ ID NO: 40) or of the human lambda subtype (CL, SEQ ID NO: 41).

Genes (including His tag and Fib Foldon domains) of the above recombinant proteins were designed and synthesized based on the amino acid sequences of the recombinant proteins in the Uniprot database. The various recombinant protein genes synthesized are cloned into a suitable eukaryotic expression vector (such as pcDNA3.1 of Invitrogen company, etc.) using conventional molecular biology techniques, and then the prepared recombinant protein expression plasmid is transfected into HEK293 cells (such as HEK293F of Invitrogen company) using liposomes (such as 293fectin of Invitrogen company, etc.) or other cationic transfection reagents (such as PEI, etc.), cultured for 3-4 days under serum-free suspension culture conditions, and then the culture supernatant is harvested by centrifugation, etc.

The recombinant proteins His-fib-hTL1A (SEQ ID NO: 42), his-fib-mfTL A (SEQ ID NO: 43), his-fib-mTL A (SEQ ID NO: 44) and His-fib-rTL1A (SEQ ID NO: 45) expressed by His tag fusion were purified in one step from the culture supernatant using a metal chelating affinity chromatography column (e.g., GE company HISTRAP FF, etc.). The recombinant antibody is purified in one step using a ProteinA/G affinity column (e.g., mabselect SURE, inc. of GE Co., ltd.). The recombinant protein preservation buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., hitrap desaulting from GE). After filtration and sterilization, the mixture is packaged and stored at-20 ℃ for standby.

EXAMPLE 2 preparation and screening of immune repertoires

2.1 Preparation of mouse immunity and antibody library

BALB/c mice of 6-8 weeks old were collected from the tail vein of the mice prior to immunization and background serum was left. First immunization His-fib-hTL1A fusion protein was emulsified with Freund's complete adjuvant and 50. Mu.g of fusion protein was injected into each mouse. Two weeks apart of booster immunization, the first and third booster immunizations were performed by emulsifying His-fib-hTL1A fusion protein with Freund's incomplete adjuvant, and 50. Mu.g of fusion protein was injected into each mouse, followed by tail-breaking blood collection. The second and fourth booster immunizations were emulsified with His-fib-mfTL A in Freund's incomplete adjuvant and 100. Mu.g of fusion protein was injected into each mouse. The sixth immunization was performed with the His-fib-hTL1A recombinant antigen without adjuvant as immunogen, and 50. Mu.g of fusion protein was injected into each mouse, and the mice were sacrificed 3 days after the impact immunization and spleen cells were collected.

Mouse spleen lymphocytes were isolated using a mouse lymphocyte isolate (Beijing daceae Biotechnology Co., ltd., CAT#DKW 33-R0100), and total lymphocyte RNA was extracted using a cell total RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd., CAT#DP430). cDNA of the heavy chain variable region and cDNA of the light chain variable region are synthesized by using the extracted total RNA as a template by using a first strand cDNA synthesis kit (Thermo scientific, CAT#K1621), and reverse transcription primers are gene-specific primers, wherein the primer pairs are respectively positioned in an antibody heavy chain constant region and an antibody light chain constant region, and specific sequences are PmCGR: TGCATTTGAACTCCTTGCC (SEQ ID NO: 46) and PmCKR: CCATCAATCTTCCACTTGAC (SEQ ID NO: 47). The synthesized cDNA was immediately stored at-70℃until use. Then, primers were synthesized according to reference ⁶ using the cDNA obtained by reverse transcription as a template, and murine antibody VH and VK genes were amplified by PCR, respectively, and then single chain antibody (scFv) genes were constructed by overlap extension PCR technique. Finally, the prepared mouse single-chain antibody gene is cloned to a vector pADSCFV-S (see China patent application No. 201510097117.0- ⁷) to construct an scFv library. The library capacity of the antibody library reaches 6.0E+8, and the accuracy is 70%.

2.2 Screening of Single chain antibody repertoires from mice

His-fib-hTL1A and His-fib-mfTL A prepared in example 1 are used as antigens, and a phage library constructed as described above and displaying mouse single-chain antibodies is screened by using a solid phase screening strategy (experimental scheme refers to phage display: general experimental guidelines, (Meclackson, T.), (Me.) Loman (Lowman, H.B.), compiled by Ma Lan et al, chemical industry Press, 2008.5) ⁸, and 3 rounds of screening are performed in a combined, eluted, neutralized, infected and amplified manner to finally obtain 3 single-chain antibodies S4D3 (SEQ ID NO: 48), S9H9 (SEQ ID NO: 49) and S7B11 (SEQ ID NO: 50) with different sequences.

Single chain antibodies S4D3, S9H9 and S7B11 were prepared as whole antibodies of the IgG1m3 subtype, respectively, using conventional molecular biology means. With reference to U.S. Pat. No. 4, 20150132311A1, ⁹, the heavy chain variable region (SEQ ID NO: 51) and the light chain variable region (SEQ ID NO: 52) of RVT-3101 were synthesized, and with reference to U.S. Pat. No. 4, 20210122828A1, ¹⁰, the heavy chain variable region (SEQ ID NO: 53) and the light chain variable region (SEQ ID NO: 54) of PRA023 were synthesized. RVT-3101 and PRA023 were prepared as IgG1m3 subtype whole antibodies used as positive controls. DP47 (germ line gene antibody, see U.S. Pat. No. 5, 20160200833A1 No. ¹¹, DP47VH, SEQ ID NO:55;DP47VK,SEQ ID NO:56) was prepared as an IgG1m3 subtype whole antibody for use as a negative control.

EXAMPLE 3 identification of anti-TL 1A murine monoclonal antibody

3.1 Affinity analysis of murine monoclonal antibodies S4D3, S9H9 and S7B11 against TL1A

The affinity of murine anti-human TL1A monoclonal antibodies was determined by surface plasmon resonance technique using Biacore T200. The relevant reagents and consumables such as amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), S series CM5 chip (14100530) and 10 XHBS-EP (BR 100669) at pH7.4 were purchased from GE HEALTHCARE. Carboxylated CM5 chip surfaces were activated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, EDC) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) according to the instructions in the kit, anti-human IgG (Fc) antibody (capture antibody) was diluted to 25. Mu.g/mL with 10mM sodium acetate pH5.0, followed by injection at a flow rate of 10. Mu.L/min to achieve a coupling of approximately 10000 Response Units (RU). After injection of the capture antibody, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, anti-TL 1A monoclonal antibody was diluted to 1. Mu.g/mL and injected at 10. Mu.L/min, ensuring that about 200RU of antibody was captured by anti-human Fc antibody. His-fib-hTL1A, his-fib-mTL A and His-fib-mfTL A were set up in a series of concentration gradients (e.g., 1.23nM, 3.7nM, 11.1nM, 33.3nM and 100 nM) and injected at a flow rate of 30. Mu.L/min from low to high concentration for 90s of binding time and 1200s of dissociation time and 3M MgCl ₂ injected at a flow rate of 10. Mu.L/min for 30s total to regenerate the chip surface. Binding rate (K _a) and dissociation rate (K _d) were calculated by fitting the binding and dissociation sensorgrams using Biacore T200 evaluation software version 3.2.1, 1 binding model. The dissociation equilibrium constant (K _D) is calculated at a ratio K _d/K_a. Fitting results are shown in tables 1,2 and 3.

TABLE 1 affinity constants for binding of TL1A monoclonal antibodies to human TL1A (His-fib-hTL 1A)

Antibodies to	K_a(M^-1s^-1))	K_d(s^-1)	K_D(M)
				RVT-3101	6.521E+5	1.001E-4	1.536E-10
PRA023	4.755E+5	1.686E-4	3.546E-10
				S4D3	5.323E+5	1.313E-4	2.467E-10
S9H9	1.340E+7	5.012E-4	3.742E-11
				S7B11	4.704E+5	1.101E-4	2.341E-10

TABLE 2 affinity constants of TL1A monoclonal antibodies to mouse TL1A (His-fib-mTL A)

Antibodies to	K_a(M^-1s^-1)	K_d(s^-1)	K_D(M)
				RVT-3101	9.039E+5	4.358E-5	4.821E-11
PRA023	NB	/	/
				S4D3	NB	/	/
S9H9	6.857E+4	2.551E-3	3.719E-8
				S7B11	6.832E+4	3.657E-3	5.353E-8

NB No binding was detected

TABLE 3 affinity constants of TL1A monoclonal antibodies to monkey TL1A (His-fib-mfTL A)

Antibodies to	K_a(M^-1s^-1)	K_d(s^-1)	K_D(M)
				RVT-3101	1.643E+6	4.515E-5	2.748E-11
PRA023	1.124E+6	9.014E-5	8.023E-11
				S4D3	1.290E+6	6.309E-5	4.893E-11
S9H9	2.900E+6	1.749E-4	6.031E-11
				S7B11	1.322E+6	6.930E-5	5.202E-11

3.2 Effect of anti-TL 1A murine monoclonal antibody inhibiting the activation of TF-1 cell caspase stimulated by TL1A

TF-1 cells (ATCC, CRL-2003, human erythroid leukemia cells) were purchased from ATCC and cell growth was completely dependent on IL-3 or GM-CSF and non-responsive to IL-5. This cell expresses the TL1A receptor DR3 and TL1A stimulates caspase 3/7 activation in this cell. Cycloheximide (CHX) are inhibitors of eukaryotic protein synthesis, which inhibit cellular protein synthesis. TF-1 cells were resuspended to 2X 10 ⁵/mL, 50. Mu.L/well in 96-well plates using RPMI 1640+5% inactivated FBS as test medium, and plated with test medium containing 2. Mu. M Cycloheximide. The antibodies were diluted with a test medium containing 800ng/mL His-fib-hTL1A, starting at 40nM, 1.7-fold gradient dilution for 10 concentration spots. The diluted test antibody was added to TF-1 cells at 50. Mu.L/well, and the total volume was 100. Mu.L/well, and incubated at 37℃for 5 hours with 5% CO ₂. Caspase 3/7 activity was detected using a Caspase-glo 3/7 kit (promega, G8093), using a multifunctional microplate reader (Molecular Devices,I3X) was scanned for full wavelength and fit analysis was performed with chemiluminescent readings (FIGS. 1 and 4) showing that anti-TL 1A murine monoclonal antibodies S4D3, S9H9 and S7B11 were effective in inhibiting His-fib-hTL 1A-induced caspase activation with activity comparable to RVT-3101.

TABLE 4 results of inhibition of caspase activation by TL1A stimulated TF-1 cells by anti-TL 1A murine monoclonal antibodies

Antibodies to	IC₅₀(nM)
		RVT-3101	3.129
S4D3	3.047
		S9H9	2.686
S7B11	2.686

3.3 Inhibition of TL1A murine monoclonal antibodies against TL1A stimulation of NF- κB activation of 293T-hDR3-NF- κB-RE-luci cells

293T-NF- κB-RE-luci (cat. CS 025) was purchased from the Biotechnology Co.Ltd. Of the England flourishing industry and was a NF- κB reporter cell strain. On the basis of the cells, the cells are transfected and expressed hDR by liposome, and 293T-hDR-NF- κB-RE-luci cell lines stably expressing hDR3 are obtained by screening and used for evaluating the activity of the anti-TL 1A antibody. The test antibody was diluted with 240ng/mL His-fib-hTL1A in a 20nM initial concentration, 1.7-fold gradient, 10 concentration points total using DMEM+2% FBS as the test medium. 293T-hDR-NF-. Kappa.B-RE-luci cells were diluted to 4X 10 ⁶/mL with test medium, 50. Mu.L of cells were mixed with 50. Mu.L of antibody and inoculated into 96-well plates, and cultured in 5% CO ₂ at 37℃for 20 hours. The activities of Firefly (Firefly) luciferase and Renilla (Renilla) luciferase were sequentially detected using the Dual-glo luciferase assay kit (Promega, E2920). Multifunctional enzyme label instrument (Molecular Devices,I3X) performing full-wave scanning, and performing fitting analysis by using the ratio of the firefly luciferase luminescence signal to the sea cucumber luciferase luminescence signal. The results (FIG. 2 and Table 5) show that the anti-TL 1A murine monoclonal antibodies S4D3, S9H9 and S7B11 effectively inhibited the His-fib-hTL1A induced NF- κB signaling, with activity comparable to RVT-3101.

TABLE 5 results of inhibition of NF- κB activation by TL 1A-stimulated 293T-hDR3-NF- κB-RE-luci cells by anti-TL 1A murine monoclonal antibody

Antibodies to	IC₅₀(nM)
		S9H9	1.284
S7B11	1.346
		RVT-3101	1.278
S4D3	1.128

EXAMPLE 4 screening of fully humanized Fab recombinant libraries

A fully human Fab phage library was screened using the recombinant His-fib-hTL1A and His-fib-mfTL A prepared in example 1 as antigens using a solid phase screening strategy (experimental protocol reference phage display: general guidelines, (Mei) Clackson (T.), (Mei) Loman (Lowman, H.B.); compiled Ma Lan et al, chemical industry Press, 2008.5) (see China patent application No. 202210871809.6). 3 rounds of screening are carried out in a combined, eluted, neutralized, infected and amplified way, and finally 1 monoclonal antibody H3F1+L28E1 (the amino acid sequence of H3F1VH is shown as SEQ ID NO:26, and the amino acid sequence of L28E1VK is shown as SEQ ID NO: 30) which specifically binds to human, cynomolgus monkey, mouse and rat TL1A is obtained.

H3f1+l28e1 was prepared as IgG1m3 subtype whole antibodies using conventional molecular biology means.

Example 5 identification of fully human anti-TL 1A monoclonal antibodies

5.1 Affinity analysis of anti-TL 1A monoclonal antibody H3F1+L28E1

Referring to example 3.1, the affinity of the TL1A monoclonal antibody h3f1+l28e1 to bind to different species of TL1A was analyzed using Biacore T200, and the results are shown in tables 6, 7, 8 and 9.

TABLE 6 affinity constant for anti-TL 1A monoclonal antibody H3F1+L28E1 binding to human TL1A (His-fib-hTL 1A)

Antibodies to	K_a(M^-1s^-1)	K_d(s^-1)	K_D(M)
				RVT-3101	7.063E+5	1.016E-4	1.438E-10
PRA023	4.755E+5	1.686E-4	3.546E-10
				H3F1+L28E1	5.381E+6	1.562E-4	2.093E-11

TABLE 7 affinity constant for anti-TL 1A monoclonal antibody H3F1+L28E1 binding to mouse TL1A (His-fib-mTL A)

Antibodies to	K_a(M^-1s^-1)	K_d(s^-1)	K_D(M)
				RVT-3101	7.635E+5	5.92E-5	7.753E-11
PRA023	NB	/	/
				H3F1+L28E1	9.643E+5	1.831E-3	1.898E-9

NB No binding was detected

TABLE 8 affinity constant for anti-TL 1A monoclonal antibody H3F1+L28E1 binding to rat TL1A (His-fib-rTL 1A)

Antibodies to	K_a(M^-1s^-1)	K_d(s^-1)	K_D(M)
				RVT-3101	9.778E+5	4.243E-4	4.34E-10
PRA023	NB	/	/
				H3F1+L28E1	8.037E+6	1.170E-3	1.456E-10

NB No binding was detected

TABLE 9 affinity constants for anti-TL 1A monoclonal antibody H3F1+L28E1 binding monkey TL1A (His-fib-mfTL A)

Antibodies to	K_a(M^-1s^-1)	K_d(s^-1)	K_D(M)
				RVT-3101	1.340E+6	6.700E-5	4.998E-11
PRA023	1.124E+6	9.014E-5	8.023E-11
				H3F1+L28E1	4.191E+6	1.330E-4	3.173E-11

5.2H3F1+L2831 inhibition of TL1A stimulation of caspase activation in TF-1 cells

Referring to example 3.2, the inhibition of Tf1+L2831 by TL1A stimulates caspase activation of TF-1 cells was evaluated. The antibodies were diluted with a test medium containing 800ng/mL His-fib-hTL1A, starting at 40nM, 1.7-fold gradient dilution for 10 concentration points. The results (FIG. 3 and Table 10) show that H2+L28E1 can effectively inhibit caspase signal caused by His-fib-TL1A, and the activity is superior to RVT-3101 and PRA023 control antibody.

TABLE 10 results of H3F1+L2831 inhibition of TL1A stimulation of caspase activation in TF-1 cells

Antibodies to	IC₅₀(nM)
		RVT-3101	3.464
PRA023	6.523
		H3F1+L28E1	1.734

Inhibition of TL1A by 3H3F1+L28E1 stimulates NF- κB activation in 293T-hDR-NF- κB-RE-luci cells

Referring to example 3.3, inhibition of TL1A by H2F1+L28E1 was evaluated for NF- κB activation by 293T-hDR3-NF- κB-RE-luci cells. The test antibody was diluted with 240ng/mL His-fib-hTL1A in test medium at an initial concentration of 20nM and 1.7-fold gradient dilution for a total of 10 concentration points. The results (FIG. 4 and Table 11) show that H2+L28E1 can effectively inhibit His-fib-hTL1A stimulated NF- κB signal, and the activity is superior to RVT-3101 and PRA023.

TABLE 11 NF- κB activation of h3F1+L28E1 inhibits TL1A stimulated 293T-hDR-NF- κB-RE-luci cells

Antibodies to	IC₅₀(nM)
		RVT-3101	1.082
PRA023	2.813
		H3F1+L28E1	0.574

5.4H3F1+L28E1 inhibits the ability of TL1A to stimulate secretion of interferon gamma by PBMC

NK cells or activated T cells express TL1A receptor DR3 at their surface, and binding of TL1A to NK cells or activated T cells with the aid of co-stimulatory factors such as IL-12/IL-18 activates downstream signaling pathways, ultimately leading to secretion of interferon gamma. Inhibition of TL1A by anti-TL 1A antibodies can be assessed by measuring the level of interferon gamma in the cell supernatant.

Blood (50 mL) was collected from normal volunteers, with the collected blood being provided by the inventors and their colleagues as volunteers, all of whom had signed a known consent. Inclusion criteria for volunteers were:

1. Age above 18 years old;

2. no infection of HIV and HBV;

3. Normal blood routine detection;

4. non-pregnant or lactating women.

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy human peripheral blood using Ficoll density gradient centrifugation. As test medium, RPMI 1640+5% inactivated FBS was used, PBMC were resuspended to 6X 10 ⁵/mL with test medium containing 0.5ng/mL IL-12 and 2ng/mL IL-18, 100. Mu.L/well was plated in 96-well plates, and incubated at 37℃in 5% CO ₂ for 3 hours. After 3 hours, the antibodies were diluted with a test medium containing 2.4. Mu.g/mL His-fib-hTL1A, starting at 200nM, 2.3-fold gradient dilution, total 8 concentration spots. Diluted antibodies were added to PBMC at 100. Mu.L/well and incubated at 37℃for 20 hours with 5% CO ₂ in total volume of 200. Mu.L/well. The interferon gamma content of the cell supernatants was measured using a human IFN-gamma pre-coat ELISA kit (Dayou 1110002). The values were read at OD450nm using a microplate reader (800 TS, biotek), the standard curves of concentration and absorbance were drawn with the interferon gamma standard contained in the detection kit, and the interferon gamma content in the supernatant was obtained by conversion and subjected to fitting analysis. The results (FIG. 5 and Table 12) show that H2+L28E1 can effectively inhibit His-fib-hTL1A from stimulating PBMC to secrete interferon gamma, and the activity is superior to RVT-3101 and PRA023.

TABLE 12 ability of h3F1+L28E1 to inhibit the stimulation of interferon gamma secretion by PBMC by TL1A

Antibodies to	IC₅₀(nM)
		RVT-3101	13.61
PRA023	6.545
		H3F1+L28E1	5.232

Example 6 Selective neutralization of the functional receptor DR3 by anti-TL 1A monoclonal antibodies

Human DR3 (Recombinant Human DR/TNFRSF 25 FC CHIMERA Protein, CF, R & D SYSTEMS, 943-D3-050) was biotinylated using a biotin labeling kit (EZ-Link ^TMSulfo-NHS-Biotin,No-Weigh^TM Format, thermo Fisher, A39256). His-fib-hTL1A was coated on 96-well ELISA plates, 4. Mu.g/mL, 100. Mu.L/well, 4℃overnight. Blocking was performed with blocking solution (3% skim milk-PBST) for 1 hour at 37 ℃. The anti-human TL1A monoclonal antibodies (RVT-3101 and H2+L28E1) were each diluted in a gradient of 10. Mu.g/mL with a starting concentration of 200. Mu.g/mL, 3-fold, 8 concentration gradients total, 100. Mu.L/well and incubated at 37℃for 1 hour in a blocked 96-well ELISA plate. ELISA plates were washed using PBST and then incubated with horseradish peroxidase-labeled Streptavidin (Strepitavidin/HRP, borson, bs-0437P-HRP) at 37℃for 1 hour. ELISA plates were washed with PBST, OPD substrate chromogenic solution was added, after 5-10 minutes, the chromogenic was stopped with 1M H ₂SO₄, and the 490nm single wavelength optical density was measured using an enzyme-labeled instrument. ELISA analysis results are shown in FIG. 6, where RVT-3101 and H2+L28E1 block binding of human DR3 to human TL1A.

Human DcR3 (Recombinant Human DcR/TNFRSF 6B FC CHIMERA Protein, CF, R & D SYSTEMS, 142-DC-100) was coated on 96-well ELISA plates, 3. Mu.g/mL, 100. Mu.L/well, 4℃overnight. Blocking was performed with blocking solution (3% skim milk-PBST) for 1 hour at 37 ℃. A3. Mu.g/mL His-fib-hTL1A was used to gradient the anti-human TL1A monoclonal antibodies (RVT-3101, PRA023, S4D3, S9H9, S7B11 and H2+L28E1) respectively, starting at a concentration of 100. Mu.g/mL, 3-fold gradient dilution, 11 concentration gradients total, 100. Mu.L/well were added to a blocked 96-well ELISA plate and incubated at 37℃for 1 hour. ELISA plates were washed using PBST, then HRP-labeled anti-His tag murine monoclonal antibody (well-known as century, cw 0285M) was added and incubated at 37℃for 1 hour. ELISA plates were washed with PBST, OPD substrate chromogenic solution was added, after 5-10 minutes, the chromogenic was stopped with 1M H ₂SO₄, and the 490nm single wavelength optical density was measured using an enzyme-labeled instrument. The ELISA assay results are shown in FIG. 7, where RVT-3101 and PRA023 block human DcR3 from binding to human TL1A, and S4D3, S9H9, S7B11 and H2+L28E1 do not block human DcR3 from binding to human TL1A.

DcR3 acts as a co-decoy receptor for TNF family cytokines (Fas-L, LIGHT and TL 1A), and has a natural antagonistic function in vivo. S4D3, S9H9, S7B11 and h3f1+l28e1 were able to neutralize DR3 instead of DcR3, suggesting that these antibodies do not destroy the natural antagonistic activity of DcR3, helping to maintain the homeostasis of DcR3 with better safety.

Example 7 Effect of anti-TL 1A monoclonal antibody on DNBS-induced acute ulcerative colitis in rats

Male Wistar rats without dosing history were purchased from Shanghai Laek laboratory animal feeding Co., ltd and acclimatized to the animal house environment 3 days in advance. Rats were fasted for 40 hours prior to DNBS molding, and 5% glucose injection (10 mL/kg) was injected subcutaneously during the fasted period as an energy supplement during the fasted period. DNBS powder was dissolved in 30% ethanol to a final concentration of 60mg/mL and fasted rats were anesthetized on day 0 with sultai (intraperitoneal injection, 25-50 mg/kg) and xylazine (intraperitoneal injection, 5-10 mg/kg). The specific animal groupings and dosing regimens are shown in table 13. Wherein the G2-G4 group extended the hose from anus into colon, and induced colitis in rats by DNBS enema. Group G1 was enemaed with 30% ethanol in the same manner. Isotype control was DP47 antibody.

TABLE 13 grouping and dosing regimen

During the test period, the fecal properties of the experimental animals were scored daily (0=normal, 1=wet/sticky, 2=soft, 3=liquid). Body weights of experimental animals were measured and recorded daily. On day 6, all experimental animals were euthanized with excess carbon dioxide asphyxiation, after which the abdominal cavity was cut, the colon was removed, split longitudinally, the colonic ulcer surface was observed after rinsing, and the colonic length, weight and ulcer area were recorded for macroscopic damage scoring of the colon. The results (FIGS. 8 and 9) show that the fecal characteristics and intestinal lesions of the experimental animals of group G4 (H23F1+L28E1) are significantly improved over those of group G2 (model-isotype control).

All patents, patent application publications, and non-patent documents mentioned and/or listed in this disclosure are incorporated herein by reference in their entirety. While exemplary embodiments of the application have been described above, modifications and improvements to the described exemplary embodiments of the application can be made by those skilled in the art without departing from the spirit and scope of the application, and the resulting variations or equivalents thereof also fall within the scope of the application.

Sequence information

SEQ ID NO:1

KYDIN

SEQ ID NO:2

WIFPGDGRTDYNEKFKG

SEQ ID NO:3

YGYALDY

SEQ ID NO:4

RSSQNIVHSNGDTYLE

SEQ ID NO:5

KVSNRFS

SEQ ID NO:6

FQGSHFPYT

SEQ ID NO:7

NYWLG

SEQ ID NO:8

DIHPGRGNIFYNEKFKG

SEQ ID NO:9

GYDTFDY

SEQ ID NO:10

KASQDVSTAVA

SEQ ID NO:11

SASYRYT

SEQ ID NO:12

QQHYSTPWT

SEQ ID NO:13

DIHPGRGNIYYNEKFKG

SEQ ID NO:14

RASKSISKYLA

SEQ ID NO:15

SGSTLQS

SEQ ID NO:16

QQHNEYPYT

SEQ ID NO:17

SYDIN

SEQ ID NO:18

WLNPNSGNTGYAQKFQG

SEQ ID NO:19

EIPESAAIEY

SEQ ID NO:20

TSSSSDIGAGLGVH

SEQ ID NO:21

GYYNRPS

SEQ ID NO:22

QSYDASLTGI

SEQ ID NO:23

QVQLKESGSDLATPGASVKLSCKVSGYTFTKYDINWVRQRPEQGLEWIGWIFPGDGRTDYNEKFKGKATLTIDTSSSTAYMQLSRLTSEDSAVYFCARYGYALDYWGQGTSVTVSS

SEQ ID NO:24

QVQLKQSGAELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIFYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAEGYDTFDYWGQGTTLTVSA

SEQ ID NO:25

QVKLQQSGGELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAEGYDTFDYWGQGTTLTVSS

SEQ ID NO:26

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWLNPNSGNTGYAQKFQGRVTMTADRSTSTAYMELSSLRSEDTAVYYCAREIPESAAIEYWGQGTLVTVSS

SEQ ID NO:27

DVVMTQTPLSLPVSLGDQASISCRSSQNIVHSNGDTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHFPYTFGGGTKLEMK

SEQ ID NO:28

DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEMK

SEQ ID NO:29

DIVMTQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIK

SEQ ID NO:30

QSVLTQPPSVSGAPGQRVTISCTSSSSDIGAGLGVHWYQQLPGTAPKLLIEGYYNRPSGVPDRFSGSKSGTSASLTITGLLPEDEGDYYCQSYDASLTGIFGGGTKLTVL

SEQ ID NO:31

LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

SEQ ID NO:32

LKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHLKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFVYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

SEQ ID NO:33

ITEERSEPSPQQVYSPPRGKPRAHLTIKKQTPAPHLKNQLSALHWEHDLGMAFTKNGMKYINKSLVIPESGDYFIYSQITFRGTTSVCGDISRGRRPNKPDSITMVITKVADSYPEPARLLTGSKSVCEISNNWFQSLYLGATFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLL

SEQ ID NO:34

VTEERSAPSAQPVYTPSRDKPKAHLTIMRQTPVPHLKNELAALHWENNLGMAFTKNRMNYTNKFLVIPESGDYFIYSQITFRGTTSECGDISRVRRPKKPDSITVVITKVADSYPEPAHLLTGTKSVCEISSNWFQPIYLGAMFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLI

SEQ ID NO:35

GYIPEAPRDGQAYVRKDGEWVLLSTFL

SEQ ID NO:36

HHHHHH

SEQ ID NO:37

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:38

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:39

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:40

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:41

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:42

HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSLKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSN

WFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

SEQ ID NO:43

HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSLKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHLKNQFPALHWEHELGLAFTKNRMNYTNKFLLIPESGDYFVYSQVTFRGMTSECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL

SEQ ID NO:44

HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSITEERSEPSPQQVYSPPRGKPRAHLTIKKQTPAPHLKNQLSALHWEHDLGMAFTKNGMKYINKSLVIPESGDYFIYSQITFRGTTSVCGDISRGRRPNKPDSITMVITKVADSYPEPARLLTGSKSVCEISNNWFQSLYLGATFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLL

SEQ ID NO:45

HHHHHHGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSVTEERSAPSAQPVYTPSRDKPKAHLTIMRQTPVPHLKNELAALHWENNLGMAFTKNRMNYTNKFLVIPESGDYFIYSQITFRGTTSECGDISRVRRPKKPDSITVVITKVADSYPEPAHLLTGTKSVCEISSNWFQPIYLGAMFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLI

SEQ ID NO:46

TGCATTTGAACTCCTTGCC

SEQ ID NO:47

CCATCAATCTTCCACTTGAC

SEQ ID NO:48

QVQLKESGSDLATPGASVKLSCKVSGYTFTKYDINWVRQRPEQGLEWIGWIFPGDGRTDYNEKFKGKATLTIDTSSSTAYMQLSRLTSEDSAVYFCARYGYALDYWGQGTSVTVSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQNIVHSNGDTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHFPYTFGGGTKLEMK

SEQ ID NO:49

QVQLKQSGAELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIFYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAEGYDTFDYWGQGTTLTVSAGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEMK

SEQ ID NO:50

QVKLQQSGGELVRPGTSVKISCKASGYAFTNYWLGWIKQRPGHGLEWIGDIHPGRGNIYYNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAEGYDTFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIK

SEQ ID NO:51

QVQLVQSGAEVKKPGASVKVSCKASGYDFTYYGISWVRQAPGQGLEWMGWISTYNGNTHYARMLQGRVTMTTDTSTRTAYMELRSLRSDDTAVYYCARENYYGSGAYRGGMDVWGQGTTVTVSS

SEQ ID NO:52

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPWTFGQGTKVEIK

SEQ ID NO:53QVQLVQSGAEVKKPGASVKVSCKASGFDIQDTYMHWVKQRPGQGLEWMGRIDPASGHTKYDPKFQVRVTITRDTSTSTVYLELSSLRSEDTAVYYCARSGGLPDVWGQGTTVTVSS

SEQ ID NO:54

EIVLTQSPGTLSLSPGERATLSCRASSSVSYMYWYQQKPGQAPRPLIYATSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWEGNPRTFGGGTKLEIK

SEQ ID NO:55

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS

SEQ ID NO:56

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK

Reference to the literature

1.Xu WD,Li R,Huang AF.Role of TL1A in Inflammatory Autoimmune Diseases:A Comprehensive Review.Front Immunol.2022Jul 14;13:891328.

2.Migone TS,Zhang J,Luo X,Zhuang L,Chen C,Hu B,Hong JS,Perry JW,Chen SF,Zhou JX,Cho YH,Ullrich S,Kanakaraj P,Carrell J,Boyd E,Olsen HS,Hu G,Pukac L,Liu D,Ni J,Kim S,Gentz R,Feng P,Moore PA,Ruben SM,Wei P.TL1A is a TNF-like ligand for DR3 and TR6/DcR3and functions as a T cell costimulator.Immunity.2002Mar;16(3):479-92.

3.Valatas V,Kolios G,Bamias G.TL1A(TNFSF15)and DR3(TNFRSF25):A Co-stimulatory System of Cytokines With Diverse Functions in Gut Mucosal Immunity.Front Immunol.2019Mar27;10:583.

4.Ramos GP,Papadakis KA.Mechanisms of Disease:Inflammatory Bowel Diseases.Mayo Clin Proc.2019Jan;94(1):155-165.

5.Li N,Ye M.Advances in biological agents in the treatment of inflammatory bowel disease[J].Yixue Xinzhi Zazhi,2022,32(4):310-320.

6.Krebber A,Bornhauser S,Burmester J,Honegger A,Willuda J,Bosshard HR,Plückthun A.Reliable cloning of functional antibody variabledomains from hybridomas and spleen cell repertoires employing a reengineered phage display system.J Immunol Methods.1997Feb14;201(1):35-55.

7.CN201510097117.0.

8. Phage display were compiled in general experimental guidelines/(Clackson, T.), (Mei) Loman (Lowman, H.B.), ma Lan et al. Chemical industry Press 2008.5.

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10.US20210122828A1

11.US20160200833A1。

Claims

1. An antibody that binds to tumor necrosis factor-like ligand 1A (TL 1A) comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3, wherein

The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 17, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 18, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 19, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 20, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 21 and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 22

Wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.

2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 26.

3. The antibody of claim 1, wherein the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID No. 30.

4. The antibody of any one of claims 1-3, wherein

5. The antibody of any one of claims 1-3, wherein the amino acid sequence of the heavy chain variable region of the antibody has at least 90% identity to SEQ ID No. 26 and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity to SEQ ID No. 30.

6. The antibody of any one of claims 1-3, wherein

The antibody is a whole antibody, a Fab fragment, a F (ab') ₂ fragment or a single chain Fv fragment (scFv), and/or

The antibody is monoclonal antibody, and/or

The antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or

The antibodies also comprise a light chain constant region selected from the kappa subtype or the lambda subtype.

7. The antibody of claim 6, wherein the antibody is a fully human antibody.

8. The antibody of claim 6, wherein the antibody heavy chain constant region is an IgG1 subtype.

9. The antibody of claim 6, wherein the antibody heavy chain constant region comprises an Fc segment sequence of an IgG1 subtype heavy chain constant region and the amino acid sequences at positions 234, 235, 331 of the Fc segment sequence are F, E, S, respectively, and/or the amino acid sequences at positions 252, 254, 256 of the Fc segment sequence are Y, T and E, respectively, wherein the antibody constant region amino acid sequence is determined according to EU numbering.

10. The antibody of any one of claims 1-3, wherein

The antibodies bind to TL1A of primate and/or rodent animals, and/or

The antibodies inhibit binding of TL1A to death receptor 3 (DR 3), and/or

The antibody inhibits caspase activation of TL1A stimulated cells, and/or

The antibodies inhibit NF- κB activation of TL1A stimulated cells, and/or

11. A nucleic acid molecule encoding the antibody of any one of claims 1-10.

12. A pharmaceutical composition comprising the antibody of any one of claims 1-10 and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition of claim 12, wherein the excipient is a diluent or carrier.

14. Use of the antibody of any one of claims 1-10, or the pharmaceutical composition of claim 12, in the manufacture of a medicament for the prevention or treatment of a TL1A associated disease, wherein said TL1A associated disease is rheumatoid arthritis, inflammatory bowel disease, psoriasis, and systemic sclerosis associated interstitial lung disease.

15. The use of claim 14, wherein the inflammatory bowel disease is crohn's disease and ulcerative colitis.