Antibodies targeting tl1a and uses thereof

Abstract

The present invention relates to antibodies targeting TL1A and uses thereof, in particular, provided are antibodies or antigen-binding portions thereof that specifically bind to free or transmembrane TL1A and uses thereof. In certain aspects, described are methods of blocking biological activity of the TL1A-DR3 signaling pathway. In certain aspects, described are antibodies and methods for treating autoimmune diseases or diseases associated with TL1A-DR3 pathway induction.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a depleting antibody or its antigen-binding portion of tumor necrosis factor-like ligand 1A (TL1A), a fusion protein comprising or expressing the antibody or its antigen-binding portion, a multispecific antibody, a nucleic acid molecule, an expression vector, recombinant cells, and pharmaceutical compositions, as well as their use in the preparation of drugs for treating or preventing autoimmune diseases and related treatment methods. This invention particularly relates to a biotechnology that enhances the specific depletion of TL1A-positive cells through Fc amino acid mutations or alterations in Fc glycosylation of antibodies to suppress the pathological progression of autoimmune diseases via complement-dependent or complement-dependent cytotoxic mechanisms. Background Technology

[0002] Autoimmune diseases are a group of chronic inflammatory diseases caused by the immune system abnormally attacking its own tissues. These include inflammatory bowel disease (IBD, such as ulcerative colitis and Crohn's disease), rheumatoid arthritis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, primary biliary cholangitis, and fibrosis-related diseases (such as intestinal fibrosis, pulmonary fibrosis, and liver fibrosis). These diseases have a high global incidence, affecting hundreds of millions of patients, severely impacting their quality of life and imposing a huge healthcare burden.

[0003] Tumor necrosis factor-like ligand 1A (TL1A, also known as TNFSF15) is a member of the tumor necrosis factor (TNF) superfamily, primarily expressed on immune cells such as endothelial cells, macrophages, dendritic cells, and activated T cells. TL1A activates downstream signaling pathways, including the TRADD-TRAF2-RIP1-mediated NF-κB, PI3K, and MAPK pathways, by binding to its functional receptor, death receptor 3 (DR3, TNFRSF25). This promotes the secretion of pro-inflammatory cytokines (such as IFN-γ, IL-4, IL-13, IL-17, and IL-23), T cell (Th1, Th2, Th17) proliferation, and enhanced effector function, while also inducing apoptosis or necroptosis. Furthermore, TL1A can bind to the trap receptor DcR3, modulating signal intensity.

[0004] Studies have shown that TL1A is abnormally overexpressed in various autoimmune diseases and plays a crucial role in disease development and progression. For example, TL1A and DR3 expression is significantly upregulated in the intestinal mucosa of IBD patients, promoting local T cell accumulation and amplifying the inflammatory cascade. Genetic studies have shown that TNFSF15 gene polymorphism is associated with susceptibility to diseases such as IBD and rheumatoid arthritis. Animal models (such as DSS-induced colitis, TNBS model, EAE multiple sclerosis model, and asthma model) have confirmed that blocking TL1A-DR3 interaction can significantly reduce inflammation, inhibit Th1 / Th2 / Th17 responses, and reverse fibrosis.

[0005] Current medications for treating autoimmune diseases mainly include glucocorticoids, immunosuppressants (such as 6-mercaptopurine), biologics (such as the anti-TNF-α antibody infliximab and adalimumab, as well as anti-IL-12 / 23 and JAK inhibitors), etc. While these therapies are effective, they have limitations such as short-lasting efficacy, drug resistance, increased risk of infection, and inability to effectively control fibrosis. Some patients do not respond or respond poorly to existing therapies, necessitating novel targeted therapy strategies.

[0006] TL1A is a key mediator of inflammation and fibrosis, and its blockers (such as anti-TL1A monoclonal antibodies) have shown potential for best-in-class efficacy. Preclinical and early clinical studies (such as PF-06480605 / Tulisokibart, RVT-3101, etc.) have demonstrated that anti-TL1A antibodies can efficiently neutralize TL1A, inhibit DR3 signaling without affecting DcR3, significantly improve endoscopic healing and clinical remission rates, and have good safety profiles. Precision medicine guided by genetic biomarkers further improves response rates.

[0007] Therefore, developing antibodies that specifically bind to TL1A and block its interaction with DR3 is of significant clinical importance, providing novel treatment options for patients with autoimmune diseases who are unresponsive to or intolerant of existing therapies. By specifically blocking the interaction between TL1A and DR3, the antibody of this invention can effectively inhibit Th1, Th2, and Th17-mediated inflammatory responses and fibrotic processes, providing new treatment options for patients who are unresponsive to or intolerant of existing therapies. Summary of the Invention

[0008] This invention provides binding proteins that target TL1A, including antigen-binding domains of antibodies, which specifically bind to TL1A and inhibit the interaction between TL1A and DR3.

[0009] Specifically, the present invention provides the following technical solutions: In a first aspect, the present invention provides an antibody or antigen-binding moiety thereof that specifically binds to TL1A or a functional fragment thereof, characterized in that the antibody or antigen-binding moiety thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). Wherein, the VH includes: I. Contains a heavy chain complementarity-determining region 1 (HCDR1) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; II. A heavy chain complementarity-determining region 2 (HCDR2) containing at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, wherein SEQ ID NO: 17: X1ISX2X3GRTYYNPSLKS; Wherein, X1 is selected from Y or S; X2 is selected from P or Y; X3 is selected from V or S; and III. Containing a heavy chain complementarity-determining region 3 (HCDR3) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 13; and The VL includes: Ⅰ) Contains a light chain complementarity-determining region 1 (LCDR1) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; II) Contains a light chain complementarity-determining region 2 (LCDR2) having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; wherein SEQ ID NO: 18: DTLKLX4S; Wherein, X4 is selected from A, F, or S; and III) Contains a light chain complementarity-determining region 3 (LCDR3) having at least about 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0010] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL). Wherein, the VH includes: i. A heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 11; or a heavy chain complementarity-determining region 1 (HCDR1) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 11. ii. A heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 17; or a heavy chain complementarity-determining region 2 (HCDR2) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 17. iii. A heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence shown in SEQ ID NO: 13; or a heavy chain complementarity-determining region 3 (HCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 13; and The VL includes: i) A light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 14; or a light chain complementarity-determining region 1 (LCDR1) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 14. ii) A light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 18; or a light chain complementarity-determining region 2 (LCDR2) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 18. iii) A light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 16; or a light chain complementarity-determining region 3 (LCDR3) having at least one, preferably 1-3, amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 16. Preferably, the amino acid difference is an amino acid substitution; more preferably, the amino acid difference is a conserved amino acid substitution.

[0011] In some implementations, A1: The VH comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 17, and 13, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14, 18, and 16, respectively; or A2: The VH comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12, and 13, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14, 18, and 16, respectively; or A3: The VH comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 17, and 13, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14, 15, and 16, respectively; or A4: The VH comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 11, 12 and 13 respectively; and the VL comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 14, 15 and 16 respectively.

[0012] In some embodiments, the heavy chain variable region of the antibody or its antigen-binding portion comprises a backbone of the amino acid sequence shown in SEQ ID NO:19, or comprises a backbone having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:19.

[0013] In some embodiments, the light chain variable region of the antibody or its antigen-binding portion comprises a backbone of the amino acid sequence shown in SEQ ID NO:20, or comprises a backbone having at least about 80%, 85%, 90%, 95%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:20.

[0014] In some embodiments, the VH has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-5; and / or The VL has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 9.

[0015] In some implementations, the VH and VL are selected from combinations of the following: S1) VH containing the amino acid sequence shown in SEQ ID NO: 1; and VL containing the amino acid sequence shown in SEQ ID NO: 6; or S2) VH containing the amino acid sequence shown in SEQ ID NO: 2; and VL containing the amino acid sequence shown in SEQ ID NO: 6; or S3) VH containing the amino acid sequence shown in SEQ ID NO: 3; and VL containing the amino acid sequence shown in SEQ ID NO: 6; or S4) VH containing the amino acid sequence shown in SEQ ID NO: 4; and VL containing the amino acid sequence shown in SEQ ID NO: 6; or S5) VH comprising the amino acid sequence shown in SEQ ID NO: 5; and VL comprising the amino acid sequence shown in SEQ ID NO: 6; or S6) VH comprising the amino acid sequence shown in SEQ ID NO: 1; and VL comprising the amino acid sequence shown in SEQ ID NO: 9; or S7) VH containing the amino acid sequence shown in SEQ ID NO: 2; and VL containing the amino acid sequence shown in SEQ ID NO: 9; or S8) VH comprising the amino acid sequence shown in SEQ ID NO: 3; and VL comprising the amino acid sequence shown in SEQ ID NO: 9; or S9) VH comprising the amino acid sequence shown in SEQ ID NO: 4; and VL comprising the amino acid sequence shown in SEQ ID NO: 9; or S10) contains VH, which contains the amino acid sequence shown in SEQ ID NO: 5; and VL, which contains the amino acid sequence shown in SEQ ID NO: 9.

[0016] In some embodiments, the antibody or its antigen-binding portion is an antibody or its antigen-binding portion of a human synthetic library, preferably a fully human antibody or its antigen-binding portion.

[0017] In some embodiments, the antibody or its antigen-binding portion further comprises an immunoglobulin Fc region or a functional variant thereof, preferably a human immunoglobulin Fc region or a functional variant thereof, more preferably the Fc region of human IgG1, IgG2, IgG3 or IgG4 or a functional variant thereof. Optionally, functional variants of the Fc region may include point mutations or modifications to reduce or weaken ADCC activity, ADCP activity, CDC activity, or prolong antibody PK.

[0018] In some embodiments, the antibody or its antigen-binding portion further comprises a constant region, preferably: I. The constant region connected to the light chain variable region is SEQ ID NO: 27 or SEQ ID NO: 28; and the constant region connected to the heavy chain variable region is selected from any one of SEQ ID NO: 29-57 and SEQ ID NO: 72; or II. The constant region connected to the light chain variable region is SEQ ID NO: 28; and the constant region connected to the heavy chain variable region is SEQ ID NO: 47, SEQ ID NO: 50, or SEQ ID NO: 29. More preferably, the variable region is directly connected to the constant region or connected through a connector.

[0019] In some implementations, (a) The antibody is a monoclonal antibody; and / or (b) The antigen-binding portion is selected from Fab, Fab', F(ab')2, Fv, scFv, nanobody, Fd and Fd'.

[0020] In a second aspect, the present invention provides a fusion protein comprising the antibody or its antigen-binding portion described in the first aspect.

[0021] Thirdly, the present invention provides a multispecific antibody comprising: (a) The antibody or its antigen-binding portion as described in the first aspect, or the fusion protein as described in the second aspect; and (b) At least one other antigen-binding region, wherein the at least one other antigen-binding region binds to a different antigen or a different epitope of the same antigen to the antibody or its antigen-binding portion.

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

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

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

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

[0026] Eighthly, the present invention provides the use of the antibody or its antigen-binding portion as described in the first aspect, the fusion protein as described in the second aspect, the multispecific antibody as described in the third aspect, the nucleic acid molecule as described in the fourth aspect, the expression vector as described in the fifth aspect, or the recombinant cell as described in the sixth aspect in the preparation of a medicament for treating or preventing TL1A-related diseases, wherein the TL1A-related diseases are selected from autoimmune diseases, including but not limited to inflammatory bowel disease (IBD), specifically including ulcerative colitis (UC), Crohn's disease (CD) and its associated intestinal fibrosis and stricture; psoriasis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), rheumatoid arthritis (RA); primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC); atopic dermatitis, AD), severe eosinophilic asthma, eosinophilic esophagitis (EoE); idiopathic pulmonary fibrosis (IPF), systemic sclerosis (SSC) and other organ fibrosis; systemic lupus erythematosus (SLE), Sjögren's syndrome, multiple sclerosis (MS); as well as immune checkpoint inhibitor-induced colitis (ICI-induced colitis), intestinal GVHD, and fibrosis-related diseases (such as intestinal fibrosis, pulmonary fibrosis, and liver fibrosis).

[0027] In a ninth aspect, the present invention provides a method for treating or preventing TL1A-related diseases, comprising administering to a subject in need a therapeutically effective amount of a substance selected from: the antibody or antigen-binding portion thereof described in the first aspect, the fusion protein described in the second aspect, the multispecific antibody described in the third aspect, or the pharmaceutical composition of the seventh aspect, wherein: (a) The subject is a mammal, preferably a human; (b) The TL1A-related diseases are selected from autoimmune diseases, preferably from inflammatory bowel disease (IBD, such as ulcerative colitis and Crohn's disease), rheumatoid arthritis, systemic lupus erythematosus, psoriasis, ankylosing spondylitis, primary biliary cholangitis, and fibrosis-related diseases (such as intestinal fibrosis, pulmonary fibrosis, and liver fibrosis).

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

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

[0030] Figure 1a According to ELISA data on antibody binding to human free TL1A, the binding activity of the candidate molecule of this invention is comparable to that of the positive reference. Figure 1b ELISA data on antibody binding to free TL1A in cynomolgus monkeys showed that the binding activity of the candidate molecule of this invention was comparable to that of the positive reference. Figure 2 Flow cytometry data on antibody binding to human transmembrane TL1A-overexpressing cells showed that the binding activity of the candidate molecule of this invention was comparable to that of Sanofi's Tevakitug and stronger than that of Merck's Tusocciabimab. Figure 3Antibody inhibition of TL1A-DR3 reporter cell activation: The inhibitory-induced activity of the candidate molecule of this invention is comparable to that of Pfizer's Afimkibart positive reference and stronger than that of Merck's Tusocciabizumab. Figure 4 The antibody inhibits the release of IFN-γ from CD4+ T cells in PBMCs induced by TL1A. The binding activity of the candidate molecule of this invention is comparable to that of Sanofi's Tevamumab and stronger than that of Merck's Tusocciabumab. Detailed Implementation

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

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

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

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

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

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

[0037] Table A - Chemical Properties of Amino Acids

[0038] Table B - Hydrophilicity Measurement Table

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

[0040] Table C Conserved Amino Acid Substitution

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0056] The terms “antagonism,” “inhibition,” “neutralization,” or “blocking,” when used to refer to an antigen-binding site or a molecule containing an antigen-binding site, mean that the binding of the antigen-binding site or molecule to its target results in the inhibition of at least some of the biological activities of the target.

[0057] The terms "Fc mutation or modification" or "Fc functional variant" refer to the substitution of amino acids in the CH2 and CH3 regions of an antibody to alter its binding to the Fc receptor or to change its function after binding to an antigen. Mutation sites include human IgG1, S298A / E333A / K334A, S298A / E333A / K334A / K326A (Lu Y, Vernes JM, Chiang N et al., J. Immunol. Methods., 2011 Feb 28; 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L, F243L / R292P / Y300L / L235V / P396L (Stavenhagen JB, Gorlatov S, Tuaillon N et al., Cancer Res., September 15, 2007; 67(18):8882-90; Nordstrom JL, Gorlatov S, Zhang W et al., Breast Cancer Res., November 30, 2011; 13(6):R123); F243L (Stewart R, Thom G, Levens M et al., Protein Eng Des Sel., September 2011; 24(9):671-8.), S298A / E333A / K334A (Shields RL, Namenuk AK, Hong K et al., J Biol Chem., March 2, 2001; 276(9):6591-604); S239D / I332E / A330L, S239D / I332E (Lazar GA, Dang W, Karki S et al., Proc Natl Acad Sci USA., March 14, 2006; 103(11):4005-10); S239D / S267E, S267E / L328F (Chu SY, Vostiar I, Karki S et al., Mol Immunol., September 2008;45(15):3926-33), and S239D / D265S / S298A / I332E, S239E / S298A / K32 6A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A3 The mutations listed in WO2011 / 120134 and WO2011 / 120135 include 30L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S267E / H268D, L234F / S267E / N325L, G237F / V266L / S267D, and others. In some embodiments, the Fc region includes one or more amino acid substitutions, wherein, compared to an Fc region without one or more substitutions, one or more substitutions result in an increased antibody half-life and a decrease in one or more of ADCC activity, ADCP activity, or CDC activity. In these embodiments, ADCC refers to antibody-dependent cell-mediated cytotoxicity; ADCP refers to antibody-dependent cell-mediated phagocytosis; and CDC refers to complement-dependent cytotoxicity. In some embodiments, compared to antibodies containing a wild-type Fc region, one or more amino acid substitutions result in prolonged antibody PK (pharmacokinetics), meaning the antibody is more stable and has a longer duration of action in vivo, preferably with a prolonged half-life. In some embodiments, compared to antibodies containing a wild-type Fc region, one or more amino acid substitutions result in an increased antibody half-life at pH 6.0. In some embodiments, the half-life of isolated antibodies containing an Fc region with one or more amino acid substitutions in humans is approximately 80 to 110 days.

[0058] As used in this invention, human antibody Fc mutations comprise one or more substitutions selected from the group consisting of: S228P (SP); M252Y, S254T, T256E, T256D, T250Q, H285D, T307A, T307Q, T307R, T307W, L309D, Q411H, Q311V, A378V, E380A, M428L, N434A, N434S, N297A, D265A, L234A, L235A, and N434W.In some embodiments, the one or more amino acid substitutions are selected from the following amino acid substitutions: M428L / N434S (LS); M252Y / S254T / T256E (YTE); T250Q / M428L; T307A / E380A / N434A; T256D / T307Q (DQ); T256D / T307W (DW); M252Y / T256D (YD); T307Q / Q311V / A378V (QVV); T256D / H285D / T307R / Q311V / A378V (DDRVV); L309D / Q311H / N434S; S228P / L235E (SPLE); L 234A / L235A (LA); M428L / N434A / L234A / G237A (LALA); L234A / L235A / G237A; L234A / L235A / P329G; N297A; D265A / YTE; LALA / YTE; LAGA / YTE; LALAGA / YTE; LALAPG / YTE; N297A / LS; D265A / LS; LALA / LS; SP / YTE; SPLE / YTE; SP / LS; SPLE / LS; SP / DHS; SPLE / DHS; N297A / LA; D265A / LA; LALA / LA; LAGA / LA; LA LA / LA; LALA / LA; LALAPG / LA; N297A / N434A; D265A / N434A; LALA / N434A; LAGA / N434A; LALAGA / N434A; LALAPG / N434A; LA / N434W; LAGA / N434W; LALAGA / N434W; LALAPG / N434W; N297A / DQ; D265A / DQ; LALA / DQ; LAGA / DQ; LALAGA / DQ; LALAPG / DQ; N297A / DW; D265A / DW; LALA / DW; LAGA / DW; LALAGA / DW; LALAPG / DW / N297A / YD; D265A / YD; LALA / YD; LAGA / YD; LALAGA / YD; LALAPG / YD; T307Q / Q311V / A378V (QVV); N297A / QVV; D265A / QVV; LALA / QVV; LAGA / QVV; LALAGA / QVV; LALAPG / QVV; DDRVV; N297A / DDRVV; D265A / DDRVV; LALA / DDRVV; LAGA / DDRVV; LALAGA / DDRVV and LALAPG / DDRVV.In some embodiments, the one or more amino acid substitutions are selected from: LALA / YTE, LAGA / YTE, LALA / LS, YTE, and LS. In some embodiments, the one or more amino acid substitutions comprise or consist of LALA / YTE. In some embodiments, the one or more amino acid substitutions comprise or consist of LAGA / YTE. In some embodiments, the one or more amino acid substitutions comprise or consist of LALA / LS. In some embodiments, the one or more amino acid substitutions comprise or consist of YTE. In some embodiments, the one or more amino acid substitutions comprise or consist of LS, and these mutations are incorporated herein by reference.

[0059] As used in this invention, the "antigen-binding fragment" of an antibody comprises a portion of the complete antibody that is still capable of binding the antigen. In some embodiments, papain digestion of the antibody yields two identical antigen-binding fragments, referred to as the "Fab" fragment, and a residual "Fc" fragment, the names reflecting its ease of crystallization. The Fab fragment consists of the entire light chain variable region domain (Vc) of the heavy chain. H ) and the first constant structural domain of a heavy chain (C H1 The Fab fragment is composed of two disulfide-linked Fab fragments. Each Fab fragment is monovalent in its antigen binding, meaning it has a single antigen-binding site. In some embodiments, treating the antibody with pepsin produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments in that they are located at C... H1 Several residues were added to the carboxyl terminus of the domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH stands for Fab', where the cysteine ​​residues of the constant domain have free thiol groups. The F(ab')2 antibody fragments were initially generated as Fab' fragment pairs with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0060] The term "antigen binding site" refers to any molecule or any part of a molecule capable of binding to an antigen. In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable region ("V") of the heavy chain ("H") and the light chain ("L"). Three highly divergent segments within the V region of the heavy and light chains are called "hypervariable regions," situated between more conserved flanking segments called "frame regions" or "FRs." Thus, the term "FR" refers to the naturally occurring amino acid sequence between and near the hypervariable regions of an immunoglobulin. In human antibodies, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged opposite each other in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of antigen binding, and the three hypervariable regions of the heavy and light chains are called "complementarity-determining regions" or "CDRs." In some animals, such as camels and cartilaginous fish, the antigen binding site may be formed by a single antibody chain, providing a "single-domain antibody." Antigen binding sites may also be present in antibody mimics.

[0061] As used in this invention, CDRs can be determined by the methods described in Kabat et al., J. biol. chem. 252, 6609-6616 (1977), Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. mol. biol. 196: 901-917 (1987), and MacCallum et al., J. mol. biol. 262: 732-745 (1996). CDRs determined according to these definitions typically include overlapping or subsets of amino acid residues when compared with each other. In some embodiments, the term "CDR" is the CDR defined by MacCallum et al., J. mol. biol. 262: 732-745 (1996) and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann, and Dübel ed., pp. 422-439, Chapter 31, Springer-Verlag, Berlin (2001). In some embodiments, the term "CDR" is the CDR defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In some embodiments, the heavy chain CDR and light chain CDR of the antibody are defined using different conventions. For example, in some embodiments, the heavy chain CDR is defined according to MacCallum's definition, and the light chain CDR is defined according to Kabat's definition. HCDR1, HCDR2, and HCDR3 represent heavy chain CDRs, while LCDR1, LCDR2, and LCDR3 represent light chain CDRs.

[0062] As used in this invention, the “binding affinity” of the molecules disclosed herein (e.g., antigen-binding sites) for a given target (e.g., TL1A, DR3, or PD-L2) can be determined by a variety of methods known to those skilled in the art. Such methods include, but are not limited to, fluorescence titration, ELISA (enzyme-linked immunosorbent assay), calorimetry (e.g., isothermal titration calorimetry (ITC)), and surface plasmon resonance (SPR).

[0063] The term "detectable affinity" refers to affinity measured by an affinity constant (usually expressed as K). D or EC 50 The ability of a given target to bind to a target (measured) typically has an affinity constant of about 10.-5 M or smaller (lower K) D or EC 50 The value reflects a better binding ability.

[0064] The term "epitope" refers to a specific antigen-binding site (composed of six complementarity-determining regions of the antibody) that interacts with a variable region of an antibody molecule. A single antigen can have multiple epitopes. Epitopes can be conformational or linear. Conformational epitopes consist of spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes consist of adjacent amino acid residues in a polypeptide chain.

[0065] The term "Fc" includes the carboxyl-terminal portion of two heavy chains linked together by disulfide bonds. The effector function of an antibody is determined by the sequence of the Fc region, which is also recognized by Fc receptors in certain cell types. A "fragment" of an antigen (e.g., TL1A, DR3, or PD-L2) refers to an N-terminal and / or C-terminal truncated portion or protein domain of the antigen. In some embodiments, the fragment of the antigen retains the ability of the full-length antigen to be recognized and / or bound by the antigen-binding fragment of this disclosure.

[0066] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced and their progeny. Host cells include “transformers” (or “transformed cells”) and “transfectants” (or “transfected cells”), each comprising primary transformed or transfected cells and their derived progeny. The nucleic acid content of these progeny cells may not be entirely identical to that of the parent cells and may contain mutations. “Recombinant host cell” or “host cell” refers to a cell containing exogenous polynucleotides, regardless of the method used for insertion (e.g., direct uptake, transduction, f-conjugation, or other methods known in the art for producing recombinant host cells).

[0067] The term "monoclonal antibody" refers to an antibody derived from a population of essentially homogeneous antibodies; that is, the individual antibodies constituting the population are identical, except for the possible presence of a small number of naturally occurring mutations. Monoclonal antibodies are highly specific (targeting a single antigenic site). Furthermore, each monoclonal antibody targets a single determinant on an antigen, unlike the various antibodies that typically contain targets against different determinants (epitopes). The modifier "monoclonal" indicates that the antibody is derived from a population of essentially homogeneous antibodies and should not be construed as requiring production by any particular method. For example, monoclonal antibodies can be prepared using hybridoma methods or recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries.

[0068] The term "reference level" generally refers to a level considered "normal" for comparative purposes, such as a properly controlled level. For example, in the context of enhancing antibody effector function, "reference level" could refer to the effector function level of an antibody containing the wild-type constant region (Fc). As another example, in the context of target binding, "reference level" could refer to the binding affinity level of the target's natural ligand (e.g., by K...). D or EC 50 (Value definition). The reference level can be determined simultaneously or predetermined, for example, it can be known or inferred from past observations.

[0069] The terms "subject" and "patient" refer to an organism that will be treated by the methods and compositions described in this invention. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cattle, pigs, canines, felines, etc.), and more preferably include humans.

[0070] The terms "therapeutic effective dose" and "effective dose" are used interchangeably, referring to the effective amount that achieves the desired therapeutic effect at the necessary dose and within a given time period. Therapeutic effective doses can vary due to a variety of factors, such as disease type, disease state, individual age, sex, and / or weight, and the ability of the antibody (or its pharmaceutical composition) to elicit the desired response in the individual. An effective dose can also be the amount by which any toxic or harmful effects of the antibody or its pharmaceutical composition are offset by the beneficial therapeutic effect.

[0071] As used in this invention, “treating” a disease (e.g., the disease described in this invention, such as an autoimmune disease) is a method of achieving a beneficial or desired outcome (e.g., clinical outcome). Beneficial or desired outcomes may include, but are not limited to, alleviating or improving one or more symptoms or conditions, reducing the severity of a disease, disorder, or condition, stabilizing (i.e., not worsening) the state of a disease, disorder, or condition, preventing the spread of a disease, disorder, or condition (e.g., primary cancer and / or secondary metastasis), delaying or slowing the progression of a disease, condition, or symptom, improving or palliating a disease, condition, or symptom, and partial or complete remission, whether detectable or undetectable. Examples of autoimmune diseases for which this invention is preferred for treatment include, but are not limited to, Behçet's disease, systemic lupus erythematosus, multiple sclerosis (systemic scleroderma and progressive systemic scleroderma), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa and microscopic polyangiitis), aortitis syndrome (Gau's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, Wegener's granulomatosis, mixed connective tissue disease, Sjögren's syndrome, adult-onset Still's disease, allergic granulomatous vasculitis, allergic vasculitis, Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia syndrome, antiphospholipid antibody syndrome, eosinophilic fasciitis, IgG4-related diseases (such as primary sclerosing cholangitis and autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, and chronic atrophic gastritis. Autoimmune hepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpass syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (hyperthyroidism), Hashimoto's thyroiditis, autoimmune hypoadrenocortical insufficiency, primary hypothyroidism, idiopathic Addison's disease (chronic adrenocortical insufficiency), type I diabetes, chronic discoid lupus erythematosus (OSUS), localized scleroderma, psoriasis, psoriatic arthritis, pemphigus, bullous pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vitiligo, Harada disease, autoimmune photoneuropathy, idiopathic azoospermia, recurrent miscarriage, and inflammatory bowel disease (ulcerative colitis and Crohn's disease).Acid reflux / heartburn, acne, acne vulgaris, allergies and sensitivities, Alzheimer's disease, asthma, atherosclerosis and occlusive vascular disease, optional atherosclerosis, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease or stent restenosis, autoimmune diseases, bronchitis, cancer, carditis, cataracts, celiac disease, chronic pain, chronic prostatitis, cirrhosis, colitis, connective tissue diseases, optional systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis or Sjögren's syndrome, corneal diseases, Crohn's disease, lens joint disease, optional gout, pseudogout, calcium pyrophosphate deposition. Incurable diseases, dementia, dermatitis, diabetes, dry eye syndrome, eczema, edema, emphysema, fibromyalgia, gastroenteritis, gingivitis, glomerulonephritis, heart disease, hepatitis, hypertension, hypersensitivity reactions, inflammatory bowel disease, inflammatory diseases including the consequences of trauma or ischemia, insulin resistance, interstitial cystitis, iridocyclitis, iritis, arthralgia, arthritis, rheumatoid arthritis, Lyme disease, metabolic syndrome (Syndrome X), multiple sclerosis, myositis, nephritis, obesity, eye diseases including uveitis, osteoporosis, osteoporosis, Parkinson's disease, pelvic inflammatory disease, periodontitis, polyarteritis, polychondritis, polymyalgia rheumatica. Psoriasis, reperfusion injury, rheumatoid arthritis, rheumatic diseases, optional rheumatoid arthritis, osteoarthritis or psoriatic arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, sinusitis, Sjögren's syndrome, spastic colon, spondyloarthritis, optional ankylosing spondylitis, reactive arthritis or Reiter's syndrome, systemic candidiasis, tendinitis, transplant rejection, vaginitis, vascular diseases, including atherosclerotic vascular diseases, vasculitis, optional polyarteritis nodosa, Wegener's granulomatosis, Chag-Strauss syndrome or vasculitis.

[0072] In some embodiments, this application provides an antigen-binding site capable of binding to a given antigen (e.g., an antigen on a cell involved in an immune dysregulation). In some embodiments, the antigen-binding site is capable of binding to an epitope within the antigen. In some embodiments, the antigen-binding site is capable of binding to an antigen on a cell involved in an autoimmune, allergic, or inflammatory disease. In some embodiments, the cell is an immune cell, and the antigen is TL1A.

[0073] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below with reference to the accompanying drawings. The embodiments described are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments described in this invention without creative effort are within the scope of protection of this invention. The technical solutions of this invention can be implemented in other specific forms without departing from their basic characteristics. Without conflict, any embodiment of this invention can be combined with the technical features of one or more other embodiments to form new embodiments. The scope of protection of this invention includes other embodiments formed by such combinations.

[0074] Example 1: Screening of a fully human synthetic library

[0075] Using the fully human antibody backbone from the therapeutic antibody TheraSabDab, a fully synthetic phage display library (VH / VLκ, VH / VLλ, library size >10e10) was constructed using a reported method (Bostrom, J., Fuh, G., 2009). Phages from the library were incubated with biotinylated human TL1A antigen protein (ACRO Biosystems, TLA-H5243), enriched with SA-Bead (Thermo, 88816), and washed with PBST (containing 0.1% Tween-20) to remove non-specifically bound phages. The bound phages were eluted with 0.2 M glycine-HCl (pH 2.2) and infected with *E. coli* for amplification, followed by three rounds of affinity panning. The top 200 clones were selected. Human TL1A protein (1 μg / mL) was coated onto 96-well plates. Selected phage clones were added, and after incubation, binding signals were detected using HRP-labeled anti-M13 antibody, and positive clones were selected. In TL1A-coated 96-well plates, the supernatant of selected phage clones and human DR3-Avi protein (ACRO Biosystems, DR3-H82E3) were added. Anti-DR3 positive signals were detected using SA antibody, and clones inhibiting TL1A / DR3 binding were selected. Phage clones were sequenced. The amino acid sequences of the light and heavy chain variable regions of representative clones are listed in Table 1, CDR sequences in Tables 2-1 and 2-2, backbone structures in Table 3, the variable region sequences of the positive control antibodies used in screening in Table 4-1, the constant region sequences of the antibodies in Table 4-2, and the extracellular sequences of the antigens used in screening in Table 5.

[0076] Table 1 Representative clones

[0077] Table 2-1 Antibody CDR Sequences

[0078] Table 2-2 Antibody CDR Sequences

[0079] Table 3 Antibody backbone sequence

[0080] in, Indicates the location of the CDR.

[0081] Table 4-1 Positive control variable region sequences

[0082] Table 4-2 Antibody Constant Region

[0083] Table 5. Antigen Amino Acid Sequence

[0084] Example 2: Antibody preparation and purification

[0085] The variable regions of antibodies VL and VH in Table 1 were synthesized using DNA. The positive reference antibody in Table 4-1 was derived from the sequence disclosed in the patent. A human homologous control antibody (Biointron, B117901) was used. The variable regions were ligated to the constant regions of antibodies in Table 4-2 using PCR to form the coding sequences for antibodies HC and LC (where the heavy chain IgG1, IgG1-LALA / LS, IgG1-LALA / YTE, hIgG1-LS, and IgG1-LAGA / LS, hIgG1-LALAPG / LS constant regions were used as weakened ADCC mutations, with IgG1-LS preferred, followed by the light chain κ; for each antibody of this invention, unless otherwise specified, the variable region is directly ligated to the constant region sequence at the C-terminus). These sequences were then subcloned into a pDNA3.0 plasmid, and the HC and LC gene sequences in the expression vector were confirmed by DNA sequencing. Approximately 5.5 x 10⁻⁶ cells were used in shake flasks. 6 / mL of CHO cells were used as the host (Baiying Biotech CRO transient expression). A mixture of 1 mg / L DNA (HC plasmid + LC plasmid) and 7 mg / L PEI was added to OptiMEM. TMThe culture medium (Invitrogen, 31985070) was added to 5.5 E6 / ml CHO cells and gently mixed to begin transfection. Cells were cultured in an incubator shaker at 110 rpm, 37°C, and 8% CO2 for 9 days. Protein purification was performed using an AKTA purification system (GE Lifesciences) via affinity chromatography and ion exchange chromatography. Conditioned medium expressing TL1A antibody was harvested by centrifugation at 4000 rpm for 50 min and filtered through a 0.22 μm filter. The harvested supernatant was loaded onto a MabSelect™ SuRe™ (GE HealthCARE) column. After washing the column with buffer A (PBS, pH 7.4), the protein was eluted with buffer B (1M glycine, pH 2.7) and immediately neutralized with 1 / 10 volume of buffer D (1M sodium citrate, pH 6.0). The affinity-purified antibody buffer was exchanged for PBS buffer. The purified samples were tested by SEC-HPLC and SDS-PAGE, and the purity was greater than 90% in both tests.

[0086] Example 3: Determination of antibody affinity by BLI

[0087] The binding kinetics of the prepared antibody to human TL1A (ACRO Biosystems, TLA-H5243) protein were detected using biomembrane interference (BLI) (Sartorius, ForteBio Red96). The antibody was immobilized onto an AHC2.0 biosensor (Cat. Nos. 18-5142). The TL1A antigen protein was diluted to four concentrations (100 nM, 50 nM, 25 nM, and 12.5 nM) with 0.02% PBST 20, and injected. Kinetic analysis was performed using a 1:1 binding model, and the binding rate (Kb) was calculated. on ) and dissociation rate (K off ), equilibrium dissociation constant (K D ) is K off / K on The ratios and antibody affinities are shown in Table 6. Further affinity tests were performed on the candidate molecule (WP02-1, i.e., clone 1; in this invention, the light chain constant region of clones 1-5 is light chain κ and the heavy chain constant region is heavy chain IgG1-LS; the constant region sequences are shown in Table 4-2) and the positive control antibody. TL1A was diluted to four concentrations: 100 nM, 50 nM, 25 nM, and 12.5 nM. The antibody affinities are shown in Table 7.

[0088] Table 6. Clonal BLI Affinity

[0089] Table 7. Multi-site BLI affinity of candidate molecules

[0090] Example 4: ELISA assay for binding of TL1A in humans and cynomolgus monkeys

[0091] The binding of antibody candidate molecules to human / cynomolgus monkey TL1A protein was detected using ELISA. Human TL1A (ACRO Biosystems, TLA-H5243) or cynomolgus monkey TL1A protein (ACRO Biosystems, TLA-C5241) was diluted with PBS to 1 μg / mL, and 100 µl was added to each well of the ELISA plate. The plate was incubated overnight at 2–8°C, followed by blocking with 200 µl of blocking buffer (0.05% Tween 20 + 2% BSA + PBS) at room temperature for 1 hour. The diluted antibody was then added to each well at 100 µl. The plate was incubated with gentle shaking at room temperature for 1 hour. The washing step was repeated three times. The enzyme-labeled secondary antibody (goat anti-human IgG-HRP, ABCAM, AB204901) was diluted 1:10000 with blocking buffer. 100 µL of the diluted secondary antibody was added to each well, and after washing, 100 µL of TMB substrate solution (Solepro, PR1200) was added to each well. Incubate the plate at room temperature in the dark for 10 minutes. Add 50 µL of sulfuric acid stop solution to each well. Within 30 minutes of adding the stop solution, measure the absorbance at 450 nm using a microplate reader (ENVISION, PerkinElmer). The results are shown in Table 8. Figure 1a and Figure 1b .

[0092] Table 8 Antibody binding to recombinant TL1A protein

[0093] Example 5: Flow Cytometry Detection of Human TL1A-CHOK1 Binding

[0094] The binding activity of the antibody to human TL1A overexpressing cells was detected by flow cytometry. TL1A cells (CHOK1, GM-C19170) at a density of 10E6 cells / ml were seeded at 100 μl / well in 96-well V plates. The antibody was initially 500 nM and diluted 1:5 eight times, and incubated at 4°C for 30 min. After washing three times with PBS, PE-labeled anti-human IgG antibody (ABCAM, ab150080) was added, and the cells were incubated at 4°C for 30 min. After washing three times with PBS, the cells were analyzed using a Cytoflex (Backman, B5300) flow cytometer. The mean fluorescence intensity (MFI) values ​​of live cells were collected, and EC50 was calculated using Graphpad software. 50 Values ​​and test results are shown in Table 9 and below. Figure 2 .

[0095] Table 9

[0096] Example 6: Inhibition of inhibitory signaling in TL1A-DR3 reporter cells by antibody

[0097] Free human TL1A (ACRO Biosystems, TLA-H5243) and Jurkat-DR3-Luc cells (1E5 cells / well) were diluted with DMEM and seeded in wells containing the test antibody. The test antibody was diluted 1:4 seven times starting at a concentration of 100 nM and incubated for 6 h. The luciferase substrate (Bright-Glo™, Luciferase Substrate, Cat. No. DD1204-03, Vazyme) was then added to the corresponding wells. The chemiluminescence signal was measured using a microplate reader (Meigu, i3). The luminescence readings were expressed as RLU (Relative Light Units), and the IC50 was calculated. 50 The results are shown in Table 10 and... Figure 3 .

[0098] Table 10

[0099] Example 7: Antibody inhibits the release of cytokines from primary PBMCs

[0100] T cells were pre-stimulated with anti-CD3 / CD28 magnetic beads and PBMCs (1:10), and the beads were removed overnight. PBMCs (Schbio, PBMNC100C 1E5 cells / well) were mixed with 10 ng / ml TL1A protein and seeded in wells containing 100 nM of the antibody to be tested. After incubation for 24 h, the cell supernatant was collected, and the IFN-γ concentration was detected by ELISA. The IFN-γ release ratio was calculated (negative control: anti-CD3 / CD28 magnetic beads; positive control: anti-CD3 / CD28 magnetic beads + 10 ng / ml TL1A). The results are as follows: Figure 4 .

[0101] Example 8: Antibody Competitive Epitope Detection

[0102] Using biomembrane interference (BLI) (Sartorius, ForteBio Red96), the competitive binding of prepared antibodies to human TL1A antigen was investigated. Using Octect HIS1K (Fotebio, 18-5077), 10ug / ml human TL1A (ACROBiosystems) was immobilized for 60s with TLA-H5243. Binding to antibody 1 (candidate antibody WP02-1, 100nM) took 200s, followed by binding to antibody 2 (100nM) for 100s (Tusocimibas, Tevamarab, and afimkibat positive control). Octet epitope mapping analysis showed that the candidate antibody and Tevamarab showed 100% inhibition, indicating competition for the same epitope; afimkibat showed 20% inhibition, indicating partial competition; and no inhibition was observed with Tusocimibas, indicating different epitopes.

[0103] The embodiments of the present invention demonstrate that the anti-TL1A antibody within the scope of the present invention blocks or inhibits TL1A-DR3 binding by binding to free or transmembrane TL1A protein. The antibody prepared by the present invention retains the killing function of IgG1 and has more significant inhibitory activity against downstream DR3 signaling. At least several aspects and embodiments of the present invention have been described thus. It is understood that various equivalent substitutions, changes, modifications, and improvements will be readily apparent to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to conform to the spirit and scope of the invention. Therefore, the above description is by way of example only, and the following claims provide a detailed description of the invention.

[0104] All references cited in this invention, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as each reference individually and expressly indicated by reference and fully listed herein. The foregoing description is intended only to illustrate the technical solutions of this invention and does not limit its scope of protection. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solutions of this invention do not depart from the essence and scope of this invention.

Claims

1. An antibody or its antigen-binding moiety that specifically binds to TL1A, characterized in that, The antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes heavy chain complementarity determination region 1, heavy chain complementarity determination region 2 and heavy chain complementarity determination region 3 as shown in SEQ ID NO: 11, 12 and 13 respectively; and the light chain variable region includes light chain complementarity determination region 1, light chain complementarity determination region 2 and light chain complementarity determination region 3 as shown in SEQ ID NO: 14, 15 and 16 respectively.

2. The antibody or its antigen-binding portion according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region are: a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 1; and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:

6.

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

4. The antibody or its antigen-binding portion according to claim 1, characterized in that, The antibody or its antigen-binding portion further comprises an immunoglobulin Fc region or a functional variant thereof.

5. The antibody or its antigen-binding portion according to claim 1, characterized in that, The antibody or its antigen-binding portion further includes a constant region.

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

7. A fusion protein comprising the antibody or its antigen-binding portion as described in any one of claims 1-6.

8. An isolated nucleic acid molecule encoding an antibody or antigen-binding portion thereof as described in any one of claims 1-6 or a fusion protein as described in claim 7.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A recombinant cell comprising the nucleic acid molecule of claim 8 or the expression vector of claim 9, wherein the recombinant cell is selected from mammalian cells or insect cells.

11. A pharmaceutical composition comprising at least one component selected from the group consisting of: an antibody or antigen-binding portion thereof as described in any one of claims 1-6, a fusion protein as described in claim 7, a nucleic acid molecule as described in claim 8, an expression vector as described in claim 9, a recombinant cell as described in claim 10; and at least one pharmaceutically acceptable carrier.

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