TL1A binding molecules and uses thereof

Abstract

The present invention provides TL1A binding molecules and uses thereof. Specifically, the invention provides an anti-TL1A single-domain antibody or an antigen binding fragment thereof, which can bind to TL1A of human and cynomolgus monkeys to block a TL1A / DR3 pathway without affecting the binding of TL1A to a natural blocker DcR3 of the TL1A / DR3 pathway. The combination of the anti-TL1A single-domain antibody or the antigen binding fragment thereof and TL1A has pH dependence, so that the anti-TL1A single-domain antibody or the antigen binding fragment thereof is more beneficial to long-term circulation in vivo, and the half-life period is prolonged. Therefore, the anti-TL1A single-domain antibody or the antigen binding fragment of the anti-TL1A single-domain antibody can be used as a novel therapeutic agent to be applied to treatment of immune inflammation related diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody drugs, and more specifically, to a TL1A binding molecule and its applications. Background Technology

[0002] TNF-like ligand 1A (TL1A), also known as TNFSF15, is a type II transmembrane protein that self-assembles into a stable trimer through interactions with TNF homologous domains. TL1A is primarily expressed in a membrane-bound form, forming a stable trimer, and is converted into soluble TL1A through selective splicing or cleavage by TNF-α convertase. TL1A is expressed in a variety of immune cells (such as monocytes, macrophages, dendritic cells, and T cells) and non-immune cells (such as synovial fibroblasts and endothelial cells).

[0003] TL1A binds to death receptor 3 (DR3) and activates MAPK, NF-κB, and effector kinase PI3K signaling, thereby regulating the proliferation, activation, apoptosis, and production of cytokines and chemokines in effector cells. Studies have found that TL1A is abnormally expressed in autoimmune diseases, including rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, and ankylosing spondylitis. In vivo and in vitro studies further demonstrate that TL1A is involved in the occurrence and pathogenesis of these diseases.

[0004] Multiple preclinical studies have demonstrated the role of the TL1A / DR3 pathway in numerous autoimmune disease models, and TL1A inhibitors can alleviate various inflammatory diseases, including inflammatory bowel disease (IBD), arthritis, and asthma. Meanwhile, several monoclonal antibody drugs targeting TL1A have shown therapeutic potential for IBD in clinical trials. However, existing treatments still carry safety and efficacy risks, such as frequent dosing cycles and the potential for drug resistance due to high immunogenicity. Therefore, a safer and more effective treatment strategy is urgently needed. Currently, no single-domain antibodies targeting TL1A have been reported, but utilizing this type of antibody may yield better therapeutic results. Summary of the Invention

[0005] The purpose of this invention is to provide a single-domain antibody targeting TL1A or an antigen-binding fragment thereof, and the application of the anti-TL1A single-domain antibody or the antigen-binding fragment thereof in disease diagnosis and treatment.

[0006] In a first aspect of the invention, an anti-TL1A single-domain antibody or an antigen-binding fragment thereof is provided, wherein the VHH chain of the anti-TL1A single-domain antibody or the antigen-binding fragment thereof comprises a complementarity-determining region (CDR) selected from the group consisting of:

[0007] (1) CDR1 shown in SEQ ID NO:86 (GYTX1X2NYY, where X1 is D, E, or T, and X2 is S, T, or A), CDR2 shown in SEQ ID NO:87 (IX3TX4GGHT, where X3 is N, Q, or D, and X4 is S or G), and CDR3 shown in SEQ ID NO:5; or

[0008] (2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:87 and CDR3 shown in SEQ ID NO:5.

[0009] In another preferred embodiment, the VHH chain includes a complementary determination region (CDR) selected from the group consisting of:

[0010] (1) CDR1 shown in SEQ ID NO:3, 21, 22, 23 or 24, CDR2 shown in SEQ ID NO:4, 18, 19 or 20 and CDR3 shown in SEQ ID NO:5; or

[0011] (2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:4, 18, 19 or 20 and CDR3 shown in SEQ ID NO:5.

[0012] In another preferred embodiment, the VHH chain includes the following complementary determination regions (CDRs):

[0013] CDR1 shown in SEQ ID NO:3 or 6, CDR2 shown in SEQ ID NO:87 and CDR3 shown in SEQ ID NO:5.

[0014] In another preferred embodiment, the VHH chain includes the following complementary determination regions (CDRs):

[0015] CDR1 shown in SEQ ID NO:3 or 6, CDR2 shown in SEQ ID NO:4 and CDR3 shown in SEQ ID NO:5.

[0016] In another preferred embodiment, the VHH chain includes the following complementary determination regions (CDRs):

[0017] CDR1 shown in SEQ ID NO:3 or 6, CDR2 shown in SEQ ID NO:18 and CDR3 shown in SEQ ID NO:5.

[0018] In another preferred embodiment, the VHH chain includes the following complementary determination regions (CDRs):

[0019] CDR1 shown in SEQ ID NO:3 or 6, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:5.

[0020] In another preferred embodiment, the VHH chain includes the following complementary determination regions (CDRs):

[0021] CDR1 shown in SEQ ID NO:3 or 6, CDR2 shown in SEQ ID NO:20 and CDR3 shown in SEQ ID NO:5.

[0022] In another preferred embodiment, the VHH chain includes the following complementary determination regions (CDRs):

[0023] CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5.

[0024] In another preferred embodiment, the VHH chain includes a complementary determination region (CDR) selected from the group consisting of:

[0025] (1) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4 and CDR3 shown in SEQ ID NO:5;

[0026] (2) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:18 and CDR3 shown in SEQ ID NO:5;

[0027] (3) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:5;

[0028] (4) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:20 and CDR3 shown in SEQ ID NO:5;

[0029] (5) CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:5;

[0030] (6) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:5;

[0031] (7) CDR1 shown in SEQ ID NO:23, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:5;

[0032] (8) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:19 and CDR3 shown in SEQ ID NO:5;

[0033] (9) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:4 and CDR3 shown in SEQ ID NO:5;

[0034] (10) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:18 and CDR3 shown in SEQ ID NO:5;

[0035] (11) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5; or

[0036] (12) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:20 and CDR3 shown in SEQ ID NO:5.

[0037] In another preferred embodiment, the VHH chain further includes a frame region (FR).

[0038] In another preferred embodiment, the frame region (FR) includes camel-derived FR and human-derived FR.

[0039] In another preferred embodiment, the frame region (FR) includes FR1, FR2, FR3 and FR4.

[0040] In another preferred embodiment, the VHH chain comprises the following FRs:

[0041] FR1 shown in SEQ ID NO:25 or 29, FR2 shown in SEQ ID NO:26, FR3 shown in SEQ ID NO:27 and FR4 shown in SEQ ID NO:28.

[0042] In another preferred embodiment, the VHH chain comprises FRs selected from the group consisting of:

[0043] (1) FR1 shown in SEQ ID NO:30, FR2 shown in SEQ ID NO:31, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33;

[0044] (2) FR1 shown in SEQ ID NO:30, FR2 shown in SEQ ID NO:34, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33; or

[0045] (3) FR1 shown in SEQ ID NO:30, FR2 shown in SEQ ID NO:35, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33.

[0046] (4) FR1 shown in SEQ ID NO:30, FR2 shown in SEQ ID NO:31, FR3 shown in SEQ ID NO:36 and FR4 shown in SEQ ID NO:33;

[0047] (5) FR1 shown in SEQ ID NO:37, FR2 shown in SEQ ID NO:38, FR3 shown in SEQ ID NO:27 and FR4 shown in SEQ ID NO:33;

[0048] (6) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:31, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33;

[0049] (7) FR1 shown in SEQ ID NO:40, FR2 shown in SEQ ID NO:31, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33;

[0050] (8) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:34, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33;

[0051] (9) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:35, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33;

[0052] (10) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:31, FR3 shown in SEQ ID NO:36 and FR4 shown in SEQ ID NO:33; or

[0053] (11) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:38, FR3 shown in SEQ ID NO:27 and FR4 shown in SEQ ID NO:33.

[0054] In another preferred embodiment, the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% (preferably at least 85%, more preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 7-11, and 12-17.

[0055] In another preferred embodiment, the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment has an amino acid sequence as shown in any one of SEQ ID NO: 1, 2, 7-11 and 12-17.

[0056] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment has one or more (e.g., 2, 3 or 4) amino acid residue mutations relative to the amino acid sequences shown in any one of SEQ ID NO: 1, 2, 7-11 and 12-17.

[0057] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment has an amino acid residue mutation at the following sites relative to the amino acid sequence shown in any one of SEQ ID NO: 1, 2, 7-11 and 12-17: position 52 and / or position 54.

[0058] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment has an amino acid residue mutation at the following sites relative to the amino acid sequence shown in any one of SEQ ID NO:1, 7-11: position 29, position 30, position 52, position 54, or a combination thereof.

[0059] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment has an amino acid residue mutation selected from the group consisting of the amino acid residues shown in any one of SEQ ID NO:1, 7-11:

[0060] (M1) Replace the asparagine (N) at position 52 with glutamine (Q) or aspartic acid (D), i.e., N52Q or N52D;

[0061] (M2) The serine (S) at position 54 is replaced with glycine (G), i.e., S54G;

[0062] (M3) Replace the aspartic acid (D) at position 29 with glutamic acid (E) or threonine (T), i.e., D29E or D29T;

[0063] (M4) The 30th serine (S) is replaced with threonine (T) or alanine (A), i.e., S30T or S30A; or

[0064] (M5) Any combination of the aforementioned (M1)-(M4).

[0065] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment is such that, relative to the amino acid sequence shown in any one of SEQ ID NO:1, 7-11, the 54th serine (S) is replaced with glycine (G), and the 29th aspartic acid (D) is replaced with glutamic acid (E) or threonine (T).

[0066] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment is such that, relative to the amino acid sequence shown in any one of SEQ ID NO:1, 7-11, the 54th serine (S) is replaced with glycine (G), and the 30th serine (S) is replaced with threonine (T) or alanine (A).

[0067] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment is such that, relative to the amino acid sequence shown in any one of SEQ ID NO:1, 7-11, the 52nd position of asparagine (N) is replaced with glutamine (Q) or aspartic acid (D).

[0068] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment is such that the 54th serine (S) is replaced with glycine (G) relative to the amino acid sequence shown in any one of SEQ ID NO:1, 7-11, i.e., S54G.

[0069] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment has an amino acid residue mutation selected from the group consisting of, relative to the amino acid sequence shown in any one of SEQ ID NO:2, 12-17:

[0070] (M1) Replace the asparagine (N) at position 52 with glutamine (Q) or aspartic acid (D), i.e., N52Q or N52D;

[0071] (M2) The serine (S) at position 54 is replaced with glycine (G), i.e., S54G; or

[0072] (M3) Any combination of (M1) and (M2) mentioned above.

[0073] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment is such that, relative to the amino acid sequence shown in any one of SEQ ID NO:2, 12-17, asparagine (N) at position 52 is replaced with glutamine (Q) or aspartic acid (D).

[0074] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment is such that, relative to the amino acid sequence shown in any one of SEQ ID NO:2, 12-17, the 54th serine (S) is replaced with glycine (G).

[0075] In another preferred embodiment, the VHH chain has an amino acid sequence as shown in any one of SEQ ID NO:41-85.

[0076] In another preferred embodiment, the anti-TL1A single-domain antibody includes a camel-derived single-domain antibody, a chimeric single-domain antibody, or a humanized single-domain antibody.

[0077] In another preferred embodiment, the anti-TL1A single-domain antibody includes a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0078] In another preferred embodiment, the anti-TL1A single-domain antibody comprises one or more VHH chains having amino acid sequences or derivative sequences as shown in SEQ ID NO:1, 2, 7-11 and 12-17, 41-85.

[0079] In another preferred embodiment, the anti-TL1A single-domain antibody or its antigen-binding fragment specifically binds to human TL1A and cynomolgus monkey TL1A.

[0080] In another preferred embodiment, the anti-TL1A single-domain antibody or its antigen-binding fragment binds only to the trimer TL1A and not to the monomer TL1A.

[0081] In another preferred embodiment, the anti-TL1A single-domain antibody or its antigen-binding fragment blocks the binding of TL1A to DR3, thereby blocking the TL1A / DR3 pathway.

[0082] In another preferred embodiment, the anti-TL1A single-domain antibody or its antigen-binding fragment does not affect the interaction between TL1A and DcR3.

[0083] In another preferred embodiment, the blocking activity (IC50 value) of the anti-TL1A single-domain antibody or its antigen-binding fragment against the binding of TL1A to DR3 is denoted as B1, and the blocking activity (IC50 value) against the binding of TL1A to DR3 is denoted as B2, wherein B2 / B1 > 1000, preferably, B2 / B1 > 5000, and more preferably, B2 / B1 > 10000.

[0084] In another preferred embodiment, the binding of the anti-TL1A single-domain antibody or its antigen-binding fragment to TL1A is pH-dependent, and its binding activity to TL1A under neutral conditions (pH 7.2-7.6) is more than 5 times, preferably more than 10 times, greater than its binding activity to TL1A under acidic conditions (pH 5.4-6.0).

[0085] In a second aspect of the invention, a fusion protein is provided, the structure of which from the N-terminus to the C-terminus is shown in formula Ia or Ib:

[0086] ALB(Ia);

[0087] BLA(Ib);

[0088] in,

[0089] A is an anti-TL1A single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0090] B is the Fc fragment of IgG; and

[0091] L represents no or flexible joints.

[0092] In another preferred embodiment, the flexible connector is a peptide connector.

[0093] In another preferred embodiment, the peptide linker has 1-50 amino acids, more preferably 1-20 amino acids.

[0094] In another preferred embodiment, the peptide linker is selected from (GS)n, (GGS)n, (GGGS)n, and (GGGGS)n, where n is a positive integer from 1 to 5.

[0095] In another preferred embodiment, the peptide linker has a (GGGGS)n structure, where n is a positive integer from 1 to 5.

[0096] In another preferred embodiment, the Fc fragment of the IgG comprises the Fc fragment of human IgG.

[0097] In another preferred embodiment, the Fc fragment of the IgG comprises the Fc fragment of human IgG.

[0098] In another preferred embodiment, the Fc fragment of the IgG is selected from the group consisting of the Fc fragments of IgG1, IgG2, IgG3, IgG4, or combinations thereof.

[0099] In another preferred embodiment, the Fc fragment of the IgG is IgG4.

[0100] In a third aspect of the invention, a multispecific antibody is provided, the multispecific antibody comprising an anti-TL1A single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, or a fusion protein as described in the second aspect of the invention.

[0101] In another preferred embodiment, the multispecific antibody includes bispecific antibodies, trispecific antibodies, etc.

[0102] In a fourth aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0103] (i) an anti-TL1A single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0104] (ii) Optional tag sequences to assist in expression and / or purification.

[0105] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, and a His tag.

[0106] In a fifth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: an anti-TL1A single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, or a recombinant protein as described in the fourth aspect of the invention.

[0107] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0108] In a sixth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the fifth aspect of the invention.

[0109] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0110] In another preferred embodiment, the expression vector includes a viral vector, such as adenovirus, AAV virus, retrovirus, lentivirus, or a combination thereof.

[0111] In a seventh aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the sixth aspect of the invention, or having a genome integrated with polynucleotides as described in the fifth aspect of the invention.

[0112] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0113] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0114] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0115] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0116] In another preferred embodiment, the eukaryotic cells shown are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0117] In another preferred embodiment, the host cell is preferably a mammalian cell, more preferably a HEK293 cell, CHO cell, BHK cell, NSO cell, or COS cell.

[0118] In another preferred embodiment, the host cell is Pichia pastoris.

[0119] In an eighth aspect of the invention, a method is provided for generating an anti-TL1A single-domain antibody or its antigen-binding fragment, or its fusion protein, the multispecific antibody described in the third aspect of the invention, or the recombinant protein described in the fourth aspect of the invention, comprising the steps of:

[0120] (a) Under conditions suitable for protein expression, host cells as described in the seventh aspect of the present invention are cultured to obtain a culture containing the anti-TL1A single-domain antibody or its antigen-binding fragment or its fusion protein, the multispecific antibody, and the recombinant protein;

[0121] (b) Isolating or recovering from the culture the anti-TL1A single-domain antibody or its antigen-binding fragment, or its fusion protein, the multispecific antibody, or the recombinant protein; and

[0122] In another preferred embodiment, the method further includes the step of: (c) purifying and / or modifying the anti-TL1A single-domain antibody or its antigen-binding fragment, or its fusion protein, multispecific antibody, or recombinant protein obtained in step (b).

[0123] In a ninth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0124] (i) an anti-TL1A single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention; and

[0125] (ii) Pharmaceutically acceptable carriers.

[0126] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0127] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for the prevention and / or treatment of diseases or conditions mediated by the TL1A / DR3 pathway.

[0128] In another preferred embodiment, the diseases or conditions mediated by the TL1A / DR3 pathway include autoimmune diseases.

[0129] In another preferred embodiment, the autoimmune diseases include, but are not limited to: rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, asthma, systemic sclerosis-associated interstitial lung disease, irritable bowel syndrome, and chronic idiopathic urticaria.

[0130] In another preferred embodiment, the pharmaceutical composition further comprises other medicines for the prevention and / or treatment of autoimmune diseases.

[0131] In a tenth aspect of the present invention, a TL1A inhibitor is provided, the inhibitor comprising an anti-TL1A single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention, and the TL1A inhibitor blocks the binding of TL1A to DR3 without affecting the binding of TL1A to DcR3.

[0132] In another preferred embodiment, the TL1A inhibitor specifically binds to human TL1A and cynomolgus monkey TL1A.

[0133] In another preferred embodiment, the TL1A inhibitor binds only to the trimer TL1A and not to the monomer TL1A.

[0134] In another preferred embodiment, the blocking activity (IC50 value) of the TL1A inhibitor against the binding of TL1A to DR3 is denoted as B1, and the blocking activity (IC50 value) against the binding of TL1A to DcR3 is denoted as B2, wherein B2 / B1 > 1000, preferably, B2 / B1 > 5000, and more preferably, B2 / B1 > 10000.

[0135] In an eleventh aspect of the invention, the use of an anti-TL1A single-domain antibody or its antigen-binding fragment as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, or a recombinant protein as described in the fourth aspect of the invention, for the preparation of:

[0136] (a) Medications used to prevent and / or treat diseases or conditions mediated by the TL1A / DR3 pathway; or

[0137] (b) Detection reagents, detection plates or detection kits for detecting TL1A molecules.

[0138] In another preferred embodiment, the diseases or conditions mediated by the TL1A / DR3 pathway include autoimmune diseases.

[0139] In another preferred embodiment, the autoimmune diseases include, but are not limited to: rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, asthma, systemic sclerosis-associated interstitial lung disease, irritable bowel syndrome, and chronic idiopathic urticaria.

[0140] In another preferred embodiment, the detection includes flow cytometry, immunofluorescence assay, and ELISA.

[0141] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0142] In a twelfth aspect of the present invention, a method for detecting TL1A protein in a sample is provided, the method comprising the steps of:

[0143] (1) Contact with an anti-TL1A single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, or a recombinant protein as described in the fourth aspect of the present invention;

[0144] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of TL1A protein in the sample.

[0145] In another preferred embodiment, the aspect is an in vitro method.

[0146] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0147] In a thirteenth aspect of the invention, a method for treating a disease or condition related to TL1A is provided, the method comprising administering to a subject in need an anti-TL1A single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, a recombinant protein as described in the fourth aspect of the invention, or a pharmaceutical composition as described in the ninth aspect of the invention.

[0148] In another preferred embodiment, the object includes mammals, such as humans.

[0149] In another preferred embodiment, TL1A is abnormally expressed within the object.

[0150] In another preferred embodiment, the diseases or conditions mediated by the TL1A / DR3 pathway include autoimmune diseases.

[0151] In another preferred embodiment, the autoimmune diseases include, but are not limited to: rheumatoid arthritis, inflammatory bowel disease (IBD), psoriasis, primary biliary cirrhosis, systemic lupus erythematosus, ankylosing spondylitis, asthma, systemic sclerosis-associated interstitial lung disease, irritable bowel syndrome, and chronic idiopathic urticaria.

[0152] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0153] Figure 1-7 The results are obtained by flow cytometry detection of the blocking activity of TL1A single-domain antibody against human TL1A and cell surface DR3.

[0154] Figure 8 The results are obtained by flow cytometry analysis of the blocking activity of TL1A single-domain antibody against TL1A and DR3 on the cell surface of cynomolgus monkeys.

[0155] Figure 9 This is the result of ELISA detection of the binding activity between TL1A single-domain antibody and human TL1A trimer protein.

[0156] Figure 10 This is the result of ELISA detection of the binding activity between TL1A single-domain antibody and human TL1A monomer protein.

[0157] Figure 11 This refers to the functional activity of TL1A single-domain antibody in human TL1A and DR3-Jurkat reporter gene cells.

[0158] Figure 12This is the inhibitory effect of TL1A single-domain antibody on apoptosis in TF-1 cells.

[0159] Figure 13 This refers to the blocking effect of TL1A single-domain antibody on human TL1A and DcR3.

[0160] Figure 14 This refers to the binding of TL1A single-domain antibodies to human TL1A under different pH conditions.

[0161] Figure 15-19 This describes the functional activity of humanized TL1A single-domain antibody in DR3 reporter cells.

[0162] Figure 20 It is the blocking effect of the modified humanized TL1A single-domain antibody on human TL1A and human DR3.

[0163] Figure 21 It is the blocking effect of the modified humanized TL1A single-domain antibody on cynomolgus monkey TL1A and human DR3.

[0164] Figure 22 It is the functional activity of the modified humanized TL1A single-domain antibody in DR3 reporter cells.

[0165] Figure 23 It is the inhibitory activity of the modified humanized TL1A single-domain antibody against apoptosis in TF-1 cells.

[0166] Figure 24 This refers to the change in body weight in a mouse enteritis model based on the drug's effects.

[0167] Figure 25 It is the DAI score of drug efficacy in a mouse enteritis model.

[0168] Figure 26 It is the intestinal immunohistochemical score of drug efficacy in a mouse enteritis model. Detailed Implementation

[0169] Through extensive and in-depth research and numerous screenings, the inventors of this invention have unexpectedly discovered a new class of anti-TL1A single-domain antibodies. These anti-TL1A single-domain antibodies not only bind to human TL1A with high affinity but also to cynomolgus monkey TL1A, facilitating animal experiments. Furthermore, these anti-TL1A single-domain antibodies selectively block the binding of TL1A to DR3 without affecting the binding of TL1A to DcR3, a natural blocker of the TL1A / DR3 pathway, thus more effectively blocking the TL1A / DR3 pathway. In vitro and in vivo experiments have demonstrated that these anti-TL1A single-domain antibodies can be used to prepare therapeutic agents for treating diseases or conditions mediated by the TL1A / DR3 pathway.

[0170] Based on this, the present invention was completed.

[0171] the term

[0172] To better understand this invention, the following terms are defined.

[0173] Unless the context clearly indicates otherwise, the term “about” includes values ​​within the standard deviation range of the stated values.

[0174] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0175] In this invention, "subject" or "patient" refers to an animal, including human patients requiring treatment for diseases mediated by the TL1A / DR3 pathway. In some aspects, the invention can also be applied in veterinary practice to any mammal or other animal requiring such TL1A-targeted therapy. This may include, for example, non-human primates, dogs, felines, pigs, horses, and any other animals requiring TL1A treatment.

[0176] As used herein, the terms “single-domain antibody of the present invention”, “TL1A single-domain antibody of the present invention”, “anti-TL1A single-domain antibody of the present invention”, “anti-TL1A single-domain antibody”, “TL1A single-domain antibody”, “anti-TL1A nanobody”, and “TL1A nanobody” have the same meaning and can be used interchangeably, all referring to the nanobody that specifically recognizes and binds to the TL1A protein and is screened and prepared by the present invention.

[0177] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0178] As used herein, the terms "nanobody" and "single-domain antibody (sdAb)" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of the antibody heavy chain to construct a single-domain antibody consisting of only one heavy chain variable region (VHH). This is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0179] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0180] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0181] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0182] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to the TL1A protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0183] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., antibody-drug conjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0184] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called complementarity-determining regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0185] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0186] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0187] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0188] The term "antibody" in this invention refers to a polypeptide having TL1A binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of this invention.

[0189] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0190] The present invention also provides other polypeptides, such as fusion proteins comprising a single-domain antibody or a fragment thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0191] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0192] Table A

[0193] The last remnant representational substitution preferred replacement Ala(A) Val;Leu;Ile Shaft Arg(R) Lys;Gln;Asn Dirty Asn(N) Gln;His;Lys;Arg Gln Asp(D) Glu Glu Cys(C) Sir Sir Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro;Ala Ala Feeling(H) Asn;Gln;Lys;Arg Arg With(I) Leu;Val;Met;Ala;Phe Leo Leu(L) Ile;Val;Met;Ala;Phe With Lys(K) Arg;Gln;Asn Arg Met(M) Leu;Phe;Ile Leo Phe(F) Leu;Val;Ile;Ala;Tyr Leo Pro(P) Ala Ala Sir(S) Thursday Thursday Thr(T) Sir Sir Trp(W) Tyr;Phe Tyre Tyre(Y) Trp;Phe;Thr;Ser Phew Shaft(V) Ile;Leu;Met;Phe;Ala Leo

[0194] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0195] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0196] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0197] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0198] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0199] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0200] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0201] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0202] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0203] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0204] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0205] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0206] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0207] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0208] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0209] TL1A / DR3 channel

[0210] TL1A, also known as vascular endothelial growth inhibitor (VEGI)-251 or TNFSF15, is a member of the tumor necrosis factor (TNF) superfamily. TL1A is expressed as a type II transmembrane trimeric protein, which is subsequently released as a soluble protein by metalloproteinases such as TNF-α convertase (TACE). It binds to its receptor DR3, initiating a downstream signaling cascade that ultimately leads to various cellular responses (Xu, WD., et al. 2022. Front Immunol. 13:891328.). TL1A binds to its receptor DR3 and activates the TRADD pathway, forming a complex that regulates downstream pathways such as TRAF2, RIP1, PI3K, MAPKs, and NF-κB, exerting a pro-inflammatory effect and thereby regulating the secretion of cytokines and chemokines. TL1A / DR3 also promotes apoptosis through the FADD, RIP3, and Caspase-8 pathways. The complex composed of FADD, RIP3, RIP1 and downstream effector molecule MLKL forms a cytoplasmic "necrosome" after phosphorylation, which then oligomerizes onto the cell membrane and leads to necrotic cell death; NF-κB can activate c-IAP protein and negatively regulate apoptosis.

[0211] Furthermore, decoy receptor 3 (DcR3), a soluble member of the TNF receptor superfamily, complexly modulates the biological functions of TL1A by acting as a competitive inhibitor, binding to TL1A and preventing its interaction with the functional receptor DR3. Through this mechanism, DcR3 effectively inhibits TL1A-mediated signaling pathways, thereby attenuating immune responses and contributing to immune regulation and tolerance.

[0212] The TL1A / DR3 pathway plays a crucial mediating role in the interaction between innate and adaptive immunity, influencing the activation, differentiation, and cytokine production of immune cells. Dysregulation of TL1A / DR3 signaling is associated with the pathogenesis of autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, highlighting its importance in immune-mediated diseases (Aiba, Y., and Nakamura, M. 2013. Mediators Inflamm. 2013:258-164).

[0213] Anti-TL1A single-domain antibody

[0214] This invention provides multiple anti-TL1A single-domain antibodies. Through extensive screening, this invention provides multiple specific anti-TL1A single-domain antibodies with high binding affinity. Furthermore, through humanization, corresponding humanized anti-TL1A single-domain antibodies are provided.

[0215] The camel-derived single-domain antibody obtained by screening in this invention is named Nb20-8. Based on Nb20-8, humanization and modification sites were performed to obtain multiple modified single-domain antibodies. The VHH chain of Nb20-8 and its modified single-domain antibodies includes CDR1 shown in SEQ ID NO:86 (GYTX1X2NYY, where X1 is D, E, or T, and X2 is S, T, or A), CDR2 shown in SEQ ID NO:87 (IX3TX4GGHT, where X3 is N, Q, or D, and X4 is S or G), and CDR3 shown in SEQ ID NO:5. Specifically, the VHH chain of Nb20-8 includes CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody includes CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:18, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody comprises CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody comprises CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:20, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody comprises CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody comprises CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody comprises CDR1 shown in SEQ ID NO:23, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody includes CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5.

[0216] Another camel-derived single-domain antibody obtained by screening in this invention is named Nb8-20. Based on Nb8-20, humanization and modification sites were performed to obtain multiple modified single-domain antibodies. The VHH chain of Nb8-20 and its modified single-domain antibodies contains CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:87 (IX3TX4GGHT, where X3 is N, Q, or D, and X4 is S or G), and CDR3 shown in SEQ ID NO:5. Specifically, the VHH chain of Nb8-20 contains CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody contains CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:18, and CDR3 shown in SEQ ID NO:5. In one embodiment, the VHH chain of the modified single-domain antibody comprises CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:5. In another embodiment, the VHH chain of the modified single-domain antibody comprises CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:20, and CDR3 shown in SEQ ID NO:5.

[0217] The CDR2 and CDR3 sequences of the VHH chain of Nb20-8 and Nb8-20 are identical, exhibiting comparable binding affinity to TL1A and possessing essentially the same biological functions and activities: (1) they bind to both human TL1A and cynomolgus monkey TL1A; (2) they block the binding of TL1A to DR3, thereby blocking the TL1A / DR3 pathway, but do not affect the binding of TL1A to DcR3; (3) they bind only to trimer TL1A, not monomeric TL1A; (4) their binding to TL1A is pH-dependent, exhibiting strong binding activity under neutral conditions and decreased activity under acidic conditions. Furthermore, the modified single-domain antibody also retains the high binding affinity to TL1A, as well as the aforementioned biological functions and activities.

[0218] pH-dependent antibodies

[0219] Human plasma is neutral (pH 7.4), while the intracellular environment is acidic (pH 5.4-6.0). pH-dependent antibodies bind to antigens in plasma. After the antigen-antibody complex is internalized into the cell, in the acidic environment of the endosome, the pH-dependent antibody dissociates from the antigen. The dissociated antibody is captured by FcRn and circulated extracellularly. In the neutral extracellular environment, the FcRn releases the antibody, which returns to the plasma and can then bind to other antigens, achieving antibody recycling. Existing research indicates that the key to transforming natural antibodies into recyclable antibodies is the introduction of histidine. Histidine is a positively charged basic amino acid and serves as both a proton donor and acceptor. Under acidic conditions, histidine undergoes protonation. When histidine is located at the antigen-antibody interaction interface, if it is a key amino acid in the interaction, protonation directly affects the binding. If histidine is a structural amino acid maintaining conformation, protonation causes a conformational change, thus disrupting the binding. Introducing an appropriate amount of histidine into the appropriate epitope of an antibody may endow it with pH-dependent antigen-binding properties. Previous studies have shown that pH dependence is more conducive to antibody cycling and prolongs its half-life in vivo (Tomoyuki Igawa, et al., Nat Biotechnol. 2010 Nov; 28(11):1203-7. and Kenta Haraya, et al., Drug Metab Pharmacokinet. 2016 Apr; 31(2):123-32.).

[0220] The binding of the single-domain antibody of the present invention to TL1A is pH-dependent, with strong binding activity under neutral conditions and reduced binding activity under acidic conditions. This is beneficial for the long-term circulation of antibody molecules in vivo and prolongs the antibody half-life.

[0221] Pharmaceutical Composition

[0222] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-described antibody or its active fragment, or its fusion protein, or its antibody-drug conjugate, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0223] The pharmaceutical compositions of the present invention can be directly used to bind to TL1A protein molecules, blocking the TL1A / DR3 pathway, and therefore can be used to treat TL1A-related diseases, such as autoimmune diseases. In addition, other therapeutic agents can be used simultaneously.

[0224] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0225] In one embodiment of the invention, when using the pharmaceutical composition, a safe and effective amount of the antibody or antibody-drug conjugate of the invention is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0226] Reagent test kit

[0227] The present invention also provides a kit containing the antibody (or fragment thereof) or detection reagent of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0228] This invention also provides a detection kit for detecting TL1A levels. The kit includes an antibody that recognizes the TL1A protein (the antibody described in this invention), a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0229] Detection methods

[0230] The present invention also relates to a method for detecting TL1A protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of TL1A protein in the dissolved samples.

[0231] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0232] application

[0233] As described above, the single-domain antibody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of TL1A-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of TL1A.

[0234] Sequence information of the present invention:

[0235] TL1A single-domain antibody and its humanized antibody sequence information:

[0236]

[0237]

[0238] The full-length amino acid sequence of the above single-domain antibody is shown below:

[0239] In the above amino acid sequence, the underlined parts are CDR1, CDR2 and CDR3 in sequence; FR1, FR2, FR3 and FR4 are separated by CDR1, CDR2 and CDR3.

[0240] Nb20-8

[0241] QVQLQESGGGSVQAGGSLRLSCVGS GYTDSNYY LGWFRQAPGKEREGVAA I NTSGGHT YYADSVKGRSTISQDNAKNTLYLEMNSLKPEDTAMYYC AAVHTPFGGL LDPLWKGNYNY WGQGTQVTVSS(SEQ ID NO:1)

[0242] MY5370(Nb20-8 Hu-1)

[0243] EVQLVESGGGLVQPGGSLRLSCSAS GYTDSNYY MGWFRQAPGKEREGVAA IN TSGGHT YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLL DPLWKGNYNY WGQGTLVTVSS(SEQ ID NO:7)

[0244] MY5371(Nb20-8 Hu-2)

[0245] EVQLVESGGGLVQPGGSLRLSCSAS GYTDSNYY MGWVRQAPGKEREGVAA INTSGGHTYYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLLDPLVKGNYNY WGQGTLVTVSS(SEQ ID NO:8)

[0246] MY5372(Nb20-8 Hu-3)

[0247] EVQLVESGGGLVQPGGSLRLSCSAS GYTDSNYY MGWFRQAPGKGLEGVAA INTSGGHT YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLLDPLVKGNYNY WGQGTLVTVSS(SEQ ID NO:9)

[0248] MY5373(Nb20-8 Hu-4)

[0249] EVQLVESGGGLVQPGGSLRLSCSAS GYTDSNYY MGWFRQAPGKEREGVAA INTSGGHT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLLDPLVKGNYNY WGQGTLVTVSS(SEQ ID NO:10)

[0250] MY5374(Nb20-8 Hu-5)

[0251] EVQLVESGGGLVQPGGSLRLSCVGS GYTDSNYY LGWFRQAPGKGLEGVAA INTSGGHT YYADSVKGRSTISQDNAKNTLYLEMNSLKPEDTAMYYC AAVHTPFGGLLDPLVKGNYNY WGQGTLVTVSS(SEQ ID NO:11)

[0252] Nb8-20

[0253] QVQLQESGGGSVQSGGSLRLSCAIS RYTGSTYY LGWFRQAPGKEREGVAA INTSGGHT YYADSVKGRSTISQDNAKNTLYLEMNSLKPEDTAMYYC AAVHTPFGGLLDPLVKGNYNY WGQGTQVTVSS(SEQ ID NO:2)

[0254] MY5393(Nb8-20 Hu-1)

[0255] EVQLLESGGGLVQPGGSLRLSCAIS RYTGSTYY MGWFRQAPGKEREGVAA INTSGGHT YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLLDPLVKGNYNY WGQGTLVTVSS(SEQ ID NO:12)

[0256] MY5394(Nb8-20 Hu-2)

[0257] EVQLLESGGGLVQPGGSLRLSCAAS RYTGSTYY MGWFRQAPGKEREGVAA INTSGGHT YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLLDPLVKGNYNY WGQGTLVTVSS(SEQ ID NO:13)

[0258] MY5395(Nb8-20 Hu-3)

[0259] EVQLLESGGGLVQPGGSLRLSCAIS RYTGSTYY MGWVRQAPGKEREGVAA INTSGGHT YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLLDPLVKGNYNY WGQGTLVTVSS(SEQ ID NO:14)

[0260] MY5396(Nb8-20 Hu-4)

[0261] EVQLLESGGGLVQPGGSLRLSCAIS RYTGSTYY MGWFRQAPGKGLEGVAA IN TSGGHT YYADSVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLL DPLWKGNYNY WGQGTLVTVSS(SEQ ID NO:15)

[0262] MY5397(Nb8-20 Hu-5)

[0263] EVQLLESGGGLVQPGGSLRLSCAIS RYTGSTYY MGWFRQAPGKEREGVAA IN TSGGHT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AAVHTPFGGLL DPLWKGNYNYWGQGTLVTVSS(SEQ ID NO:16)

[0264] MY5398(Nb8-20 Hu-6)

[0265] EVQLLESGGGLVQPGGSLRLSCAIS RYTGSTYY LGWFRQAPGKGLEGVAA INT OSHHT YYADSVKGRSTISQDNAKNTLYLEMNSLKPEDTAMYYC AAVHTPFGGLLD PLWKGNYNY WGQGTLVTVSS(SEQ ID NO:17)

[0266] In the above amino acid sequence, the underlined parts are CDR1, CDR2 and CDR3 in sequence; FR1, FR2, FR3 and FR4 are separated by CDR1, CDR2 and CDR3.

[0267] The main advantages of this invention include:

[0268] (1) The TL1A single-domain antibody described in this invention has better TL1A / DR3 pathway blocking activity compared with existing reported monoclonal antibodies, and has extremely strong reporter gene cell inhibitory activity and TF-1 cell apoptosis inhibitory activity.

[0269] (2) The TL1A single-domain antibody described in this invention does not affect the interaction between TL1A and DcR3, and retains the natural blocking function of DcR3 on the TL1A / DR3 pathway.

[0270] (3) The TL1A single-domain antibody described in this invention has good binding activity with TL1A proteins of various genera, including TL1A derived from humans and cynomolgus monkeys.

[0271] (4) The binding of the TL1A single-domain antibody to TL1A described in this invention is pH dependent. The binding activity is strong under neutral conditions and decreases under acidic conditions, which is beneficial for the long-term circulation of antibody molecules in vivo.

[0272] (5) The TL1A single-domain antibody described in this invention has a good therapeutic effect on DSS-induced enteritis model, and therefore can be used to treat diseases mediated by the TL1A / DR3 pathway.

[0273] The following specific embodiments further illustrate the invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0274] Example 1: Preparation of TL1A single-domain antibody

[0275] The TL1A protein sequence was derived from the Uniprot website (ID O95150). A 6xhis tag was fused to the N-terminus of its extracellular segment sequence AA57-251, and expression was performed using Expi293 cells. After 5 days of transient culture, the supernatant of Expi293 cells was incubated with a nickel column, and the eluted protein was collected and its purity was determined by SEC-HPLC. The obtained high-purity TL1A trimer antigen was mixed with an adjuvant and injected into four healthy Bactrian camels from Xinjiang. Immunization was performed weekly for a total of seven times. Subsequently, 100 mL of peripheral blood was collected from each camel. Peripheral blood mononuclear cells (PBMCs) were extracted, and total RNA was isolated. After reverse transcription into cDNA, the VHH gene fragment was amplified using two rounds of PCR, cloned into the pMECS vector, and then electroporated into *E. coli* TG1 competent cells to generate a phage-displaying TL1A nanobody library.

[0276] TL1A-specific nanobodies were screened using liquid chromatography. Biotinylated human TL1A-His and mouse TL1A-His were incubated with streptavidin-conjugated magnetic beads, allowing the TL1A protein to bind to the beads. Subsequently, they were incubated with a library of phages at room temperature for 1 hour, followed by 10 PBST washes to remove non-specific phages. After washing, trypsin incubation for 20 minutes was used to elute the specifically bound antibodies and display the phages. The eluted phages were then used to infect *E. coli* TG1 cells in the logarithmic growth phase to expand the phage culture for the next round of screening. This screening process was repeated four times. In rounds 1-2, biotinylated human TL1A protein was used as the antigen, and in rounds 3-4, biotinylated mouse TL1A protein was used.

[0277] Example 2: Flow Cytometry Screening and Identification of Blocking TL1A Single-Domain Antibodies

[0278] Flow cytometry was used to detect the blocking effect of candidate clones on TL1A and DR3 on the surface of 293 cells. More than 2,000 single clones were randomly selected from 2-4 rounds of enriched specific phages and inoculated into TB medium containing Amp in 96-well plates. After reaching the logarithmic growth phase, IPTG was added for overnight induction, followed by crude antibody extraction using an osmotic shock assay. The crude extract expressing TL1A antibody was mixed with a certain amount of biotinylated TL1A and co-incubated with DR3-293T cells. Cells were then washed and incubated with diluted SA-PE secondary antibody. Finally, after washing with PBS, the cells were analyzed by flow cytometry. A total of 282 nanobodies with TL1A / DR3 blocking activity were obtained. Sequencing analysis revealed that these antibodies could be classified into 82 nanobodies with different sequences.

[0279] Example 3: Identification of the blocking activity of candidate antibodies

[0280] Eighty-two nanobodies with distinct sequences were expressed and purified using E. coli for further quantitative analysis of their blocking activity. The purified antibodies were mixed with a certain amount of biotinylated human TL1A protein and co-incubated with a 293T cell line overexpressing human DR3 (293T-hDR3). The cells were then washed and incubated with diluted SA-PE secondary antibody. Finally, the cells were washed with PBS and analyzed by flow cytometry. Results are as follows: Figure 1-7 As shown, most candidate antibodies maintained good human TL1A / DR3 blocking activity, among which 9 antibodies (Nb20-08, Nb08-20, Nb08-52, Nb08-34, Nb08-14, Nb08-28, Nb08-20, Nb08-92, Nb18-13) showed superior blocking activity compared to the control TL1A monoclonal antibody RVT-3101.

[0281] Nine nanobodies with different sequences were mixed with a certain amount of biotinylated cynomolgus monkey TL1A protein and co-incubated with 293T-hDR3 cell lines. The cells were then washed and incubated with diluted SA-PE secondary antibody. Finally, the cells were washed with PBS and analyzed by flow cytometry. Results are as follows: Figure 8 As shown, compared with the control TL1A monoclonal antibodies RVT-3101 (self-made by Luoqi Biotechnology, antibody sequence derived from patent CN105814081B) and PRA023 (self-made by Luoqi Biotechnology, antibody sequence derived from patent CN114901311A), all nine TL1A nanobodies showed good cynomolgus monkey TL1A / DR3 blocking activity.

[0282] Example 4: Identification of the binding activity of candidate antibodies

[0283] To detect the binding of candidate antibodies to trimer TL1A: Human TL1A antigen was coated overnight at 2 μg / mL and blocked with 1% BSA at room temperature for 2 h; serially diluted versions of the above 9 antibodies were added to the blocked ELISA plates and incubated at 37°C for 1 h; the plates were washed three times with PSBT, and then diluted secondary antibody (mouse anti-HA antibody) was added and incubated at 37°C for 30 min; after washing three times with PSBT, anti-mouse-HRP antibody was added and incubated at 37°C for 30 min; after washing three times with PSBT, TMB was added for color development and the reaction was terminated with 2M sulfuric acid; the OD was measured using an ELISA reader. 450nm Absorbance value.

[0284] Detection of the binding of candidate antibody to monomeric TL1A: Human TL1A antigen was reduced with TCEP, coated overnight at 2 μg / mL, blocked with 1% BSA at room temperature for 2 h, and then serially diluted candidate antibody was added and incubated at 37°C for 1 h. After washing with PSBT, mouse anti-HA antibody was added and incubated at 37°C for 30 min. After washing, anti-mouse-HRP antibody was added and incubated at 37°C for 30 min. After washing three times with PSBT, TMB was added for color development and the reaction was terminated with 2M sulfuric acid. OD was detected using a microplate reader. 450nm Absorbance value.

[0285] The results are as follows Figure 9-10 As shown: the nine candidate nanobodies have good binding ability to human TL1A trimer protein, while some nanobodies (Nb20-08 and Nb08-20) do not bind to TL1A monomers.

[0286] Example 5: Inhibition of functional activity of candidate TL1A single-domain antibody on DR3 reporter gene cells

[0287] The candidate antibody and human TL1A protein mixture was prepared using 1640 medium: serially diluted nanobodies were mixed with a fixed concentration of human T1LA protein and incubated for 30 min; then added to the Jurkat-DR3 reporter gene cell line, 40 μL per well in a 96-well plate at a density of 1.25 E6 / mL; after incubation for 6 h, 80 μL of fluorescent detection reagent was added to each well, and after lysis for 5 min, 100 μL of supernatant was taken from each well and added to a 96-well plate for absorbance detection using a microplate reader.

[0288] The results are as follows Figure 11 As shown, all candidate TL1A single-domain antibodies can effectively inhibit the activity of DR3 reporter cells, demonstrating good functional activity.

[0289] Example 6: Inhibition of apoptosis in TF-1 cells by candidate TL1A single-domain antibody

[0290] A mixture of TL1A single-domain antibody, human TL1A protein, and Cycloheximide (CHX) at a 2-fold concentration was prepared using 1640 medium: the antibody was serially diluted starting at 200 nM; simultaneously, the human TL1A protein and CHX were incubated at 37°C for 30 min. TF-1 cells were cultured to a density of 3E5 / mL using 1640 medium, and 100 μL was added to each well of a 96-well plate. The prepared antibody mixture was then added to each well at a concentration of 100 μL. After incubation for 4 h, 40 μL of Caspase 3 / 7 reagent (Promega) was added to each well, and after lysis for 5 min, 100 μL of supernatant was collected from each well and transferred to a 96-well plate for absorbance analysis using a microplate reader.

[0291] The results are as follows Figure 12 As shown, the candidate TL1A single-domain antibody exhibited good TF-1 cell apoptosis inhibitory activity.

[0292] Example 7: Identification of the blocking activity of candidate TL1A single-domain antibodies against TL1A and DcR3

[0293] The blocking activity of candidate TL1A single-domain antibodies against TL1A / DcR3 was detected by ELISA. DcR3 (purchased from Acro) was coated onto an ELISA plate at 300 ng / mL overnight, and then blocked with 1% BSA at room temperature for 2 h. Serially diluted antibodies and a fixed concentration of TL1A-Biotin were mixed and incubated together, then added to the blocked ELISA plate and incubated at 37°C for 1 h. After washing the plate three times with PSBT, secondary antibody SA-HRP was added and incubated at 37°C for 30 min. After a second wash, TMB was added for color development, and the reaction was terminated with 2M sulfuric acid. OD was measured using an ELISA reader. 450nm Absorbance value.

[0294] The results are as follows Figure 13 As shown, candidate TL1A single-domain antibodies Nb20-08 and Nb08-20 do not block the interaction between TL1A and DcR3, while the other 7 candidate antibodies show strong TL1A / DcR3 blocking activity.

[0295] Example 8: Binding activity of candidate TL1A single-domain antibody under different pH conditions

[0296] The binding activity of candidate TL1A single-domain antibodies to TL1A under different pH conditions was detected by ELISA. Human TL1A antigen was coated overnight at 2 μg / mL and blocked with 1% BSA at room temperature for 2 h. TL1A nanobodies were serially diluted with different pH buffers (pH 5.6 and pH 7.0) and added to the blocked ELISA plates, then incubated at 37°C for 1 h. The plates were washed three times with PSBT, and then diluted mouse anti-HA antibody was added and incubated at 37°C for 30 min. After washing three times with PSBT, anti-mouse-HRP antibody was added and incubated at 37°C for 30 min. After washing three times with PSBT, TMB was added for color development, and the reaction was terminated with 2M sulfuric acid. OD was measured using an ELISA reader. 450nm Absorbance value.

[0297] The results are as follows Figure 14 As shown, candidate TL1A single-domain antibodies Nb20-08 and Nb08-20 exhibit good binding activity to TL1A under neutral pH conditions, while their binding activity significantly decreases under acidic conditions. Nb08-52 shows strong TL1A binding activity under both neutral and acidic conditions. Therefore, the binding of candidate TL1A single-domain antibodies Nb20-08 and Nb08-20 to TL1A is pH-dependent, which is more conducive to the long-term circulation of antibody molecules in vivo.

[0298] Example 9: Humanization and Engineering of Antibodies

[0299] The candidate TL1A single-domain antibody was humanized. On one hand, the CDR region was labeled using the IMGT annotation method, keeping the CDR unchanged, while the backbone region was humanized. On the other hand, homology modeling was performed on the unhumanized sequence, and appropriate reversion mutations were performed on the CDR-transplanted sequence, selecting sites that might maintain CDR-antigen interaction for reversion mutation. Furthermore, potential sugar modification sites in the variable region of the candidate TL1A single-domain antibody were mutated, and multiple mutation schemes were designed to remove N-sugar modifications in the CDR region, which were then combined with the humanized sequence. The modified single-domain antibodies with different sequences were expressed using E. coli, and the activity of the humanized antibodies was evaluated using DR3 reporter cells. The modified sequences are shown in Table 1 below.

[0300] Table 1. Amino acid sequences of humanized antibodies

[0301]

[0302]

[0303]

[0304]

[0305] The different TL1A single-domain antibodies expressed and purified above were used to assess DR3 reporter gene cell activity, and the detection method was the same as in Example 4. The results are as follows: Figure 15-19 As shown, the modified single-domain antibodies all maintained good functional activity.

[0306] Example 10: Determination of Tm value of humanized TL1A single-domain antibody

[0307] The Tm value of the modified TL1A single-domain antibody in Example 9 was analyzed using the SDF method.

[0308] Mix the 1 mg / mL antibody with SYPRO Orange protein Gel stain and incubate on ice in the dark for 15 min. After 15 min of incubation on ice in the dark, add 10 μL to a 96-well plate, with three replicates for each sample. Seal the plate and place it in a qPCR instrument: incubate at 25°C for 30 s, and scan the melting curve from 25°C to 98°C, increasing by 0.5°C every 5 s. Analyze the results in FRET scan mode.

[0309] The test results are shown in Table 2.

[0310] Table 2. Tm values ​​of humanized TL1A single-domain antibodies

[0311]

[0312] It is evident that the humanized TL1A single-domain antibody exhibits good stability.

[0313] Example 11: Modification of humanized TL1A single-domain antibody

[0314] The candidate humanized TL1A single-domain antibody was further mutated to reduce the risk of poor activity or stability caused by antibody modification. Based on the antibody's structural analysis, different mutation schemes were designed (Table 3), and the high-purity modified antibody was expressed using E. coli for further testing and analysis.

[0315] Table 3 Modified humanized antibodies

[0316]

[0317]

[0318] The Tm of the modified antibody was tested again using the same method as in Example 10. The DSF detection results are shown in Table 4.

[0319] Table 4. Tm values ​​of modified antibodies

[0320] no Temperature (℃) no Temperature (℃) MY5531 80.0 MY5532 64.5 MY5534 80.0 MY5535 63.0 MY5537 80.5 MY5538 65.0 MY5540 80.5 MY5541 69.0

[0321] The results showed that the modified candidate antibody still maintained good thermal stability.

[0322] Example 12: Blocking effect of modified TL1A single-domain antibody on the TL1A / DR3 pathway

[0323] The blocking activity of the modified antibody against the TL1A / DR3 pathway was tested using flow cytometry.

[0324] The modified antibody, purified by expression, was mixed with a certain amount of biotinylated TL1A and co-incubated with a 293T cell line overexpressing human DR3. The cells were then washed and incubated with diluted SA-PE secondary antibody. Finally, the cells were washed with PBS and analyzed by flow cytometry.

[0325] The results are as follows Figure 20-21 As shown, the modified antibody maintained good human TL1A / hDR3 blocking activity and cynomolgus monkey TL1A / hDR3 blocking activity.

[0326] Example 13: Inhibition of functional activity of modified TL1A single-domain antibody on DR3 reporter gene cells

[0327] The modified antibodies were subjected to activity analysis again. The functional activity of the antibodies was detected using the DR3 reporter gene assay. The specific experimental methods were the same as in Example 5.

[0328] The results are as follows Figure 22 As shown, the modified antibody can effectively inhibit the activity of DR3 reporter gene cells and maintain good biological function.

[0329] Example 14: Inhibition of apoptosis in TF-1 cells by modified TL1A single-domain antibody

[0330] The modified antibodies were then subjected to TF-1 cell apoptosis inhibitory activity assays. The specific experimental methods were the same as in Example 6.

[0331] The results are as follows Figure 23 As shown, the modified antibody can effectively inhibit apoptosis of TF-1 cells and maintain good biological function.

[0332] Example 15: Inhibitory effect of TL1A single-domain antibody on DSS-induced mouse enteritis model

[0333] To confirm the efficacy of the blocking TL1A single-domain antibody of the present invention in treating intestinal inflammation, a blocking nanobody targeting mouse TL1A with activity equivalent to the aforementioned blocking TL1A single-domain antibody blocking the human TL1A / DR3 pathway was used to study the efficacy of dextran sulfate sodium (DSS)-induced enteritis in mice.

[0334] Seven-week-old female BALB / c mice were administered 2% DSS (600 μL) via gavage from day 0 to 3, 3% DSS as a substitute for drinking water from day 4 to 6, and normal drinking water as a substitute on day 7 to establish the mouse model. On days 0, 3, and 6, mice were intraperitoneally injected with anti-mouse TL1A nanobody (2 mg / kg), RVT-3101 (5 mg / kg, TL1A antibody), and PBS (model group), respectively. During the experiment, body weight, fecal viscosity, and fecal hematoma were observed and scored, and the Disease Activity Index (DAI) score was calculated. On day 10, the animals were euthanized with excessive CO2, and serum and colon samples were collected for cytokine detection. Histopathological examination of intestinal tissue was performed to assess tissue inflammation. The raw data were analyzed, and the results are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed on the results, with p < 0.05 considered statistically significant. The analysis of results must consider both statistical and biological significance.

[0335] The results showed that anti-TL1A treatment with a blocking TL1A single-domain antibody effectively regulated the occurrence of DSS-induced acute enteritis. Compared with the model group, mice treated with the antibody on days 0, 3, and 6 showed significant changes in body weight (…). Figure 24 DAI score Figure 25 ) and intestinal pathology score ( Figure 26 All showed a significant decrease.

[0336] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-TL1A single-domain antibody or its antigen-binding fragment, characterized in that, The VHH chain of the anti-TL1A single-domain antibody or its antigen-binding fragment contains a complementarity-determining region (CDR) selected from the group consisting of: (1) CDR1 shown in SEQ ID NO:86, CDR2 shown in SEQ ID NO:87, and CDR3 shown in SEQ ID NO:5; or (2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:87 and CDR3 shown in SEQ ID NO:

5.

2. The anti-TL1A single-domain antibody or its antigen-binding fragment as described in claim 1, characterized in that, The VHH chain contains complementary determination regions (CDRs) selected from the following group: (1) CDR1 shown in SEQ ID NO:3, 21, 22, 23 or 24, CDR2 shown in SEQ ID NO:4, 18, 19 or 20 and CDR3 shown in SEQ ID NO:5; or (2) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:4, 18, 19 or 20 and CDR3 shown in SEQ ID NO:

5.

3. The anti-TL1A single-domain antibody or its antigen-binding fragment as described in claim 1, characterized in that, The VHH chain also includes frame regions (FRs), which include camel-derived FRs and human-derived FRs.

4. The anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3, characterized in that, The VHH chain has an amino acid sequence that is at least 80%, 85%, or 90% sequence identical to the amino acid sequence shown in any one of SEQ ID NO:1, 2, 7-11, and 12-17.

5. The anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3, characterized in that, The VHH chain has one or more amino acid residue mutations relative to the amino acid sequence shown in any one of SEQ ID NO:1, 2, 7-11 and 12-17.

6. The anti-TL1A single-domain antibody or its antigen-binding fragment as described in claim 5, characterized in that, The amino acid sequence of the VHH chain has an amino acid residue mutation at the following sites relative to the amino acid sequence shown in any one of SEQ ID NO:1, 2, 7-11 and 12-17: position 52 and / or position 54.

7. The anti-TL1A single-domain antibody or its antigen-binding fragment as described in claim 5, characterized in that, The amino acid sequence of the VHH chain has an amino acid residue mutation at the following sites relative to the amino acid sequence shown in any one of SEQ ID NO:1, 7-11: position 29, position 30, position 52, position 54, or a combination thereof.

8. The anti-TL1A single-domain antibody or its antigen-binding fragment as described in claim 4, characterized in that, The VHH chain has an amino acid sequence as shown in any of SEQ ID NO:1, 2, 7-11, 12-17, 41-85.

9. A fusion protein, characterized in that, The structure of the fusion protein from the N-terminus to the C-terminus is shown in formula Ia or Ib: ALB(Ia); BLA(Ib); in, A is the anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-8; B is the Fc fragment of IgG; and L represents no or flexible joints.

10. A multispecific antibody, characterized in that, The multispecific antibody comprises an anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-8, or a fusion protein as described in claim 9.

11. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-8; and (ii) Optional tag sequences to assist in expression and / or purification.

12. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: an anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-8, a fusion protein as described in claim 9, a multispecific antibody as described in claim 10, or a recombinant protein as described in claim 11.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-8, the fusion protein as described in claim 9, the multispecific antibody as described in claim 10, or the recombinant protein as described in claim 11; and (ii) Pharmaceutically acceptable carriers.

14. A TL1A inhibitor, characterized in that, The inhibitor comprises an anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-8, a fusion protein as described in claim 9, a multispecific antibody as described in claim 10, or a recombinant protein as described in claim 11, and the TL1A inhibitor blocks the binding of TL1A to DR3 without affecting the binding of TL1A to DcR3.

15. Use of the anti-TL1A single-domain antibody or its antigen-binding fragment as described in any one of claims 1-8, the fusion protein as described in claim 9, the multispecific antibody as described in claim 10, or the recombinant protein as described in claim 11, for the preparation of: (a) Medications used to prevent and / or treat diseases or conditions mediated by the TL1A / DR3 pathway; or (b) Detection reagents, detection plates or detection kits for detecting TL1A molecules.

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