TL1A antibody as well as preparation method and application thereof

Abstract

The invention relates to a TL1A antibody as well as a preparation method and application thereof. The amino acid sequence of the TL1A antibody is selected from any one or more of SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41 or SEQ ID NO. 42. Compared with Yangshen, the TL1A antibody provided by the invention can better block the interaction between TL1A and the receptor DR3 thereof, the TL1A plays an important role in the aspects of inflammatory reaction and the like due to the unique action mechanism of the TL1A, and the TL1A blocking agent has become a potential new therapy for treating inflammatory and fibrotic diseases at present.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to a TL1A antibody and a preparation method and application thereof. Background Art

[0002] TL1A (TNF-like ligand 1A), also known as TNFSF15, is a member of the tumor necrosis factor (TNF) family. TL1A is a type II transmembrane protein that self-assembles into stable trimers through interaction with the TNF homology domain (THD). TL1A is expressed in a variety of immune cells (such as monocytes, macrophages, dendritic cells, and T cells) as well as non-immune cells (such as synovial fibroblasts and endothelial cells).

[0003] The main biological function of TL1A is to regulate the proliferation, activation, apoptosis and production of cytokines and chemokines of effector cells by binding to its receptor death receptor 3 (DR3) and activating downstream signaling pathways, playing a key role in maintaining innate and adaptive immune homeostasis.

[0004] Studies have shown that TL1A is abnormally highly expressed in a variety of autoimmune diseases and is deeply involved in the pathogenesis of diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel diseases (such as ulcerative colitis and Crohn's disease). Given its unique mechanism of action in inflammatory responses, TL1A has become an important new target for the treatment of these diseases. Currently, inhibitors that target and block the TL1A signaling pathway (such as TL1A antibodies) are considered potential new therapies for inflammatory and fibrotic diseases.

[0005] However, existing TL1A-targeted therapeutics may still have limitations in terms of efficacy and species cross-reactivity, limiting their potential and effectiveness in clinical application. Therefore, the development of novel TL1A antibodies with higher affinity, enhanced blocking potency, good species cross-reactivity, and specificity is of great scientific significance and clinical application value.

[0006] Chinese patent CN109476742A discloses a TL1A antibody and its uses, including an isolated antibody or antigen-binding fragment thereof, which binds to human TL1A (TNF-like ligand 1A) and comprises: a) a heavy chain variable domain comprising: CDRH1 set forth in SEQ ID NO:7, CDRH2 set forth in SEQ ID NO:8, and CDRH3 set forth in SEQ ID NO:9; and b) a light chain variable domain comprising: CDRL1 set forth in SEQ ID NO:12, CDRL2 set forth in SEQ ID NO:13, and CDRL3 set forth in SEQ ID NO:14. Therefore, this patent only protects a single CDR combination and only verifies that the antibody blocks TL1A-DR3 binding. Summary of the Invention

[0007] The present invention aims to provide a TL1A antibody, its preparation method, and its use. Compared to Yangshen (Ginseng), the TL1A antibody described herein is more effective in blocking the interaction between TL1A and its receptor, DR3. Due to its unique mechanism of action, TL1A plays a crucial role in inflammatory responses and other aspects. TL1A blockers have emerged as a potential new treatment for inflammatory and fibrotic diseases.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The first object of the present invention is to provide a TL1A antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:

[0010] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4 to SEQ ID NO.6; or

[0011] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.7, SEQ ID NO.2 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.9; or

[0012] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1, SEQ ID NO.10 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.9; or

[0013] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1, SEQ ID NO.10 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.11; or

[0014] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.12 to SEQ ID NO.14; or

[0015] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1, SEQ ID NO.10 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.9; or

[0016] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.15 to SEQ ID NO.17, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.18 to SEQ ID NO.20; or

[0017] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.21 to SEQ ID NO.23, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.24 to SEQ ID NO.26; or

[0018] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.29 to SEQ ID NO.31; or

[0019] The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.7, SEQ ID NO.2 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.29, SEQ ID NO.30 and SEQ ID NO.32.

[0020] Furthermore, the TL1A antibody or antigen-binding fragment thereof has an amino acid sequence selected from any one of the following: SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.42.

[0021] As a preferred technical solution, the TL1A antibody or antigen-binding fragment thereof has an amino acid sequence selected from any one of the following: SEQ ID NO.33.

[0022] Furthermore, the nucleotide sequence encoding the amino acid sequence of the TL1A antibody or the antigen-binding fragment thereof is one of the following sequences: SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.52.

[0023] As a preferred technical solution, the TL1A antibody or antigen-binding fragment thereof has an amino acid sequence selected from any one of the following: SEQ ID NO.43.

[0024] Furthermore, the TL1A antibody or antigen-binding fragment thereof substantially inhibits the binding of human TL1A to DR3 and / or DCR3.

[0025] The second object of the present invention is to provide a molecular expression vector, which comprises one of the nucleotide sequences of SEQ ID NO.43 to SEQ ID NO.52.

[0026] The third object of the present invention is to provide a host cell containing the above-mentioned molecular expression vector, wherein the host cell is a mammalian cell.

[0027] Furthermore, the mammalian cells are selected from CHO cells.

[0028] A fourth object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned isolated TL1A antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0029] A fifth object of the present invention is to provide a use of the isolated TL1A antibody or antigen-binding fragment that inhibits the binding of human TL1A to DR3 and / or DCR3 for preparing a pharmaceutical composition for treating a subject suffering from inflammatory and fibrotic diseases.

[0030] Furthermore, the inflammatory and fibrotic diseases include ulcerative colitis, Crohn's disease, rheumatoid arthritis and psoriasis.

[0031] In addition, the present invention also provides a method for preparing a TL1A antibody, the specific steps of which are as follows:

[0032] S1. Based on the protein sequence and gene sequence information of TL1A, express the screening antigen and connect a His-tag to its C-terminus to obtain a modified nucleic acid sequence;

[0033] S2. Cloning the nucleic acid sequence obtained in step S1 into an expression vector, and expressing the antigen using mammalian cells to obtain the TL1A-His antigen;

[0034] S3. Using spleen cells from immunized mice as raw materials, RNA was extracted and reverse transcribed into cDNA. Antibody gene fragments were obtained by PCR and cloned into phage vectors for library construction.

[0035] S4, using the TL1A-His antigen obtained in step S2 to perform antibody screening on the gene fragment library constructed in step S3;

[0036] S5. Expressing the antibody sequence obtained in step S4 through a mammalian cell system, and further obtaining an antibody against TL1A through screening. The antibody against TL1A has high sensitivity and specificity.

[0037] Furthermore, in step S4, the sequence of the antibody gene fragment includes the nucleotide sequence shown as SEQ ID NO.43 to SEQ ID NO.52.

[0038] Furthermore, in step S5, further screening experiments include ELISA and FACS detection.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] 1. Compared with Yangshen, the TL1A antibody of the present invention can better block the interaction between TL1A and its two receptors DR3 and DCR3. TL1A plays an important role in inflammatory response and other aspects due to its unique mechanism of action. TL1A blockers have now become a potential new therapy for the treatment of inflammatory and fibrotic diseases.

[0041] 2. The TL1A antibody described herein exhibited significantly lower EC50 values ​​than the positive control antibody Tulisokibart in binding activity, species testing, species cross-reactivity, and specificity assays, indicating that the TL1A antibody has a higher affinity for cell binding. The TL1A antibody also binds to CYNOMOLGUS but not to MOUSE, demonstrating a high degree of species specificity that can reduce potential off-target effects, which is crucial for preclinical efficacy and safety evaluations.

[0042] 3. In blocking experiments, the TL1A antibody of the present invention can more effectively prevent the binding of TL1A to its decoy receptor DCR3 and the binding of TL1A to its functional receptor DR3. Its blocking efficacy is significantly stronger than that of the positive control antibody Tulisokibart, which is the core mechanism by which it exerts its therapeutic effect.

[0043] 4. Based on the above-mentioned excellent binding activity and strong receptor blocking ability, the TL1A antibody provided by the present invention has significant advantages in blocking TL1A-mediated inflammatory and immune signaling pathways, providing a more effective candidate drug for the development of inflammatory and fibrotic diseases such as ulcerative colitis, Crohn's disease, rheumatoid arthritis, and psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagram of the results of binding experiments between HUMAN-TL1A cell lines (TL1A antigen on the cell surface) and antibodies (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, and TL1A-90);

[0045] Figure 2Schematic diagram of the results of the binding experiment between CYNO-TL1A cell line and antibodies (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90);

[0046] Figure 3 Schematic diagram of the results of the binding experiment between the MOUSE-TL1A cell line and antibodies (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, and TL1A-90);

[0047] Figure 4 Schematic diagram of the results of the cell-level antibody (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90) and DCR3 blocking assay;

[0048] Figure 5 Schematic diagram of the test results of cell-level antibodies (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90) and DR3 blocking experiments. DETAILED DESCRIPTION

[0049] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0050] As used herein, the terms "VH" and "VL" refer to the variable regions of the heavy and light chains of antibodies. The variable region is composed of discrete, well-defined subregions called "complementarity determining regions" (CDRs, also known as HVRs (hypervariable regions)) and "framework regions" (FRs). CDRs refer to the amino acids within the variable region of an antibody that confer antigen specificity and / or binding affinity, separated by FRs. There are three CDRs (VLCDR1, VLCDR2, and VLCDR3) in each antibody light chain variable region, and three CDRs (VHCDR1, VHCR2, and VHCDR3) in each antibody heavy chain variable region; the "complementarity determining regions" (CDRs) of the VH and VL regions alternate with more conserved regions of the "framework regions" (FRs); each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0051] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0052] In the following examples, the amino acid sequences are shown in Table 1:

[0053] Table 1 Amino acid sequence of the CDR region of the TL1A antibody of the present invention

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] In the following examples, the sources of the materials are as follows:

[0066] The PBMC cells of the immunized mice were purchased from Miaoshun Biotechnology Co., Ltd.; the pComb3XSS was purchased from Addgene; the SS320 bacterial solution was purchased from Lucigen; and the secondary antibody Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (imported) was purchased from Thermo.

[0067] If no specific techniques or conditions are specified in the present examples, the operations were performed according to conventional techniques and instrument specifications in the art; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0068] The following describes some embodiments of the present invention in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0069] Example 1

[0070] This example provides a method for preparing a TL1A antibody, and the specific steps are as follows:

[0071] S1. Based on the protein and gene sequence information of TL1A, its extracellular sequence was expressed, and a His-tag was connected to its C-terminus. The antigen was cloned into the pCDNA3.4 expression vector and expressed in mammalian cells Expi 293F to obtain the TL1A-His antigen for subsequent screening and detection.

[0072] S2. Using PBMC cells from immunized mice as raw materials, RNA from the obtained cells was extracted using the Trizol method and converted into cDNA using oligo(dT); gene fragments were amplified by PCR and then cloned into the phage vector pComb3XSS for library construction (PHAGE);

[0073] S3. Antibody screening is performed using the TL1A-His antigen obtained in step S1. The specific steps are as follows:

[0074] S3-1. First round of screening

[0075] S3-1-1. Prepare two labeled immunotubes (one for positive screening and one for negative screening) and set them aside in a clean bench. Wash them twice with PBST and twice with PBS.

[0076] Coating antigen: Add 30 μg of TL1A-His antigen expressed in step S1 to the washed immunotube, dilute and dissolve with 2 mL of PBS, and rotate at 4°C overnight to allow the antigen protein to adsorb (coat) on the inner wall of the immunotube.

[0077] S3-1-2. On the second day, the antigen was recovered, washed 3 times with PBST, washed twice with PBS, and 5×10 12 (5×10 12 / 1.5×10 13 ) The packaged PHAGE obtained in step S2 was then blocked with 1% BSA to a volume of 4 mL. Simultaneously, only 4 mL of 1% BSA was added to the positive screening tube for blocking and incubated with rotation at room temperature for 1 hour.

[0078] S3-1-3. After blocking, take the supernatant PHAGE from the negative screening tube and add it to the positive screening immunotube. Incubate with rotation at room temperature for 1 hour. Remove the supernatant and wash 8 times with PBST and twice with PBS.

[0079] S3-1-4. Elute the bound phage by adding 1 mL of trypsin and incubate with rotation at room temperature for 30 minutes.

[0080] S3-1-5. Infection: Pipette 500 μL of the phage elution mixture into 5 mL of shaken SS320 bacterial suspension (OD600: 0.4-0.6), mix thoroughly, and incubate at 37°C for 30 min. Simultaneously, take 10 μL of the remaining 500 μL of phage elution and add it to the shaken SS320 bacterial suspension. Perform a ten-fold serial dilution, 8-9 times, and incubate at 37°C for 30 min.

[0081] S3-1-6, centrifuge the infected bacterial solution at 3000 rpm for 5 minutes to enrich the bacteria, discard the supernatant, and resuspend the bacteria in the remaining 300 μL culture medium. + and T + The plate was inverted and placed in a 37°C incubator overnight, and titration was performed at the same time.

[0082] S3-2. Potential positive phage clones were obtained after the second round of panning. The specific steps were the same as those in the first round of screening. The differences are shown in Table 2 below:

[0083] Table 2 Differences between the two rounds of solid phase screening

[0084] Round Input phage(pfu) Coating antigen amount (μg) 1 round <![CDATA[~5×10 12 ]]> 30 2 rounds <![CDATA[~5×10 10 ]]> 15

[0085] S4. Screen the positive clones prepared in step S3 by ELISA to obtain the antibody sequence. The specific detection process is as follows:

[0086] S4-1. Coat the ELISA plate with the TL1A-His antigen expressed in step S1 at a concentration of 1 μg / mL at 4°C overnight.

[0087] S4-2. The next day, the cells were washed three times with PBST and blocked with 1% BSA at room temperature for 1 h.

[0088] S4-3, wash three times with PBST, add 200 μL of the supernatant of overnight shaken monoclonal bacteria to each well, and incubate at 37°C for 1.5 h;

[0089] S4-4, wash three times with PBST, add 1:10000 diluted Anti-FLAG-HRP secondary antibody to each well and incubate at 37°C for 1 h;

[0090] S4-5, wash three times with PBST, add 100 μL TMB substrate, incubate at 37°C for 10 min, add 50 μL 0.1 M H2SO4 to stop the reaction, and measure OD 450nm.

[0091] S5. Plasmid construction and extraction: construct the positive antibody sequence and clone it into the expression vector.

[0092] The antibody sequences (SEQ ID NO. 43 to SEQ ID NO. 52) obtained by screening were cloned into the pCDNA3.4 expression vector to obtain antibodies (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90).

[0093] S6. Cell transfection: The day before, seed cells to an appropriate density and transfect with an antibody heavy chain to light chain molar ratio of 2:3. Culture in a 37°C, 5% CO2 cell culture incubator for 12 consecutive days.

[0094] S7. To further verify the binding of positive antibodies, the antibodies obtained in step S6 were subjected to CELL BINDING ASSAY (cell binding assay) detection with the CHOK1 cell line overexpressing the full-length TL1A antigen. The screened antibodies were diluted 3-fold starting at 200 nM and added to the pre-plated cells. The cells were incubated at 4°C for 1 h, washed twice with MACS BUFFER (PBS + 10% FBS + 2 mM EDTA), and the secondary antibody (Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488) was added. The cells were incubated at 4°C for 30 min, washed twice with MACS BUFFER, and detected by flow cytometry.

[0095] The results show Figure 1 Through CELL BINDING ASSAY screening, we finally obtained that the TL1A antibody had better binding effect with cells than the positive control antibody (Tulisokibart).

[0096] S8. Select the antibody sequences (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90) with lower EC50 than Yang Shen in CELL BINDING ASSAY results for species experiments. Dilute the screened antibodies starting at 200 nM in a 3-fold gradient and add them to the pre-plated cells (CYNO-TL1A-CHOK1 CELL LINE). Incubate at 4°C for 1 hour, wash twice with MACS BUFFER, add secondary antibody (same as the secondary antibody in step S7), incubate at 4°C for 30 minutes, wash twice with MACS BUFFER, and detect on a flow cytometer.

[0097] The results show Figure 2 , the antibody sequences TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, and TL1A-90 with EC50 lower than that of Tulisokibart can all bind to CYNOMOLGUS.

[0098] S9. Select the antibody sequences (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90) with lower EC50 than Yang Shen in CELL BINDING ASSAY results for species cross-experimentation. Dilute the screened antibodies starting at 200 nM in a 3-fold gradient and add them to the pre-plated cells (MOUSE-TL1A-CHOK1 CELL LINE). Incubate at 4°C for 1 hour, wash twice with MACS BUFFER, add secondary antibody (same as the secondary antibody in step S7), incubate at 4°C for 30 minutes, wash twice with MACS BUFFER, and detect on a flow cytometer.

[0099] The results show Figure 3 , TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90 and Tulisokibart do not bind to MOUSE.

[0100] S10. Select the antibody sequences (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90) with better CELL BINDING ASSAY results than Tulisokibart to perform DCR3 cell-level blocking experiments. Start with 100 nM of the screened antibodies, dilute them 3-fold, and add them to the pre-plated cells (HUMAN-TL1A-CHOK1 CELL LINE). Incubate at 4°C for 30 min, wash twice with MACS BUFFER, add the ligand DCR3 (gene sequence from UNIPROT) at a working concentration of 10 μg / mL to the cells, incubate at 4°C for 1 h, wash twice with MACS BUFFER, add the secondary antibody (same as the secondary antibody in step S7), incubate at 4°C for 30 min, and incubate with MACS. Wash twice with buffer and detect on flow cytometer.

[0101] The results show Figure 4 The blocking effects of the screened antibody sequences TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, and TL1A-90 were all stronger than Tulisokibart.

[0102] S11. Select the antibody sequences (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-70, TL1A-78, TL1A-90) with better CELL BINDING ASSAY results than Tulisokibart to perform DR3 cell-level blocking experiments. The screened antibodies were diluted starting at 33.3 nM in a 3-fold gradient and added to the pre-plated cells (HUMAN-TL1A-CHOK1 CELL LINE). The cells were incubated at 4°C for 1 hour and washed twice with MACS BUFFER. The ligand DR3 (gene sequence from UNIPROT) was added to the cells at a working concentration of 60 μg / mL and incubated at 4°C for 1 hour. The cells were washed twice with MACS BUFFER. The secondary antibody (same as the secondary antibody in step S7) was added and incubated at 4°C for 1 hour. The cells were washed twice with MACS BUFFER and detected by flow cytometry.

[0103] The results show Figure 5The blocking effect of the screened sequences (TL1A-10, TL1A-11, TL1A-23, TL1A-30, TL1A-39, TL1A-57, TL1A-63, TL1A-78, and TL1A-90) was stronger than that of Tulisokibart.

[0104] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A TL1A antibody or an antigen-binding fragment thereof, characterized in that: It comprises a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4 to SEQ ID NO.6; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.7, SEQ ID NO.2 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.9; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1, SEQ ID NO.10 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.9; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1, SEQ ID NO.10 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.8 and SEQ ID NO.11; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.12 to SEQ ID NO.14; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.1, SEQ ID NO.10 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.9; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.15 to SEQ ID NO.17, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.18 to SEQ ID NO.20; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.21 to SEQ ID NO.23, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.24 to SEQ ID NO.26; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.29 to SEQ ID NO.31; or The heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.7, SEQ ID NO.2 and SEQ ID NO.3, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in the amino acid sequences of SEQ ID NO.29, SEQ ID NO.30 and SEQID NO.

32.

2. The TL1A antibody or antigen-binding fragment thereof according to claim 1, wherein: The TL1A antibody or antigen-binding fragment thereof has an amino acid sequence selected from any one of the following: SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.

42.

3. The TL1A antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The nucleotide sequence encoding the amino acid sequence of the TL1A antibody or its antigen-binding fragment is one of the following sequences: SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49, SEQ ID NO.50, SEQ ID NO.51, SEQ ID NO.

52.

4. The TL1A antibody or antigen-binding fragment thereof according to claim 1, wherein: The TL1A antibody or antigen-binding fragment thereof substantially inhibits the binding of human TL1A to DR3 and / or DCR3.

5. A molecular expression vector, characterized in that: The vector comprises one of the nucleotide sequences of SEQ ID NO.43 to SEQ ID NO.

52.

6. A host cell containing the molecular expression vector according to claim 5, characterized in that: The host cell is a mammalian cell.

7. The host cell according to claim 6, characterized in that The mammalian cells are selected from CHO cells.

8. A pharmaceutical composition, characterized in that It comprises the isolated TL1A antibody or antigen-binding fragment according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

9. Use of the isolated TL1A antibody or antigen-binding fragment of any one of claims 1 to 4 that inhibits binding of human TL1A to DR3 and / or DCR3 for the preparation of a pharmaceutical composition for treating a subject suffering from an inflammatory and fibrotic disease.

10. The use according to claim 9, characterized in that Such inflammatory and fibrotic diseases include ulcerative colitis, Crohn's disease, rheumatoid arthritis, and psoriasis.

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