Anti-human tigit monoclonal antibody and application thereof

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to an anti-human TIGIT monoclonal antibody and application thereof. The anti-human TIGIT monoclonal antibody provided by the application comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 with the amino acid sequences shown in SEQ ID NO:1-3, and the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 with the amino acid sequences shown in SEQ ID NO:4-6. The application also provides application of the anti-human TIGIT monoclonal antibody in tumor immunotherapy, preparation of a TIGIT protein detection reagent or preparation of a product combined with human TIGIT protein. The technical effect of the application is as follows: the application screens a new anti-human TIGIT monoclonal antibody, and the antigen binding activity of the new anti-human TIGIT monoclonal antibody is better than that of the existing anti-human TIGIT monoclonal antibody. The antibody development method provided by the application shortens the development time of the target antibody, and has high throughput, high positive rate and better antigen binding activity.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an anti-human TIGIT monoclonal antibody and its applications. Background Technology

[0002] TIGIT (T cell immunoglobulin with Ig and ITIM domains) is a protein widely present in effector CD8 cells. + The co-inhibitory receptor on the surface of lymphocytes such as T cells and NK cells has a functional domain that includes an immunoglobulin variable domain (IgV), a transmembrane domain, and an immunoreceptor tyrosine inhibitory motif.

[0003] Studies have shown that TIGIT possesses multiple ligands and can compete for or share the same ligands with other inhibitory and stimulatory receptors. CD155 (PVR / NECL-5) has a high affinity for TIGIT, as does the co-stimulatory receptor CD226 expressed on both T and NK cells. TIGIT binding to ligands CD155 and CD122 inhibits T and NK cell activation, thereby suppressing the anti-tumor immune response. When TIGIT binds to its ligand PVR / PVRL2 on tumor cells, it inhibits the immunocytotoxic activity of CD8+ T cells or NK cells through signaling pathways, thus promoting tumor cell escape. Blocking TIGIT can restore NK cell function, reduce Treg cells, increase antigen-specific CD8 memory cell responses, and induce the production of new antigen-specific CD8+ T cells. Therefore, anti-TIGIT monoclonal antibodies play a crucial role in limiting adaptive and innate immunity in tumors.

[0004] In recent years, TIGIT has been increasingly studied in tumor immunotherapy, autoimmune diseases and other immune-related fields. However, most TIGIT antibodies on the market have shortcomings such as weak competitive activity, low throughput and low positive rate.

[0005] Therefore, there is a need to provide a novel anti-human TIGIT monoclonal antibody. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-human TIGIT monoclonal antibody and its application, thereby solving one or more of the problems in the prior art.

[0007] The technical solution of this invention to solve the technical problem is as follows:

[0008] In a first aspect of the present invention, an anti-human TIGIT monoclonal antibody is provided, the antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising three heavy chain complementarity-determining regions, the three heavy chain complementarity-determining regions being denoted by HCDR1, HCDR2 and HCDR3 respectively; the light chain variable region comprising three light chain complementarity-determining regions, the three light chain complementarity-determining regions being denoted by LCDR1, LCDR2 and LCDR3 respectively;

[0009] The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:2, and the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:3.

[0010] The amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:6.

[0011] Furthermore, the aforementioned anti-human TIGIT monoclonal antibody comprises:

[0012] a. A heavy chain variable region having an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO. 7; and

[0013] b. Light chain variable region having an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO.8.

[0014] Furthermore, the aforementioned anti-human TIGIT monoclonal antibody comprises:

[0015] The amino acid sequence is as shown in the heavy chain variable region of SEQ ID NO.7; and

[0016] The amino acid sequence is shown in the light chain variable region of SEQ ID NO.8.

[0017] In a second aspect of the invention, a polynucleotide molecule is also provided, said polynucleotide molecule encoding the amino acid sequence of the anti-human TIGIT monoclonal antibody as described in the first aspect.

[0018] In a third aspect of the invention, a cloning or expression vector is also provided, the cloning or expression vector comprising the polynucleotide molecule as described in the third aspect.

[0019] In a fourth aspect of the invention, a host cell comprising the vector described in the third aspect is also provided, preferably, the host cell being a mammalian cell.

[0020] Furthermore, the mammalian cell is either HEK-293 cell or CHO cell.

[0021] In a fifth aspect of the present invention, a method for preparing an anti-human TIGIT monoclonal antibody is also provided, comprising the following steps:

[0022] S1, expressing Human TIGIT / His Protein;

[0023] S2. Animal preparation and immunization, collection of spleen and bone marrow cells;

[0024] S3. Screen for cells that secrete positive antibodies, construct an antibody library and sequence it;

[0025] S4. Screen and extract antibody sequences, and clone them into a vector;

[0026] S5, high-throughput expression of anti-Human TIGIT antibody;

[0027] S6. Antibody detection.

[0028] In a sixth aspect of the invention, a medicament or pharmaceutical composition is also provided, the medicament or pharmaceutical composition comprising the anti-human TIGIT monoclonal antibody as described in the first aspect.

[0029] In a seventh aspect of the invention, the use of the anti-human TIGIT monoclonal antibody as described in the first aspect in the preparation of tumor immunotherapy drugs, reagents for detecting TIGIT protein, or products that bind to human TIGIT protein is also provided.

[0030] Compared with the prior art, the technical effects of the present invention are as follows:

[0031] 1) This invention screened and obtained a new anti-human TIGIT monoclonal antibody, which has better antigen-binding activity and better cell-binding activity than existing anti-human TIGIT monoclonal antibodies.

[0032] 2) The antibody development method provided by this invention shortens the development time of the target antibody, and has high throughput, high positive rate, better antigen binding activity, and better cell binding activity; it provides certain technical support for the application of TIGIT immunotherapy. Attached Figure Description

[0033] Figure 1 The result is the serum titer test result after animal immunization in Example 1.

[0034] Figure 2 This is the result of flow cytometry detection.

[0035] Figure 3This refers to the results of an antigen binding test.

[0036] Figure 4 This is the result of a cell binding assay. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments. However, the present invention is not limited to the examples given. Unless otherwise specified, all methods used are conventional methods, and all reagents and materials used are commercially available unless otherwise specified.

[0038]

[0039]

[0040] Example 1: Preparation of anti-Human TIGIT monoclonal antibody

[0041] 1.1 Expression and labeling of Human TIGIT / His Protein

[0042] Based on the sequence information of Human TIGIT (Gene ID: 201633), the antigen was expressed and a His-tag was attached to its C-terminus to obtain the modified Human TIGIT / His protein. The Human TIGIT / His protein was fluorescently labeled using the Alexa Fluor 488 antibody labeling kit (purchased from Thermo Fisher) for subsequent mouse cell screening.

[0043] 1.2 Construction of CHOK1-hTIGIT-cell overexpression cell line

[0044] The pLVX-IRES-puro-hTIGIT plasmid was synthesized through gene recombination. psPAX2 plasmid, PMD2G plasmid, pLVX-IRES-puro-hTIGIT plasmid, and PEI transfection reagent were co-transfected into 293T cells. Lentiviral particle supernatant was collected after 72 hours and filtered. The lentiviral particles were then inoculated into CHOK1 cells, and 1 / 100 volume of F108 infection enhancement medium was added. After 6-8 hours, the medium was replaced with fresh medium. After 48 hours, the cells were expanded and 10 μg / mL puromycin was added for resistance selection. Cells that could continuously grow and be passaged were designated as CHOK1-hTIGIT-cells, which were subsequently used for antibody expression and FACS binding detection.

[0045] 1.3 Animal Immunization

[0046] The fluorescently labeled Human TIGIT / His protein obtained in step (1) was diluted with a water-soluble adjuvant and injected subcutaneously into mice at multiple sites on the abdomen and back for primary immunization. On day 10, mice were immunized a second time with the fluorescently labeled Human TIGIT / His protein obtained in step (1). Three days later, blood samples were collected to detect the serum titer of Anti-Human TIGIT / His. Once the serum titer was qualified, the spleen and bone marrow cells of the mice were collected for single-cell screening.

[0047] Serum titer of anti-Human TIGIT / His Protein was detected by ELISA. The procedure was as follows: ELISA plates were coated with 2 μg / mL Human TIGIT / his protein and incubated at 37°C for 2 h; washed with 0.05% PBST and blocked with 1% BSA at room temperature for 2 h; washed with 0.05% PBST, and mouse serum diluted 3-fold before and after immunization was added to the first two wells (100-fold and 1000-fold dilutions), and incubated at room temperature for 1 h; washed with 0.05% PBST, and Anti-mouse IgG (Fc specific) HRP (purchased from Sigma-Aldrich) was added, and incubated at room temperature for 30 min; washed with 0.05% PBST, and Beyotime TMB chromogenic solution was added, reacting at room temperature for 10 min, then Beyotime TMB stop solution was added to immediately stop the reaction, and OD450 was read immediately. Results are as follows: Figure 1 As shown, the antiserum titer after two immunizations was 656100. This indicates that the antigen can induce mice to produce high-titer antiserum specifically targeting Human TIGIT / HisProtein.

[0048] 1.4 Isolation of spleen and bone marrow cells

[0049] Spleen and bone marrow cells from immunized mice were collected, centrifuged at 400g for 5 min, resuspended in 1 mL of erythrocyte lysis buffer, and the lysis was terminated with a large volume of Macs buffer. After centrifugation at 400g for 5 min, the cells were resuspended in 1 mL of Macs buffer for later use.

[0050] 1.5-segment B cells

[0051] Add fluorescent antibodies containing AF488-Human TIGIT / his protein and B cell-specific molecular markers, incubate at 4°C for 1 hour, resuspend in large volume Macs buffer, centrifuge at 400g for 5 minutes, discard the supernatant, resuspend cells in the buffer, and sort cells using a flow cytometer.

[0052] The results are as follows Figure 2 As shown, the antigen positivity rate is approximately 1%.

[0053] 1.6 Single-cell library construction

[0054] The positive mouse cells were constructed using a single-cell library construction platform, and the constructed library was subjected to next-generation sequencing. The antibodies with natural pairing of light and heavy chains were obtained through data analysis. Ten antibody sequences were randomly selected, expressed and purified using a high-throughput antibody expression platform to obtain 10 antibodies, namely SH18-01 to SH18-10.

[0055] Example 2 Antibody Detection and Identification

[0056] 2.1 ELISA detection of antigen-binding activity of 10 antibodies

[0057] 1) Coat the microplate with 2 μg / mL Human TIGIT / his protein and incubate overnight at 4°C;

[0058] 2) Wash with 0.05% PBST and block with 1% BSA at room temperature for 2 hours; wash with 0.05% PBST and add 10 antibodies, positive control (Anti-Human TIGIT (Tiragolumab)), and isotype control (Anti-HEL HumanIgG1-Kappa Isotype) diluted 3-fold, respectively, and incubate at room temperature for 1 hour.

[0059] 3) Wash with 0.05% PBST, add Anti-human IgG (Fc specific) HRP (purchased from Sigma-Aldrich), and incubate at room temperature for 30 min;

[0060] 4) Wash with 0.05% PBST, add Beyotime TMB colorimetric solution, react at room temperature for 10 min, then add Beyotime TMB stop solution to immediately stop the reaction, and read OD450 immediately.

[0061] Figure 3 The results showed that the positivity rate of the 10 antibodies was 100% (10 / 10), with SH18-02 showing the strongest positivity.

[0062] 2.2 FACS detection of the cell-binding activity of 10 antibodies

[0063] 1) Adjust the CHOK1-hTIGIT-cell cell count to a concentration of 1x10e6 cells / ml and add 50 μL of cell suspension per well to a 96-well plate.

[0064] 2) Take the antibody to be tested, adjust the concentration of the first well to 100 nM, and perform a 5-fold serial dilution with MACS buffer.

[0065] 3) Add 50 μL of antibody dilution buffer to the cell suspension, mix thoroughly, and incubate at 4°C in the dark for 1 h.

[0066] 4) Wash twice with 200 μL MACS buffer.

[0067] 5) Preparation of fluorescent secondary antibody: Dilute with MACS buffer. Resuspend cells in 100 μL of fluorescent secondary antibody. Incubate at 4°C in the dark for 30 min.

[0068] 6) Wash twice with 200 μL MACS buffer.

[0069] 7) Resuspend the cells in 200 μL MACS buffer and perform analysis.

[0070] Figure 4 The results showed that all 10 antibodies bound to CHOK1-hTIGIT-cell overexpressing cells, with SH18-09 exhibiting the strongest binding activity.

[0071] Table 1. CDR amino acid sequence of anti-TIGIT monoclonal antibody SH18-09

[0072]

[0073] Table 2. Amino acid sequence of the variable region of the anti-TIGIT monoclonal antibody numbered SH18-09

[0074]

[0075] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An anti-human TIGIT monoclonal antibody, characterized in that, The antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes three heavy chain complementarity-determining regions, which are denoted by HCDR1, HCDR2 and HCDR3 respectively; the light chain variable region includes three light chain complementarity-determining regions, which are denoted by LCDR1, LCDR2 and LCDR3 respectively. The amino acid sequence of the heavy chain complementarity-determining region HCDR1 is shown in SEQ ID No:1, the amino acid sequence of the heavy chain complementarity-determining region HCDR2 is shown in SEQ ID No:2, and the amino acid sequence of the heavy chain complementarity-determining region HCDR3 is shown in SEQ ID No:

3. The amino acid sequence of the light chain complementarity-determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementarity-determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementarity-determining region LCDR3 is shown in SEQ ID No:

6.

2. The anti-human TIGIT monoclonal antibody according to claim 1, characterized in that: The antibodies include: The amino acid sequence is shown in the heavy chain variable region of SEQ ID NO. 7; and The amino acid sequence is shown in the light chain variable region of SEQ ID NO.

8.

3. A polynucleotide, characterized in that, The polynucleotide encodes the anti-human TIGIT monoclonal antibody as described in any one of claims 1-2.

4. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in claim 3.

5. A host cell, characterized in that, It contains the expression vector as described in claim 4.

6. The host cell as described in claim 5, characterized in that, The host cell is a mammalian cell.

7. A drug or drug composition, characterized in that, The drug or the drug composition comprises an anti-human TIGIT monoclonal antibody as described in any one of claims 1-2.

8. The use of the anti-human TIGIT monoclonal antibody as described in any one of claims 1-2 in the preparation of reagents for detecting TIGIT protein.

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