Anti-human TIGIT monoclonal antibody and application thereof

Abstract

The invention 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 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 of which the amino acid sequences are as shown in SEQ ID NO: 1-3; the light chain variable region comprises a VLCDR1, a VLCDR2 and a VLCDR3 of which the amino acid sequences are shown as SEQ ID NO: 4-6. The invention also provides an application of the anti-human TIGIT monoclonal antibody in tumor immunotherapy, preparation of a reagent for detecting the TIGIT protein or preparation of a product combined with the human TIGIT protein. The anti-human TIGIT monoclonal antibody has the technical effects that the novel anti-human TIGIT monoclonal antibody is obtained through screening, and the antigen binding activity of the anti-human TIGIT monoclonal antibody is better than that of the existing anti-human TIGIT monoclonal antibody. According to the antibody development method provided by the invention, the development time of the target antibody is shortened, the flux is high, the positive rate is high, and the antigen binding activity is better.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to an anti-human TIGIT monoclonal antibody and an application thereof. Background Art

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

[0003] Studies have shown that TIGIT has multiple ligands and can compete with other inhibitory and stimulatory receptors or share the same ligands. CD155 (PVR / NECL-5) has a high affinity for TIGIT, as well as the co-stimulatory receptor CD226 expressed on T cells and NK cells. After TIGIT binds to the ligands CD155 and CD122, it inhibits the activation of T cells and NK cells and inhibits the anti-tumor immune response. When TIGIT binds to the ligand PVR / PVRL2 on tumor cells, it transmits through the signal pathway and inhibits the immune killing activity of CD8+T cells or NK cells, thereby promoting tumor cell escape. After TIGIT is blocked, the function of NK cells can be restored, Treg cells can be reduced, the response of antigen-specific CD8 memory cells can be increased, and the generation of new antigen-specific CD8+T cells can be induced. Therefore, anti-TIGIT monoclonal antibodies play a key role in limiting the adaptive immunity and innate immunity of tumors.

[0004] In recent years, the research on TIGIT in immune-related fields such as tumor immunotherapy and autoimmune diseases has gradually increased, but most TIGIT antibodies on the market currently have shortcomings such as weak competitive activity, low flux, and low positive rate.

[0005] In view of this, there is a need to provide a new anti-human TIGIT monoclonal antibody. Summary of the invention

[0006] The object of the present invention is to provide an anti-human TIGIT monoclonal antibody and its application to solve one or more of the above-mentioned prior art problems.

[0007] The technical solution of the present 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 comprises three heavy chain complementary determining regions, the three heavy chain complementary determining regions are represented by HCDR1, HCDR2 and HCDR3 respectively; the light chain variable region comprises three light chain complementary determining regions, the three light chain complementary determining regions are represented by LCDR1, LCDR2 and LCDR3 respectively;

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

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

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

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

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

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

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

[0019] In the fourth aspect of the present invention, a host cell containing the vector described in the third aspect is also provided. Preferably, the host cell is a mammalian cell.

[0020] Furthermore, the mammalian cell is one of HEK-293 cells and CHO cells.

[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, expression of Human TIGIT / His Protein;

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

[0024] S3, screening positive antibody-secreting cells, constructing antibody library and sequencing;

[0025] S4, screening and extracting antibody sequences, and cloning them into vectors;

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

[0027] S6. Antibody testing.

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

[0029] In the seventh aspect of the present invention, there is also provided the use of the anti-human TIGIT monoclonal antibody as described in the first aspect in the preparation of tumor immunotherapy drugs, TIGIT protein detection reagents, or the preparation of products binding to human TIGIT protein.

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

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

[0032] 2) The antibody development method provided by the present 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results are shown in Example 1, in which the serum titer was detected after animal immunization.

[0034] Figure 2 The results of flow cytometry.

[0035] Figure 3Antigen binding test results.

[0036] Figure 4 Results of cell binding assay. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below with reference to the examples. However, the present invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the 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] According to the sequence information of Human TIGIT (Gene ID: 201633), the antigen was expressed and His-tag was connected 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 by gene recombination, and the psPAX2 plasmid, PMD2G plasmid, pLVX-IRES-puro-hTIGIT plasmid and transfection reagent PEI were co-transfected into 293T cells. The supernatant of lentiviral particles was collected and filtered after 72 hours. The lentiviral particles were inoculated into CHOK1 cells, and 1 / 100 volume of F108 infection enhancement solution was added. After 6-8 hours, the culture medium was discarded and replaced with fresh medium. After 48 hours, the culture was expanded and 10 μg / mL puromycin was added for resistance screening. The cells that could continue to grow and be passaged were CHOK1-hTIGIT-cells, which were subsequently used for antibody expression 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 at multiple points on the abdomen and back of mice for primary immunization. On the 10th day, the fluorescently labeled Human TIGIT / His protein obtained in step (1) was used again for the second immunization. After 3 days, blood was collected to detect the anti-human TIGIT / His serum titer; after the serum titer was qualified, the spleen and bone marrow cells of the mice were collected for single cell screening.

[0047] The titer of Anti-Human TIGIT / His Protein serum was detected by ELISA. The detection process was as follows: the ELISA plate was coated with 2μg / mL Human TIGIT / his protein and incubated at 37℃ for 2h; washed with 0.05% PBST and blocked with 1% BSA at room temperature for 2h; washed with 0.05% PBST, and 3-fold gradient dilutions of mouse sera before and after immunization were added, with the first two wells being 100 and 1000-fold dilutions, and incubated at room temperature for 1h; washed with 0.05% PBST, and Anti-mouse IgG (Fc specific) HRP (purchased from Sigma-Aldrich) was added, and incubated at room temperature for 30min; washed with 0.05% PBST, and Bio-Tec TMB colorimetric solution was added, reacted at room temperature for 10min, and then Bio-Tec TMB stop solution was added to immediately stop the reaction, and OD450 was read immediately. The results are as follows Figure 1 As shown, the titer of the antiserum after 2 immunizations was 656100. This shows that the antigen can induce mice to produce high-titer antiserum specific to Human TIGIT / HisProtein.

[0048] 1.4 Isolation of spleen and bone marrow cells

[0049] The spleen and bone marrow cells of the immunized mice were collected, centrifuged at 400 g for 5 min, resuspended in 1 mL of red blood cell lysis buffer, and the red blood cell lysis was terminated by a large volume of Macs buffer. The cells were centrifuged at 400 g for 5 min, and resuspended in 1 mL of Macs buffer for later use.

[0050] 1.5 B cell sorting

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

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

[0053] 1.6 Single cell library construction

[0054] The positive mouse cells sorted were constructed using a single-cell library construction platform, and the constructed library was sequenced by the next-generation sequencing. Antibodies with natural pairings 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] 2.1ELISA to detect antigen binding activity of 10 antibodies

[0057] 1) ELISA plate was coated with 2 μg / mL Human TIGIT / his protein at 4°C overnight;

[0058] 2) Wash with 0.05% PBST, block with 1% BSA at room temperature for 2 h; wash with 0.05% PBST, add 10 antibodies diluted 3 times, positive control (Anti-Human TIGIT (Tiragolumab)), isotype control (Anti-HELHumanIgG1-Kappa Isotype), and incubate at room temperature for 1 h;

[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 minutes;

[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 terminate the reaction, and read OD450 immediately.

[0061] Figure 3 The results showed that the positive rate of the 10 antibodies was 100% (10 / 10), among which SH18-02 was the most positive.

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

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

[0064] 2) Take the antibody to be tested, adjust the concentration of the first well to 100 nM, and make a 5-fold gradient 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) Prepare fluorescent secondary antibody: dilute with MACS buffer. Resuspend cells with 100 μL 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 with 200 μL MACS buffer and detect on the instrument.

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

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

[0072]

[0073] Table 2. Amino acid sequences of variable regions of anti-TIGIT monoclonal antibodies numbered SH18-09

[0074]

[0075] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An anti-human TIGIT monoclonal antibody, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three heavy chain complementary determining regions, which are represented by HCDR1, HCDR2 and HCDR3 respectively; the light chain variable region comprises three light chain complementary determining regions, which are represented by LCDR1, LCDR2 and LCDR3 respectively; The amino acid sequence of the heavy chain complementary determining region HCDR1 is shown in SEQ ID No: 1, the amino acid sequence of the heavy chain complementary determining region HCDR2 is shown in SEQ ID No: 2, and the amino acid sequence of the heavy chain complementary determining region HCDR3 is shown in SEQ ID No: 3; The amino acid sequence of the light chain complementary determining region LCDR1 is shown in SEQ ID No:4, the amino acid sequence of the light chain complementary determining region LCDR2 is shown in SEQ ID No:5, and the amino acid sequence of the light chain complementary 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: 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 b. A light chain variable region having an amino acid sequence that is at least 90% homologous to the sequence of SEQ ID NO.

8.

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

8.

4. A polynucleotide sequence or combination, characterized in that: The polynucleotide sequence or combination encodes the anti-human TIGIT monoclonal antibody according to any one of claims 1 to 3.

5. A cloning or expression vector, characterized in that: The cloning or expression vector comprises the polynucleotide sequence or combination as claimed in claim 4.

6. A host cell, characterized in that Containing the cloning or expression vector according to claim 5.

7. The host cell according to claim 6, characterized in that The host cell is a mammalian cell.

8. The method for preparing an anti-human TIGIT monoclonal antibody according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, expression of Human TIGIT / His Protein; S2, animal preparation and immunization, collection of spleen and bone marrow cells; S3, screening positive antibody-secreting cells, constructing antibody library and sequencing; S4, screening and extracting antibody sequences, and cloning them into vectors; S5, high-throughput expression of anti-Human TIGIT antibody; S6. Antibody testing.

9. A drug or a pharmaceutical composition, characterized in that The drug or the pharmaceutical composition comprises the anti-human TIGIT monoclonal antibody or related biological material as described in any one of claims 1 to 6.

10. Use of the anti-human TIGIT monoclonal antibody according to any one of claims 1 to 3 in the preparation of tumor immunotherapy drugs, TIGIT protein detection reagents, or products binding to human TIGIT protein.

Images