A tight junction protein antibody G4 and its application

Abstract

The present invention provides a tight junction protein antibody G4 and its application. The antibody G4 is a single-domain antibody, and the heavy chain variable region of the single-domain antibody includes CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 includes SEQ ID NO.1; the amino acid sequence of CDR2 includes SEQ ID NO.2; the amino acid sequence of CDR3 includes SEQ ID NO.3. The tight junction protein antibody G4 obtained in the present invention has a higher affinity for CLDN6 and a lower affinity for CLDN9, that is, it has higher specificity for CLDN6. At the same time, the tumor killing effects mediated by ADCC and CAR-T are better, and the chimeric antigen receptor molecule constructed based on it can better mediate the killing of tumors by immune cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a tight junction protein antibody G4 and its application. Background Art

[0002] Claudin 6 (CLDN6) is a four-transmembrane intercellular adhesion protein that participates in the formation of tight junctions around epidermal and endothelial cells. CLDN6 is highly expressed in tissues and organs such as the stomach, pancreas, lung, and kidney during fetal development, but is hardly expressed in the corresponding tissues and organs of adult organisms. However, studies have found that abnormal upregulation of CLDN6 expression can be detected in a variety of solid tumors, including breast cancer, endometrial cancer, ovarian cancer, testicular cancer, lung cancer, gastric cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, head and neck cancer, etc. In addition, CLDN6 also has the function of promoting tumorigenesis and development, and is associated with a poor prognosis of related cancers. For example, CLDN6 can enhance the tolerance of breast cancer to chemotherapeutic drugs through the GSTP1 and AF-6 / ERKs pathways, enhance the proliferation, migration and invasion abilities of liver cancer and endometrial cancer cells through the AKT / mTOR pathway, enhance the tolerance of liver cancer cells to chemotherapeutic drugs through ZO-2 / YAP1, and enhance the metastasis and invasion abilities of gastric cancer cells through the YAP1 / SNAIL pathway, etc.

[0003] CLDN6 has been considered as a therapeutic target with great application potential for a variety of solid tumors. Therapeutic monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies (BsAbs), and chimeric antigen receptor T (CAR-T) cells against CLDN6 have all been developed, and some drugs have entered clinical research.

[0004] CN106519035A discloses the use of an antibody that binds to CLDN6 to treat and / or prevent tumor diseases associated with cells expressing CLDN6, particularly cancer and cancer metastasis. It demonstrates that the binding of the antibody to CLDN6 on the surface of tumor cells is sufficient to inhibit tumor growth and sufficient to prolong the survival and lifespan of tumor patients. In addition, the binding of the antibody to CLDN6 effectively inhibits the growth of CLDN6-positive germ cell tumors (such as teratocarcinoma or embryonal carcinoma, particularly germ cell tumors of the testis).

[0005] CN101918450A discloses an antibody that binds to CLDN6 expressed on the cell membrane. The antibody of the present invention recognizes human CLDN6 existing in a natural form on the cell membrane and shows cytotoxicity generated by ADCC and / or CDC activities against cancer cell lines highly expressing human CLDN6. In addition, the antibody of the present invention shows an inhibitory effect on the growth of cancer cell lines highly expressing human CLDN6 by binding to toxins.

[0006] The above-mentioned prior arts are all developed based on antibodies targeting CLDN6. However, the extracellular domain structures of CLDN6 and CLDN9 are highly similar (both the first extracellular domain contains 53 amino acids, but there is only a difference at 1 amino acid site; both the second extracellular domain contains 22 amino acids, but there are only differences at 2 amino acid sites), resulting in the above-mentioned antibodies not being able to have both high affinity and high specificity for CLND6. Antibodies with high affinity for CLDN6 often can also significantly bind to CLDN9; antibodies that can effectively distinguish between CLDN6 and CLDN9 often have relatively weak affinity for CLDN6. Therefore, there is still room for improvement in the therapeutic effects of related drugs. In addition, the CLDN6 antibodies in the prior art are all murine / humanized antibodies. When applied to antibody drugs, their relatively large molecular weight is not conducive to improving the penetration rate of drugs into solid tumor lesions; when applied to cell drugs, they are usually expressed in the form of single-chain antibodies (scFv), and their relatively long coding sequences are not conducive to the construction and modification of expression vectors, and may also cause non-specific activation of cell drugs due to cross-pairing of VH and VL of adjacent scFv molecules. Therefore, CLDN6 antibodies with both high affinity and high specificity and small molecular weight have extremely high clinical application value and are crucial for improving the efficacy and safety of related tumor treatment drugs. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a tight junction protein antibody G4 and its application. To solve the problem that existing CLDN6 antibodies cannot have both affinity and specificity, the present invention screened and obtained a CLDN6 antibody with higher affinity, better specificity and smaller molecular weight than existing CLDN6 antibodies. At the same time, its mediated antibody-dependent cell-mediated cytotoxicity (ADCC) effect is better, and the CAR molecule constructed based on it can better mediate the killing of tumors by immune cells. The antibody of the present invention can improve the effectiveness of related antibody and cell drugs and has important application value in the development of tumor treatment drugs.

[0008] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a Claudin antibody G4, wherein the antibody G4 is a single-domain antibody, and the heavy-chain variable region of the single-domain antibody comprises CDR1, CDR2, and CDR3;

[0010] The amino acid sequence of the CDR1 comprises that shown in SEQ ID NO.1;

[0011] The amino acid sequence of the CDR2 comprises that shown in SEQ ID NO.2;

[0012] The amino acid sequence of the CDR3 comprises that shown in SEQ ID NO.3.

[0013] Preferably, the heavy-chain variable region sequence of the single-domain antibody comprises that shown in SEQ ID NO.4.

[0014] In a second aspect, the present invention provides a heavy-chain antibody, which comprises the heavy-chain variable region sequence described in the first aspect and the full-length or partial amino acid sequence of the crystallizable fragment (Fc) of human immunoglobulin (Ig).

[0015] Preferably, the Ig Fc comprises the full-length or partial sequence of the Fc segment of IgG1, IgG2, IgG3, or IgG4, or a combination thereof.

[0016] Preferably, the amino acid sequence of the IgG1 Fc comprises that shown in SEQ ID NO.5.

[0017] Preferably, the amino acid sequence of the heavy-chain antibody comprises that shown in SEQ ID NO.6.

[0018] The present invention immunizes alpacas with HEK293T cells overexpressing CLDN6. After determining the titer of CLDN6-specific antibodies in alpaca serum by ELISA and flow cytometry, alpaca peripheral blood mononuclear cells (PBMC) are isolated, RNA is extracted and reverse transcribed to obtain cDNA. Using alpaca single-domain antibody-specific primers, the VHH sequence is amplified and cloned into a phage expression plasmid to construct a phage display library. CHO-S cells overexpressing CLDN9 are incubated with the phage display library to eliminate antibody clones that bind to CLDN9 cells. Then, the phage display library is subjected to 3-4 rounds of enrichment panning with virus-like particles (VLP) containing CLDN6. Monoclonal clones are picked from the enrichment product, and the specific binding of the candidate antibodies to CLDN6-VLP is detected by ELISA. After the positive antibodies are expressed and purified, their affinities for CHO-S cells overexpressing CLDN6 or CLDN9 are detected respectively. Antibody clones with strong affinity for CLDN6 and weak affinity for CLDN9 are selected and co-cultured with NK cells and CLDN6-positive Huh-7 liver cancer cells to detect the antibody-dependent cell-mediated cytotoxicity (ADCC) of each clone. Meanwhile, the CLDN6 single-domain antibody sequence is cloned into a CAR expression vector to construct CAR-T cells with the CLDN6 single-domain antibody as the antigen-binding domain, and its killing ability against CLDN6-positive OVCAR3 cells in vitro and in vivo is detected. The results show that, compared with the prior art, the CLDN6 single-domain antibody obtained in the present invention has higher affinity for CLDN6 and lower affinity for CLDN9, that is, higher specificity for CLDN6, and at the same time, the tumor killing effects mediated by its ADCC and CAR-T are better.

[0019] In a third aspect, the present invention provides a nucleic acid molecule encoding the tight junction protein antibody G4 described in the first aspect or the heavy chain antibody described in the second aspect.

[0020] In a fourth aspect, the present invention provides an expression vector containing the nucleic acid molecule described in the third aspect; and after the expression vector is transfected / transduced / transformed into a host cell, the host cell expresses the tight junction protein antibody G4 described in the first aspect or the heavy chain antibody described in the second aspect.

[0021] In a fifth aspect, the present invention provides a host cell containing at least one copy of the expression vector described in the fourth aspect, or at least one copy of the nucleic acid molecule described in the third aspect.

[0022] In a sixth aspect, the present invention provides a composition for detecting claudin, the composition comprising the claudin antibody G4 described in the first aspect or the heavy chain antibody described in the second aspect.

[0023] In a seventh aspect, the present invention provides an anti-claudin chimeric antigen receptor, the chimeric antigen receptor recognizing claudin, and the chimeric antigen receptor being sequentially composed of the following structures in series: a signal peptide, the claudin antibody G4 described in the first aspect, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a human CD3 intracellular signal transduction domain.

[0024] Preferably, the signal peptide is selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα, TCRβ, or a combination thereof.

[0025] Preferably, the amino acid sequence of the signal peptide comprises that shown in SEQ ID NO.8.

[0026] Preferably, the hinge region is selected from the hinge regions of the following proteins: CD8, CD28, CD137, IgG1, IgG4, TCRα, TCRβ, or a combination thereof.

[0027] Preferably, the amino acid sequence of the hinge region comprises that shown in SEQ ID NO.9.

[0028] Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314, or a combination thereof.

[0029] Preferably, the amino acid sequence of the transmembrane domain comprises that shown in SEQ ID NO.10.

[0030] Preferably, the intracellular co-stimulatory domain is selected from the co-stimulatory domains of the following proteins: CD3ε, CD3γ, CD3δ, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (also known as CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 / CD11a / CD18, ICOS (also known as CD278), NKG2D, GITR, OX40L, or a combination thereof.

[0031] Preferably, the amino acid sequence of the intracellular co-stimulatory domain comprises that shown in SEQ ID NO.11.

[0032] Preferably, the human CD3 intracellular signal transduction domain is selected from the following proteins: CD3ζ, CD3ε, CD3γ, CD3δ, or a combination thereof.

[0033] Preferably, the amino acid sequence of the human CD3 intracellular signal transduction domain comprises that shown in SEQ ID NO.12.

[0034] Preferably, the amino acid sequence of the anti-occludin chimeric antigen receptor comprises that shown in SEQ ID NO.7.

[0035] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the anti-occludin chimeric antigen receptor described in the seventh aspect.

[0036] In a ninth aspect, the present invention provides an expression vector containing the nucleic acid molecule described in the eighth aspect; and after transfection / transduction / transformation of a host cell with the expression vector, the host cell expresses the anti-occludin chimeric antigen receptor described in the seventh aspect.

[0037] In a tenth aspect, the present invention provides a cell expressing an anti-occludin chimeric antigen receptor, which is obtained by transfection / transduction / transformation of a host cell with the expression vector described in the ninth aspect or the nucleic acid molecule described in the eighth aspect, and expresses the anti-occludin chimeric antigen receptor described in the seventh aspect.

[0038] Preferably, the host cell comprises a T cell.

[0039] In an eleventh aspect, the present invention provides a pharmaceutical composition comprising the cell expressing the anti-occludin chimeric antigen receptor described in the tenth aspect.

[0040] In a twelfth aspect, the present invention provides the use of any one or a combination of at least two of the occludin antibody G4 described in the first aspect, the heavy chain antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the host cell described in the fifth aspect, the composition for detecting occludin described in the sixth aspect, the anti-occludin chimeric antigen receptor described in the seventh aspect, the nucleic acid molecule described in the eighth aspect, the expression vector described in the ninth aspect, the cell expressing the anti-occludin chimeric antigen receptor described in the tenth aspect, or the pharmaceutical composition described in the eleventh aspect in the preparation of a drug for treating or detecting tumors.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The single-domain antibody of the present invention has a higher affinity for CLDN6, a lower affinity for CLDN9, and a smaller molecular weight. Based on it, the antibody drugs developed have better effectiveness, stronger tissue penetration, better in vivo stability, and better drug efficacy; based on it, the engineered immune cells developed have a higher exogenous gene expression level, better effectiveness, better drug efficacy, and lower modification difficulty; based on it, the detection reagents developed have higher sensitivity and lower false positives. Description of the Drawings

[0043] Figure 1 It is the detection result of the titer of 72# alpaca serum.

[0044] Figure 2 It is the detection result of the titer of 110# alpaca serum.

[0045] Figure 3 It is the amplification result of the VHH region of the alpaca antibody; among them, A is the heavy chain antibody fragment amplified by the first-round PCR, and B is the VHH region fragment amplified by the second-round PCR.

[0046] Figure 4 It is the result of detecting the specificity of the recombinant single-domain antibody by flow cytometry.

[0047] Figure 5 It is the result of detecting the EC 50 by flow cytometry.

[0048] Figure 6 It is the result of detecting the EC 50 by flow cytometry; among them, A is the concentration-effect curve detected with CHO-S-CLDN6 cells, and B is the concentration-effect curve detected with CHO-S-CLDN9 cells.

[0049] Figure 7 It is the result of detecting the binding of the recombinant single-domain antibody to ovarian cancer cells by IHC and flow cytometry.

[0050] Figure 8 It is the result of detecting the expression of CLDN6 in liver cancer cells by flow cytometry.

[0051] Figure 9 It is the IncuCyte real-time quantitative live cell imaging to analyze the ADCC function mediated by the recombinant single-domain antibody.

[0052] Figure 10 It is the structure of the CAR molecule targeting CLDN6 and its expression result in T cells; among them, A is the structure of the CAR molecule, and B is the expression of BVHCN6-006 in T cells detected by flow cytometry.

[0053] Figure 11It is to detect and analyze the function and specificity of IFN-γ secreted by CAR-T cells targeting CLDN6 by ELISA. Detailed implementation manners

[0054] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0055] For those not specifying specific techniques or conditions in the embodiments, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments not specifying the manufacturers, they are all conventional products that can be obtained through regular channels.

[0056] The sources of the following experimental materials are as follows:

[0057] HEK293T: ATCC, CRL-3216. CHO-S cells: Gibco, R80007. FITC-labeled Rabbit anti-Llama IgG(H+L) antibody: Invitrogen, A16155. anti M13-HRP antibody: SinoBiological, 11973-MM05T-H. PE-labeled anti-human IgG antibody: Biolegend, 410708. AF647-labeled Anti-human IgG antibody: Jackson ImmunoResearch, 109-606-170. Positive control antibody A-4: Santa Cruz, sc-393671. Anti-VHH antibody: GenScript, A02017. AF647-labeled anti-human CLDN6 antibody: Novus, FAB3656R. CLDN6-VLP: CUSABIO, CSB-MP005508HU(A4).

[0058] The sequences involved in the present invention and their numbers are as follows:

[0059] 89AVL amino acid sequence: SEQ ID NO.13.

[0060] 89A VH amino acid sequence: SEQ ID NO.14.

[0061] 72-2-C01 amino acid sequence: SEQ ID NO.15.

[0062] 72-2-E02 amino acid sequence: SEQ ID NO.16.

[0063] 72-1-G4 amino acid sequence: SEQ ID NO.4.

[0064] The amino acid sequence of CDR1 of 72-1-G4: SEQ ID NO.1.

[0065] The amino acid sequence of CDR2 of 72-1-G4: SEQ ID NO.2.

[0066] The amino acid sequence of CDR3 of 72-1-G4: SEQ ID NO.3.

[0067] The amino acid sequence of human IgG1 Fc: SEQ ID NO.5.

[0068] The amino acid sequence of the heavy chain antibody: SEQ ID NO.6.

[0069] The amino acid sequence of the anti-Claudin chimeric antigen receptor is shown in SEQ ID NO.7.

[0070] The amino acid sequence of human CD8 signal peptide: SEQ ID NO.8.

[0071] The amino acid sequence of human IgG4 hinge: SEQ ID NO.9.

[0072] The amino acid sequence of human CD8 transmembrane domain: SEQ ID NO.10.

[0073] The amino acid sequence of human 4-1BB intracellular domain: SEQ ID NO.11.

[0074] The amino acid sequence of human CD3ζ intracellular domain: SEQ ID NO.12.

[0075] Example 1: Alpaca immunization and antibody titer determination

[0076] (1) Construction of CLDN6- and CLDN9-overexpressing cells

[0077] According to the amino acid sequence information of human CLDN6 (UniProt Accession: P56747), a lentiviral expression vector was constructed and used to infect HEK293T and CHO-S cells to obtain the cell lines 293T-CLDN6 and CHO-S-CLDN6 overexpressing CLDN6, which were used for alpaca immunization, serum titer detection, and antibody affinity verification, respectively.

[0078] According to the amino acid sequence information of human CLDN9 (UniProt Accession: O95484), a lentiviral expression vector was constructed and used to infect CHO-S cells to obtain the cell line CHO-S-CLDN9 overexpressing CLDN9, which was used for antibody panning and affinity verification.

[0079] (2) Alpaca immunization and serum titer detection

[0080] Two alpacas (No. 72 and No. 110) were immunized with the above constructed 293T-CLDN6 cells, once every three weeks for a total of 5 times. 5 mL of peripheral blood was collected before each immunization. The centrifuge tube containing the blood sample was placed in a centrifuge and centrifuged at 800 g for 10 min. Then the supernatant was transferred to a new sterile centrifuge tube to collect the immune serum. The serum was diluted with PBS in gradient. 100 μL of the serially diluted serum (PBS was used instead in the control wells) and 2×10 5 CHO-S-CLDN6 cells or CHO-S cell lines were added to each well of a 96-well plate. After incubation at room temperature for 1 h, the plate was centrifuged at 800 g for 3 min, and the supernatant was discarded. The cells were washed 3 times with PBS. 100 μL of FITC-labeled Rabbit anti-Llama IgG (H+L) antibody (diluted 1:1000) was added to each well. After incubation at room temperature for 1 h, the plate was centrifuged at 800 g for 3 min, and the supernatant was discarded. The cells were washed 3 times with PBS and detected by flow cytometry.

[0081] The results are shown in Figure 1 、 Figure 2 、Table 1, Table 2, Table 3 and Table 4. Among them, Table 1 shows the flow cytometry detection results (expressed as MFI) of the serum of alpaca No. 72 after the third immunization; Table 2 shows the flow cytometry detection results (expressed as MFI) of the serum of alpaca No. 72 after the fourth immunization; Table 3 shows the flow cytometry detection results (expressed as MFI) of the serum of alpaca No. 110 after the third immunization; Table 4 shows the flow cytometry detection results (expressed as MFI) of the serum of alpaca No. 110 after the fourth immunization.

[0082] The mean fluorescence intensity (MFI) detected by flow cytometry after incubating the sera of the two alpacas before immunization with CHO-S cells and CHO-S-CLDN6 cells was similar, indicating that the binding ability of the two alpacas to the two types of cells before immunization was comparable, suggesting that the content of CLDN6-specific antibodies in them was extremely low. On the contrary, the binding ability of the sera after the third and fourth immunizations to CHO-S-CLDN6 cells was significantly higher than that to CHO-S cells, indicating that the content of CLDN6-specific antibodies in the sera of the two alpacas increased significantly after multiple immunizations, and the immunization was successful, which can be used for the construction of a phage surface display library.

[0083] Table 1

[0084]

[0085] Table 2

[0086]

[0087] Table 3

[0088]

[0089] Table 4

[0090]

[0091] Example 2: Construction of a single-domain antibody phage display library

[0092] (1) Cloning of VHH antibody fragments

[0093] After confirming that the sera of two alpacas contained CLDN6-specific antibodies, 100 mL of peripheral blood was collected, and PBMCs were isolated using lymphocyte separation medium, and RNA was extracted. Reverse transcription was performed using PrimeScript TM II 1 st Strand cDNA Synthesis Kit to obtain cDNA. Using PBMC cDNA as a template, the alpaca heavy chain antibody sequence was PCR-amplified with specific primers (the upstream primer binds to the signal peptide of the VHH antibody ORF, and the downstream primer binds to the CH2 region). The PCR products were analyzed by electrophoresis using 1% agarose, and the target fragment with a molecular weight of about 750 bp was recovered and separated ( Figure 3 in A). Then, using the first-round PCR product as a template, the heavy chain antibody VHH fragment was amplified with specific primers (the upstream primer binds to the antibody FR1 region, with an SfiI restriction site GGCCCAGCCGGCC at the 5' end, SEQ ID NO. 17; the downstream primer binds to the antibody Hinge and FR4 regions, with an SfiI restriction site GGCCACGAAGGCC at the 5' end, SEQ ID NO. 18). The PCR products were analyzed by electrophoresis using 1% agarose, and the target fragment with a molecular weight of about 400 bp was recovered and separated ( Figure 3 in B). The target fragment was stored at -80 °C (the storage solution contained 1 / 10 volume of 3 M sodium acetate, 1 μg / μL glycogen, and 80% absolute ethanol).

[0094] (2) Electroporation of the library vector and detection of library capacity and diversity

[0095] Digest the phage surface display vector pComf and the above-obtained VHH fragment library with SfiI endonuclease. Ligate the linearized pComf vector and the VHH fragment library overnight at 16°C using T4 ligase. Store the ligation product at -80°C (the storage solution contains 1 / 10 volume of 3M sodium acetate, 1 μg / μL glycogen, and 80% absolute ethanol). Add the mixture of 300 μL of the above ligation product and Escherichia coli SS320 competent cells to a pre-chilled electroporation cuvette, and transform the ligation product into Escherichia coli by electroporation (2500 V, 5 ms). Then, add 20 mL of SOC medium to resuspend the cells and incubate at 37°C on a shaker for 1 h. Take 15 mL of the bacterial solution for subsequent phage production and enrichment, and add an equal volume of 50% glycerol to the remaining 5 mL of the electroporation product. Mix well and store at -80°C. Additionally, take 20 μL of the bacterial solution, dilute it serially with 2YT medium, and spread it evenly on an LB plate containing ampicillin. Incubate at 37°C overnight, calculate the number of clones that can be generated from each ligation reaction, and obtain the capacity of the single-domain antibody phage surface display library. The results show that the library capacity of the single-domain antibody phage surface display library obtained from alpaca PBMC #72 is 1.33×10 9 , and the library capacity of the single-domain antibody phage surface display library obtained from alpaca PBMC #110 is 1.18×10 9 . Pick 20 monoclonal colonies on the plate and perform Sanger sequencing using the M13R primer. The results show that the phage library sequences have large differences, no repeated sequences, and good diversity.

[0096] (3) Enrichment of the phage surface display library

[0097] Take 15 mL of the bacterial solution after the above electroporation and resuscitation culture, dilute it with 2YT to adjust the OD 600 to about 0.25, add ampicillin with a final concentration of 100 μg / mL, and place it in a constant-temperature shaker. Incubate at 37°C on a shaker at 225 rpm. When the OD 600 reaches 0.6, add M13KO7 helper phage (the volume of M13KO7 helper phage added = 10 × the volume of the bacterial solution × OD 600 × 5 × 10 8 / M13KO7 titer), shake well and leave it standing at 37 °C for 30 min, then culture it on a shaker at 225 rpm at 37 °C for 1 h. After the helper phage completes the infection of the target strain, centrifuge at 6000 rpm for 10 min and discard the supernatant, resuspend it with 2YT-AK medium, and culture it overnight at 200 rpm on a shaker at 25 °C. Then centrifuge the bacterial solution at 10000 rpm for 15 min, transfer the supernatant containing phage particles to a new centrifuge tube (add PEG / NaCl with a volume of 1 / 5 of the bacterial solution volume into the tube), mix well and place it at 4 °C. After standing for 2 h, centrifuge at 10000 rpm for 30 min at 4 °C, collect the phage precipitate, and resuspend it with PBS with a volume of 1 / 50 of the original volume. Transfer the resuspended phage to a 1.5 mL EP tube, centrifuge at 12000 g for 5 min at 4 °C to remove insoluble impurities. Then transfer the supernatant to a new 1.5 mL EP tube, add 250 μL of PEG / NaCl to each tube, mix well and leave it standing at 4 °C for 10 min, then centrifuge at 12000 g for 10 min at 4 °C and discard the supernatant. After resuspending with 1 mL of PBS, centrifuge at 12000 g for 5 min at 4 °C, transfer the supernatant to a new 1.5 mL EP tube, and obtain the original library of single-domain antibody phage surface display.

[0098] Take 10 μL of the precipitate for 10-fold serial dilution, and add 200 μL of OD 600 ER2738 Escherichia coli with a value of 0.5, mix well and place it in a 37 °C water bath, leave it standing for 10 min, and then coat it on an LB plate. After culturing overnight at 37 °C, count the plaques to obtain the titer of the phage surface display library.

[0099] Example 3: Panning of the target antibody library

[0100] (1) First-round panning and product amplification

[0101] Block a 1.5 mL centrifuge tube with 3% MPBS and leave it overnight at 4 °C. Dilute the CLDN6-VLP antigen with CBS solution to 50 μg / mL, add it to a 96-well solid-phase plate, and coat it overnight at 4 °C. Take 1×10 7CHO-S-CLDN9 cells were washed 3 times with PBS, resuspended in 3% PBSA, and incubated at 37 °C for 1 h on a low-speed rotary mixer at 360°. Meanwhile, 150 μL of the precipitate of the original library of single-domain antibody phage display was taken, added with 350 μL of 1% PBSA, and incubated at 4 °C for 1 h on a low-speed rotary mixer at 360° as a premix. The blocked CHO-S-CLDN9 cells were centrifuged at 500 g for 10 min, the supernatant was removed, and the phage premix was added. The mixture was incubated at 4 °C for 1 h on a low-speed rotary mixer at 360° to remove phage clones that non-specifically bind to CLDN9. The CLDN6-VLP protein in the 96-well plate was discarded, and 200 μL of 3% MPBS buffer was added and left standing at room temperature. After blocking for 1 h, 3% MPBS buffer. The CHO-S-CLDN9 cells were centrifuged at 500 g for 10 min, the supernatant was taken and added to the wells with CLDN6-VLP protein respectively, and the mixture was incubated with shaking at room temperature for 1 h. The phage supernatant in the wells with CLDN6-VLP protein was discarded, washed 6 times with 0.05% PBST, and 4 times with PBS. 100 μL of Gly-HCl eluent with pH 2.2 was added to each well, and the mixture was incubated with shaking at 37 °C for 8 min. The specifically bound phages were eluted 2 more times. The obtained eluate was stored at 4 °C in a pre-blocked centrifuge tube (washed 2 times with PBS after blocking).

[0102] Take 20 mL of 2YT medium, add tetracycline at a final concentration of 100 μg / mL and 20 μL of Escherichia coli ER2738, and culture in an incubator at 37 °C and 225 rpm until the OD 600 reaches 0.5. The above-mentioned eluted phage product was added to the ER2738 bacterial solution, and after mixing, the mixture was incubated at 37 °C for 30 min. Then 20 mL of 2YT medium was added, and the culture was continued at 37 °C and 225 rpm for 30 min. When the OD of the bacterial solution 600 reaches 0.5 again, M13KO7 helper phage was added (the added volume of M13KO7 = 10 × the volume of the bacterial solution × OD 600 × 5 × 10 8 / M13KO7 titer), shake well and leave it standing at 37 °C for 30 min. Add ampicillin with a final concentration of 100 μg / mL to the bacterial solution, culture it at 37 °C at 225 rpm for 45 min, then centrifuge at 8000 rpm for 20 min, discard the supernatant and collect the bacteria. Resuspend with 40 mL of 2YT-AK medium and culture overnight at 30 °C at 210 rpm. Transfer the phage suspension that has been infected and amplified overnight to a 50 mL centrifuge tube, centrifuge at 8000 rpm at 4 °C for 30 min, then divide the supernatant into 40 mL centrifuge tubes. Add 10 mL of PEG / NaCl to each tube, mix well and place on ice. After standing for 1 h to precipitate the phages, centrifuge at 8000 rpm at 4 °C for 30 min. Discard the supernatant and resuspend the phages with 1 mL of sterile PBS, centrifuge at 12000 g at 4 °C for 5 min to remove insoluble impurities. Transfer 1 mL of the phage suspension to a new 1.5 mL centrifuge tube, add 250 μL of PEG / NaCl, mix well and leave it standing at 4 °C for 10 min to precipitate the phages. After centrifuging at 12000 g at 4 °C for 10 min, discard the supernatant. Resuspend the phages with 1 mL of PBS, centrifuge at 12000 g at 4 °C for 5 min to remove insoluble impurities. This is the phage product obtained from the first round of panning and amplification.

[0103] (2) The second to fourth rounds of panning and product amplification

[0104] Dilute the CLDN6-VLP antigen with CBS solution to 10 μg / mL, add it to a 96-well solid-phase plate, and coat it overnight at 4 °C. Use the phage product amplified from the first round of panning and perform the second to fourth rounds of panning in the same steps as above to obtain a phage library that specifically binds to CLDN6. The results are shown in Table 5 and Table 6. There is no significant difference in the enrichment index between the fourth round and the first round of the 72# alpaca single-domain antibody phage surface display library. The enrichment index of the fourth round of the 110# alpaca single-domain antibody phage surface display library decreased by about 27 times compared with the first round, indicating that the 110# alpaca single-domain antibody phage surface display library may contain more single-domain antibody clones specific to CLDN6. Table 5 shows the panning and enrichment results of the 72# alpaca single-domain antibody phage surface display library; Table 6 shows the panning and enrichment results of the 110# alpaca single-domain antibody phage surface display library.

[0105] Table 5

[0106]

[0107] Table 6

[0108]

[0109] (3) ELISA detection and sequencing of monoclonal phages

[0110] Infect Escherichia coli ER2738 with the single-domain antibody phage surface display library after the above multi-round panning. Mix well and place in a 37°C water bath, let stand for 10 min, then spread on an LB plate and incubate overnight at 37°C. Add 2YT-A medium to a 96-well deep well plate at 200 μL per well, pick monoclonal colonies from the plate, and culture overnight at 37°C at 225 rpm. Add 2YT-A medium to a 96-well deep well plate at 150 μL per well, add 20 μL of the above overnight cultured bacterial solution to each well, and culture at 37°C at 225 rpm until OD 600 is approximately 0.5. Add M13KO7 helper phage, mix well and let stand at 37°C for 15 min (M13KO7 volume = 10 × bacterial solution volume × OD 600 × 5 × 10 8 / M13KO7 titer), then culture at 37°C at 225 rpm for 45 min. Centrifuge at 3900 rpm for 10 min, discard the supernatant, resuspend each well with 500 μL of 2YT-AK medium, and culture overnight at 30°C at 220 rpm. Centrifuge at 3900 rpm for 10 min, and the obtained supernatant is the monoclonal phage particles.

[0111] While amplifying the phage monoclonal, coat the CLDN6-VLP antigen protein onto the ELISA plate with CBS at pH 9.6 (2 μg / mL, 100 μL / well). After coating overnight at 4°C, discard the antigen, wash three times with PBST, then add 250 μL of 3% MPBS to each well and block overnight at 4°C. After discarding the blocking solution, add 200 μL of 0.05% PBST to each well and wash 4 times, then add 50 μL of 0.1% PBST, and then add 50 μL of the above monoclonal phage supernatant in one-to-one correspondence. After incubating at 4°C for 1 h, wash 5 times with 0.05% PBST. Dilute the anti-M13-HRP antibody (1:5000) with 0.05% PBST and add 100 μL to each well, incubate at 4°C for 45 min. After washing 5 times with 0.05% PBST, add 100 μL of TMB and develop color at room temperature for 10 min, then add 50 μL of 0.2 M hydrochloric acid to terminate, and read OD 450 on the microplate reader. Calculate the value of sample / negative control, and select the clones with a ratio significantly greater than that of the positive serum control group for sequencing to obtain antibodies 72-2-C01, 72-2-E02, and 72-1-G4.

[0112] Example 4: Expression of recombinant single-domain antibody and detection of its binding to the target protein

[0113] The sequences of the candidate VHHs were amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.4, enabling their C-terminal fusion expression with the human IgG1 Fc fragment. After transient transfection of the obtained expression plasmid into HEK293 cells, the cell culture supernatants harvested contained the respective recombinant single-domain antibodies. In the same manner, the CLDN6-specific antibody 89A was expressed as a positive control.

[0114] The binding specificities of the respective recombinant single-domain antibodies were detected. The culture supernatants containing the respective recombinant single-domain antibodies were incubated with 3×10 5 CHO-S, CHO-S-CLDN6, or CHO-S-CLDN9 cells at room temperature for 1 hour. After centrifugation at 800 g for 5 min at room temperature, the supernatant was discarded, and the cells were washed 3 times with PBS. 100 μL of PE-labeled anti-human IgG antibody (diluted 1:500) was added, and the mixture was incubated in the dark at room temperature for 45 min. After centrifugation at 800 g for 5 min at room temperature, the supernatant was discarded, and the cells were washed 3 times with PBS. The cells were resuspended in 500 μL of PBS for flow cytometry analysis.

[0115] The results were as Figure 4 shown. The expression supernatants of the negative control group did not show significant binding to either CHO-S-CLDN6 or CHO-S-CLDN9; the positive control antibody 89A, and the recombinant single-domain antibodies 72-2-C01 and 72-2-E02 showed significant binding to both CHO-S-CLDN6 and CHO-S-CLDN9, and their binding levels to CHO-S-CLDN9 cells were slightly weaker than those to CHO-S-CLDN6 cells, indicating that these antibodies could bind CLDN6 but had poor specificity. In contrast, 72-1-G4 showed strong binding to CHO-S-CLDN6 cells and almost no binding to CHO-S-CLDN9 cells, indicating that its specificity for CLDN6 was higher than that of other antibodies.

[0116] Example 5: Purification of the recombinant antibody and determination of the half-maximal effective concentration (EC 50 )

[0117] To determine the EC 50, transiently transfect the relevant expression plasmid into 293F cells and culture them with shaking in a flask for antibody expression and purification. Since the target recombinant antibody contains human IgG fragments, Protein A magnetic beads can be used for affinity purification. Wash the Protein A magnetic beads twice with 30 mL of PBS buffer, 0.1 M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads to the 293F cell flask according to the sample demand (calculated as 20 mg IgG / mL Protein A magnetic beads). Incubate at 120 rpm at room temperature for 1 - 4 h or overnight at 4 °C in an orbital shaker incubator. Collect the Protein A magnetic beads with a magnetic separator and transfer them to a 50 mL centrifuge tube. Wash twice with 30 mL of PBS buffer and deionized water respectively, and then resuspend with 1 mL of elution buffer. Incubate at room temperature for 5 min, collect the magnetic beads with a magnetic separation rack, and transfer the supernatant containing the target antibody to a 15 mL centrifuge tube. Repeat the elution of the Protein A magnetic beads twice, combine the eluates, and add neutralization buffer to adjust the pH of the solution. Dialyze the eluted sample with PBS at least 100 times the volume of the sample. First, dialyze at 18 - 25 °C for 2 h, change the solution once, and then dialyze at 2 - 8 °C for 14 - 16 h. Finally, measure the protein concentration, filter the sample with a 0.22 μm sterile filter membrane, aliquot, and store at -80 °C in the refrigerator for later use.

[0118] Gradiently dilute the target antibody and incubate it with 3×10 5 CHO-S-CLDN6 and CHO-S-CLDN9 cells at room temperature for 1 h. Centrifuge at 800 g at room temperature for 5 min. After discarding the supernatant containing the antibody, wash the cells 3 times with PBS. Add 100 μL of PE-labeled Anti-human IgG antibody (diluted 1:500), mix well, and incubate at room temperature in the dark for 45 min. Centrifuge at 800 g at room temperature for 5 min, discard the supernatant containing the antibody, wash the cells 3 times with PBS, and then resuspend the cells with 500 μL of PBS for flow cytometry analysis.

[0119] The results are as Figure 5 , Figure 6 and Table 7 show. Table 7 is the EC 50 result of detecting the recombinant single-domain antibody by flow cytometry. The EC 50 value of the positive control antibody 89A (PC in the table) for CHO-S-CLDN6 is 1.26 μg / mL, and the EC 50 value of 72-1-G4 for CHO-S-CLDN6 is 0.3009 μg / mL, which is 76.1% lower than the EC 50 value of the positive antibody, indicating that 72-1-G4 has a higher affinity for CLDN6. The EC 50The value was 14.49 μg / mL, while the EC of 72-1-G4 against CHO-S-CLDN9 50 The value was 13.77 μg / mL, indicating that the affinity of 72-1-G4 for CLDN9 was similar to that of the positive control antibody. The above results show that, compared with the prior art, 72-1-G4 has higher affinity and specificity for CLDN6.

[0120] Table 7

[0121]

[0122] Example 6: Detection of CLDN6 expression in tumor cells

[0123] (1) Detection of expression in ovarian cancer cells

[0124] The positive control antibody A-4 is an antibody that binds to the intracellular domain of CLDN6 and does not bind to CLDN9, and can effectively distinguish CLDN6 and CLDN9. The expression of CLDN6 in ovarian cancer cells (OVCAR-3, SK-OV-3, and CAOV-3 cells overexpressing firefly luciferase and red fluorescent protein, respectively) was detected by immunohistochemistry (IHC) using the A-4 antibody. The results are as Figure 7 shown. Only OVCAR-3-Luc-mCherry cells were positive for CLDN6, and SK-OV-3-Luc-mCherry and CAOV-3-Luc-mCherry cells were negative for CLDN6. The above three cells were incubated with the 72-1-G4 antibody at room temperature for 1 h. After centrifugation at 800 g for 5 min at room temperature, the supernatant containing the antibody was discarded, and the cells were washed 3 times with PBS. 100 μL of AF647-labeled Anti-human IgG antibody was added, and after thorough mixing, the cells were incubated in the dark at room temperature for 45 min. After centrifugation at 800 g for 5 min at room temperature, the supernatant containing the antibody was discarded, and after washing the cells 3 times with PBS, the cells were resuspended in 200 μL of PBS for flow cytometry analysis. As Figure 7 shown, the 72-1-G4 antibody could only detect significant expression of CLDN6 in OVCAR-3-Luc-mCherry cells, indicating that the 72-1-G4 antibody also has good affinity and specificity for CLDN6 in tumor cells.

[0125] (2) Detection of expression in liver cancer cells

[0126] The anti-human CLDN6 antibody labeled with AF647 was incubated with the liver cancer cell line HuH-7-Luc-mCherry overexpressing firefly luciferase and red fluorescent protein for 1 h. After centrifugation at 800 g for 5 min and discarding the supernatant containing the antibody, the cells were washed three times with PBS. The cells were resuspended in 200 μL of PBS for flow cytometry analysis. As Figure 8 shown, CLDN6 was significantly expressed in HuH-7-Luc-mCherry cells, indicating that this liver cancer cell line can be used for subsequent functional evaluation experiments.

[0127] Example 7: Detection of ADCC function

[0128] HuH7-Luc-Mcherry cells were selected to detect the ADCC function of the target antibody. After washing the target cells three times with OptiVitro NK cell expansion medium, the target cells were seeded in 96-well plates at a density of 1×10 4 / well and cultured overnight. NK cells were added at an effector-to-target ratio of 8:1, and the CLDN6 positive control antibody 89A or the target antibody 72-1-G4 at a final concentration of 5 μg / mL was added simultaneously.

[0129] Detection was performed using the real-time quantitative live cell imaging and analysis platform IncuCyte. The fluorescence signal changes of the target cells in each group were calculated by IncuCyte SX5 software. The lower the signal value, the fewer the cells in that group, indicating better killing effect of NK cells. The results are as Figure 9 shown. Compared with the NK cell group without antibody addition, both the 72-1-G4 antibody and the positive control antibody could significantly improve the killing effect of NK cells on tumor cells, indicating that 72-1-G4 has a good function of mediating ADCC.

[0130] Example 8: Construction of CAR-T cells based on the target antibody

[0131] (1) Design of CAR molecule

[0132] The target gene structure of the lentiviral vector involved in this example is as Figure 10 shown in A.

[0133] BVHCN6-006 is composed of the following structures in series: human CD8 signal peptide (abbreviated as SP), CLDN6 antibody G4 [72-1-G4 abbreviated as VHH(72-1-G4)], human IgG4 hinge region (abbreviated as IgG4 hinge), human CD8 transmembrane domain (abbreviated as CD8 TM), human 4-1BB intracellular co-stimulatory domain (abbreviated as 4-1BB ICD), and human CD3ζ intracellular signal transduction domain (abbreviated as CD3ζICD).

[0134] (2) Lentivirus preparation

[0135] The above-mentioned CAR molecule expression sequence was synthesized by gene synthesis and then ligated into the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning to express it under the regulation of the human EF-1α promoter and Kozak sequence. Using the transfection reagent Lipofectamine 3000, the above lentiviral vector expression plasmids were co-transfected into 293T cells with the lentiviral packaging plasmids pRSV-Rev, pMDLg / pRRE, and pMD2.G according to the relevant instructions. The viral supernatant was collected 48 h after transfection, centrifuged at 3000 rpm for 10 - 15 min at 4°C, filtered through a 0.45 μm pore size filter membrane, and finally ultracentrifuged at 25000 rpm for 2 - 3 h at 4°C. The obtained virus concentrate was stored at -80°C and named BVHCN6-006. Finally, using Jurkat cells as the material, the activity titer of the above lentivirus was detected.

[0136] (3) CAR-T cell preparation

[0137] The PBMCs of healthy donors were resuscitated in AIM V medium, added with 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, and 300 IU / mL recombinant hIL-2, and cultured in a cell incubator for 24 h (culture temperature was 37°C, carbon dioxide concentration was 5%). The obtained T cells were washed, and lentivirus was added for transduction at a dosage of MOI = 5 TU / mL. At the same time, 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody, and 300 IU / mL recombinant hIL-2 were supplemented, and the cells were cultured in a cell incubator (culture temperature was 37°C, carbon dioxide concentration was 5%). After 24 h, the cell density was adjusted to (1.5 - 2)×10 6 / mL, and 300 IU / mL of hIL-2 was supplemented. On the 4th day after transduction, the cells were washed to remove the residual lentivirus particles in the supernatant and continued to be cultured in a cell incubator for 5 days (culture temperature was 37°C, carbon dioxide concentration was 5%), and the cell density was maintained at (1 - 2)×10 6 / mL during this period. The cells were harvested on the 10th day after transduction and stored in liquid nitrogen for later use. The obtained CAR-T cells were named according to the corresponding CAR molecule, and the T cells not transduced with lentivirus were named Ctrl T.

[0138] (4) Detection of CAR molecule expression

[0139] The BVHCN6-006 CAR-T cells to be detected were washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). The FITC-labeled anti-VHH antibody was incubated with the CAR-T cells for 1 h according to the antibody instruction manual. Subsequently, the supernatant was removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Ctrl T cells as a negative control, the CAR molecule expression rate of BVHCN6-006 cells was detected by flow cytometry. The results are as Figure 10 shown in B, the expression rate of BVHCN6-006 CAR was 66.8%.

[0140] Example 9: Study on the function of CAR-T cells

[0141] To study the killing function and specificity of BVHCN6-006 cells, 293T cells, OVCAR-3-Luc-mCherry cells, SK-OV-3-Luc-mCherry and CAOV-3-Luc-mCherry cells were used as target cells, and Ctrl T cells and BVHCN6-006 cells were added respectively. After overnight co-culture, the culture supernatant was collected, and the content of IFN-γ was detected by ELISA. The results are as Figure 11 shown, BVHCN6-006 was significantly activated only by CLDN6-positive OVCAR-3-Luc-mCherry cells and released a high level of IFN-γ. The activation of BVHCN6-006 by CLDN6-negative target cells was significantly reduced (SK-OV-3-Luc-mCherry cells), or even hardly activated (CAOV-3-Luc-mCherry cells). This result further shows that the CAR-T cells constructed based on the 72-1-G4 antibody have good specificity for the killing function of CLDN6-positive tumor cells.

[0142] In summary, the tight junction protein antibody G4 described in the present invention has a higher affinity for CLDN6 and a lower affinity for CLDN9, that is, it has higher specificity for CLDN6. At the same time, its mediated antibody-dependent cell-mediated cytotoxicity is better. The chimeric antigen receptor molecule constructed based on it can better mediate the killing of tumors by immune cells.

[0143] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. An anti-CLDN6 antibody G4, characterized in that, The antibody G4 is a single-domain antibody, and the variable region of the heavy chain of the single-domain antibody comprises CDR1, CDR2 and CDR3; The amino acid sequence of the CDR1 is as shown in SEQ ID NO.1; The amino acid sequence of the CDR2 is as shown in SEQ ID NO.2; The amino acid sequence of the CDR3 is as shown in SEQ ID NO.

3.

2. The anti-CLDN6 antibody G4 according to claim 1, wherein The variable region sequence of the heavy chain of the single-domain antibody comprises that shown in SEQ ID NO.

4.

3. A chimeric heavy chain antibody against CLDN6, characterized in that, The chimeric heavy chain antibody comprises the variable region sequence of the heavy chain as shown in SEQ ID NO.4 and the full-length or partial amino acid sequence of the crystallizable fragment Ig Fc of human immunoglobulin.

4. The chimeric heavy chain antibody against CLDN6 according to claim 3, characterized in that, The Ig Fc comprises the full-length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

5. The anti-CLDN6 chimeric heavy chain antibody according to claim 4, characterized in that, The amino acid sequence of the IgG1 Fc is as shown in SEQ ID NO.

5.

6. The chimeric heavy chain antibody against CLDN6 according to claim 3, characterized in that, The amino acid sequence of the chimeric heavy chain antibody is as shown in SEQ ID NO.

6.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CLDN6 antibody G4 as claimed in claim 1 or 2 or the anti-CLDN6 chimeric heavy chain antibody as claimed in any one of claims 3-6.

8. An expression vector, characterized in that, The expression vector contains the nucleic acid molecule as claimed in claim 7; and after transfection / transduction / transformation of the host cell, the host cell expresses the anti-CLDN6 antibody G4 as claimed in claim 1 or 2 or the anti-CLDN6 chimeric heavy chain antibody as claimed in any one of claims 3-6.

9. A host cell, characterized in that, The host cell contains at least one copy of the expression vector as claimed in claim 8 or at least one copy of the nucleic acid molecule as claimed in claim 7.

10. A composition for detecting CLDN6, characterized in that, The composition comprises the anti-CLDN6 antibody G4 as claimed in claim 1 or 2 or the anti-CLDN6 chimeric heavy chain antibody as claimed in any one of claims 3-6.

11. An anti-CLDN6 chimeric antigen receptor, characterized in that, The chimeric antigen receptor recognizes claudin, and the chimeric antigen receptor is composed of the following structures in series: a signal peptide, the anti-CLDN6 antibody G4 as claimed in claim 1 or 2, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signal transduction domain of human CD3.

12. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein The signal peptide is selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, IgG, IgE, TCRα, TCRβ.

13. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein The amino acid sequence of the signal peptide is as shown in SEQ ID NO.

8.

14. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein, The hinge region is selected from the hinge regions of the following proteins: CD8, CD28, CD137, IgG1, IgG4, TCRα, TCRβ.

15. The anti-CLDN6 chimeric antigen receptor according to claim 11, characterized in that, The amino acid sequence of the hinge region is as shown in SEQ ID NO.

9.

16. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein The transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD3ε, CD3ζ, CD3γ, CD3δ, TCRα, TCRβ, CD40, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, CD314.

17. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein, The amino acid sequence of the transmembrane domain includes that shown in SEQ ID NO.

10.

18. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein The intracellular co-stimulatory domain is selected from the co-stimulatory domains of the following proteins: CD3ε, CD3γ, CD3δ, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB, PD1, Dap10, ICAM-1, LFA-1 / CD11a / CD18, ICOS, NKG2D, GITR, OX40L, or a combination thereof.

19. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein The amino acid sequence of the intracellular co-stimulatory domain includes that shown in SEQ ID NO.

11.

20. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein The human CD3 intracellular signal transduction domain is selected from the following proteins: CD3ζ, CD3ε, CD3γ, CD3δ.

21. The anti-CLDN6 chimeric antigen receptor according to claim 11, characterized in that, The amino acid sequence of the human CD3 intracellular signal transduction domain includes that shown in SEQ ID NO.

12.

22. The anti-CLDN6 chimeric antigen receptor according to claim 11, wherein The amino acid sequence of the anti-CLDN6 chimeric antigen receptor includes that shown in SEQ ID NO.

7.

23. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CLDN6 chimeric antigen receptor according to any one of claims 11-22.

24. An expression vector, characterized in that, The expression vector contains the nucleic acid molecule according to claim 23; and after transfection / transduction / transformation of the host cell, the host cell expresses the anti-CLDN6 chimeric antigen receptor according to any one of claims 11-22.

25. A cell expressing a chimeric antigen receptor against CLDN6, characterized in that, The cell is obtained by transfecting / transducing / transforming The host cell with the expression vector according to claim 24 or the nucleic acid molecule according to claim 23, and expresses the anti-CLDN6 chimeric antigen receptor according to any one of claims 11-22.

26. The cell expressing the chimeric antigen receptor against CLDN6 according to claim 25, wherein The host cell includes T cells.

27. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the cell expressing the anti-CLDN6 chimeric antigen receptor according to claim 25 or 26.

28. The use of any one or a combination of at least two of the anti-CLDN6 antibody G4 according to claim 1 or 2, the anti-CLDN6 chimeric heavy chain antibody according to any one of claims 3-6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the composition for detecting CLDN6 according to claim 10, the anti-CLDN6 chimeric antigen receptor according to any one of claims 11-22, the nucleic acid molecule according to claim 23, the expression vector according to claim 24, the cell expressing the anti-CLDN6 chimeric antigen receptor according to claim 25 or 26, or the pharmaceutical composition according to claim 27 in the preparation of a drug for treating or detecting ovarian cancer or liver cancer.

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