An anti-human CLDN6 single-domain antibody and its application

Abstract

The present invention provides an anti-human CLDN6 single-domain antibody and its application. The heavy chain variable region of the single-domain antibody includes CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 includes SEQ ID NO.1; the amino acid sequence of the CDR2 includes SEQ ID NO.2; the amino acid sequence of the CDR3 includes SEQ ID NO.3. The anti-human CLDN6 single-domain antibody obtained by the present invention has a higher affinity for CLDN6 and a lower affinity for CLDN9, that is, it has a higher specificity for CLDN6, and at the same time, it mediates better antibody-dependent cell-mediated cytotoxicity, and the chimeric antigen receptor molecule constructed based on it can better mediate immune cells to kill tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-human CLDN6 single-domain antibody and an application thereof. 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 the stomach, pancreas, lungs, kidneys and other tissues and organs during the fetal period, but is almost not expressed in the corresponding tissues and organs in the adult body. 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, bile duct cancer, colorectal cancer, esophageal cancer and head and neck cancer. In addition, CLDN6 also has the function of promoting the occurrence and development of tumors, which is associated with the poor prognosis of related cancers. For example, CLDN6 can enhance the tolerance of breast cancer to chemotherapy drugs through the GSTP1 and AF-6 / ERKs pathways, enhance the proliferation, migration and invasion of liver cancer and endometrial cancer cells through the AKT / mTOR pathway, enhance the tolerance of liver cancer cells to chemotherapy drugs through ZO-2 / YAP1, and enhance the metastasis and invasion 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 targeting CLDN6 have all been developed, and some drugs have been put into clinical research. The above-mentioned existing technologies are all developed based on antibodies targeting CLDN6. However, the extracellular domain structures of CLDN6 and CLDN9 are extremely similar (the first extracellular domain of both contains 53aa, but differs only at one amino acid site; the second extracellular domain contains 22aa, but differs only at two amino acid sites), resulting in the above-mentioned CLDN6 antibodies or drugs developed based on CLDN6 antibodies not having both high affinity and high specificity for CLDN6. Antibodies with high affinity for CLDN6 can often also significantly bind to CLDN9; antibodies that can effectively distinguish CLDN6 from CLDN9 often have weaker affinity for CLDN6. Therefore, the therapeutic effect of related drugs still has room for improvement. In addition, the CLDN6 antibodies in the prior art are all mouse / humanized antibodies. When applied to antibody drugs, their larger molecular weight is not conducive to improving the drug's penetration into solid tumor lesions; when applied to cell drugs, they are usually expressed in the form of single-chain antibodies (scFv), and their longer coding sequences are not conducive to the construction and modification of expression vectors. They may also cause non-specific activation of cell drugs due to cross-pairing of VH and VL adjacent to scFv molecules. Therefore, CLDN6 antibodies with both high affinity and high specificity and small molecular weight have extremely high clinical application value, which is of key significance to improving the efficacy and safety of related tumor treatment drugs. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide an anti-human CLDN6 single-domain antibody and its application. The CLDN6 single-domain antibody obtained by the present invention has a higher affinity for CLDN6 and a lower affinity for CLDN9, that is, it has a higher specificity for CLDN6, and at the same time, it mediates better antibody-dependent cell-mediated cytotoxicity (ADCC), and the CAR molecule constructed based on it can better mediate immune cells to kill tumors.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-human CLDN6 single-domain antibody, wherein the heavy chain variable region of the single-domain antibody comprises CDR1, CDR2 and CDR3;

[0007] The amino acid sequence of the CDR1 includes SEQ ID NO.1;

[0008] The amino acid sequence of the CDR2 includes SEQ ID NO.2;

[0009] The amino acid sequence of the CDR3 includes SEQ ID NO.3.

[0010] Preferably, the heavy chain variable region sequence of the heavy chain antibody comprises SEQ ID NO.4.

[0011] In a second aspect, the present invention provides a heavy chain antibody, comprising the heavy chain variable region sequence described in the first aspect and the full-length or partial amino acid sequence of a human immunoglobulin (Ig) crystallizable fragment (Fc) Ig Fc.

[0012] Preferably, the Ig Fc comprises the full-length or partial sequence of the Fc fragment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

[0013] Preferably, the IgG1 Fc amino acid sequence includes that shown in SEQ ID NO.5.

[0014] Preferably, the amino acid sequence of the heavy chain antibody comprises SEQ ID NO.6.

[0015] There is a special antibody lacking light chains in the blood of camelids and chondrichthyes, namely heavy chain antibodies. Compared with ordinary antibodies, it only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. By cloning its variable region, a single domain antibody (also known as nanobody) consisting only of the heavy chain variable region can be obtained, which has the advantages of small molecular weight, strong tissue penetration, high stability, and low immunogenicity.

[0016] The present invention uses HEK293T cells overexpressing CLDN6 to immunize alpacas. After determining the titer of CLDN6-specific antibodies in alpaca serum by ELISA and flow cytometry, alpaca peripheral blood mononuclear cells (PBMC) are separated, RNA is extracted and reverse transcribed to obtain cDNA. Alpaca single-domain antibody-specific primers are used to amplify the VHH sequence and clone it into a phage expression plasmid to construct a phage display library. CHO-S cells overexpressing CLDN9 are used to incubate the phage display library to eliminate antibody clones that bind to CLDN9 cells. Then, virus-like particles (VLPs) containing CLDN6 are used to enrich and select the phage display library for 3-4 rounds. Monoclones are picked from the enriched products, and the specific binding of candidate antibodies to CLDN6-VLPs is detected by ELISA. After the positive antibodies are expressed and purified, their affinity to CHO-S cells overexpressing CLDN6 or CLDN9 is detected respectively. Antibody clones with strong affinity to CLDN6 and weak affinity to CLDN9 were selected, co-cultured with NK cells and CLDN6-positive Huh-7 liver cancer cells, and antibody-dependent cell-mediated cytotoxicity (ADCC) of each clone was detected. At the same time, the CLDN6 single-domain antibody sequence was cloned into the CAR expression vector, and CAR-T cells with CLDN6 single-domain antibodies as antigen binding domains were constructed to detect their killing ability against CLDN6-positive OVCAR3 cells in vivo and in vitro. The results show that compared with the prior art, the CLDN6 single-domain antibody obtained by the present invention has a higher affinity for CLDN6 and a lower affinity for CLDN9, that is, it has a higher specificity for CLDN6, and the tumor killing effect of ADCC and CAR-T mediated by it is better.

[0017] In a third aspect, the present invention provides a nucleic acid molecule encoding the anti-human CLDN6 single domain antibody of the first aspect or the heavy chain antibody of the second aspect.

[0018] In a fourth aspect, the present invention provides an expression vector, comprising the nucleic acid molecule described in the third aspect; and after transfection / transduction / transformation of a host cell, the expression vector enables the host cell to express the anti-human CLDN6 single domain antibody described in the first aspect or the heavy chain antibody described in the second aspect.

[0019] In a fifth aspect, the present invention provides a host cell, wherein the host cell contains 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.

[0020] In a sixth aspect, the present invention provides a composition for detecting human CLDN6, wherein the composition comprises the anti-human CLDN6 single domain antibody described in the first aspect or the heavy chain antibody described in the second aspect.

[0021] In the seventh aspect, the present invention provides an anti-human CLDN6 chimeric antigen receptor, which recognizes human CLDN6 protein, and is composed of the following structures in series: a signal peptide, the anti-human CLDN6 single domain antibody described in the first aspect, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and a human CD3 intracellular signal transduction domain.

[0022] 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.

[0023] Preferably, the amino acid sequence of the signal peptide includes that shown in SEQ ID NO.8.

[0024] 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.

[0025] Preferably, the amino acid sequence of the hinge region includes that shown in SEQ ID NO.9.

[0026] 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.

[0027] Preferably, the amino acid sequence of the transmembrane domain includes that shown in SEQ ID NO.10.

[0028] 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 (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, or a combination thereof.

[0029] Preferably, the amino acid sequence of the intracellular co-stimulatory domain includes that shown in SEQ ID NO.11.

[0030] Preferably, the human CD3 intracellular signal transduction domain is selected from the following proteins: CD3ζ, CD3ε, CD3γ, CD3δ, or a combination thereof.

[0031] Preferably, the amino acid sequence of the human CD3 intracellular signal transduction domain includes that shown in SEQ ID NO.12.

[0032] Preferably, the amino acid sequence of the anti-human CLDN6 chimeric antigen receptor includes that shown in SEQ ID NO.7.

[0033] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the anti-human CLDN6 chimeric antigen receptor according to the seventh aspect.

[0034] In a ninth aspect, the present invention provides an expression vector, comprising the nucleic acid molecule described in the eighth aspect; and after transfection / transduction / transformation of a host cell, the expression vector enables the host cell to express the anti-human CLDN6 chimeric antigen receptor described in the seventh aspect.

[0035] Preferably, the expression vector comprises a lentiviral expression vector.

[0036] In the tenth aspect, the present invention provides a cell expressing an anti-human CLDN6 chimeric antigen receptor, wherein the cell expressing the anti-human CLDN6 chimeric antigen receptor is obtained by transfecting / transducing / transforming 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-human CLDN6 chimeric antigen receptor described in the seventh aspect.

[0037] Preferably, the cells comprise T cells.

[0038] In an eleventh aspect, the present invention provides a pharmaceutical composition, comprising the cell expressing the anti-human CLDN6 chimeric antigen receptor according to the tenth aspect.

[0039] In a twelfth aspect, the present invention provides the use of any one or a combination of at least two of the anti-human CLDN6 single domain antibody 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 human CLDN6 described in the sixth aspect, the anti-human CLDN6 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-human CLDN6 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.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The single-domain antibody of the present invention has a higher affinity for CLDN6, a lower affinity for CLDN9, and a smaller molecular weight. Antibody drugs developed based on it have better effectiveness, stronger tissue penetration, better in vivo stability, and better efficacy; engineered immune cells developed based on it have higher exogenous gene expression levels, better effectiveness, better efficacy, and lower transformation difficulty; detection reagents developed based on it have higher sensitivity and lower false positives. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the test result of 72# alpaca serum titer.

[0043] Figure 2 This is the result of the 110# alpaca serum titer test.

[0044] 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 of PCR; B is the VHH region fragment amplified by the second round of PCR.

[0045] Figure 4 It is the specific result of recombinant single domain antibody in expression supernatant detected by flow cytometry.

[0046] Figure 5 EC of purified recombinant single domain antibody was detected by flow cytometry 50 result.

[0047] Figure 6 EC of recombinant single domain antibodies detected by flow cytometry 50 The results are as follows; A is a concentration-effect curve detected by CHO-S-CLDN6 cells; B is a concentration-effect curve detected by CHO-S-CLDN9 cells.

[0048] Figure 7 The results of IHC and flow cytometry detection of the binding of recombinant single domain antibodies to ovarian cancer cells.

[0049] Figure 8 This is the result of flow cytometry detection of CLDN6 expression in liver cancer cells.

[0050] Fig. 9 IncuCyte real-time quantitative live cell imaging analyzes the functional results of recombinant single domain antibody-mediated ADCC.

[0051] Fig.10It is the molecular structure of CAR targeting CLDN6 and its expression results in T cells; A is the molecular structure of CAR; B is the expression of BVHCN6-007 in T cells detected by flow cytometry; C is the expression of BN124 in T cells detected by flow cytometry.

[0052] Fig.11 IncuCyte real-time quantitative live cell imaging was used to analyze the killing function and specificity of CAR-T cells targeting CLDN6.

[0053] Fig.12 It is the result of ELISA analysis of the function and specificity of IFN-γ secreted by CAR-T cells targeting CLDN6.

[0054] Fig.13 It is the tumor-suppressing effect of CAR-T cells targeting CLDN6 on ovarian cancer in vivo. DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0056] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0057] The sources of the following experimental materials are as follows: 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] Example 1 Alpaca immunization and antibody titer determination

[0059] (1) Construction of CLDN6 and CLDN9 overexpressing cells

[0060] Based on 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 cell lines 293T-CLDN6 and CHO-S-CLDN6 overexpressing CLDN6, which were used for alpaca immunization, serum titer detection and antibody affinity verification, respectively.

[0061] 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 CLDN9-overexpressing cell line CHO-S-CLDN9 cells for antibody screening and affinity verification.

[0062] (2) Alpaca immunization and serum titer testing

[0063] The 293T-CLDN6 cells constructed above were used to immunize two alpacas (72# and 110#), once every three weeks, for a total of 5 times. Before each immunization, 5 mL of peripheral blood was collected, and the centrifuge tube containing the blood sample was placed in a centrifuge. After centrifugation at 800 g for 10 minutes, the supernatant was transferred to a new sterile centrifuge tube to collect the immune serum. The serum was diluted with PBS gradient, and 100 μL of gradient diluted serum (PBS was used instead of control wells) and 2×10 5 CHO-S-CLDN6 cells or CHO-S cell lines. After incubation at room temperature for 1 hour, centrifuge at 800g for 3 minutes, discard the supernatant, and wash three times with PBS. Add 100 μL FITC-labeled Rabbit anti-Llama IgG (H+L) antibody (1:1000 dilution) to each well. After incubation at room temperature for 1 hour, centrifuge at 800g for 3 minutes, discard the supernatant, and wash three times with PBS, and detect using flow cytometry.

[0064] The results are as follows Figure 1 , Figure 2 , Table 1, Table 2, Table 3 and Table 4, wherein Table 1 is the flow cytometry results of the serum of 72# alpaca after the third immunization (expressed in MFI); Table 2 is the flow cytometry results of the serum of 72# alpaca after the fourth immunization (expressed in MFI); Table 3 is the flow cytometry results of the serum of 110# alpaca after the third immunization (expressed in MFI); Table 4 is the flow cytometry results of the serum of 110# alpaca after the fourth immunization (expressed in MFI).

[0065] The mean fluorescence intensity (MFI) detected by flow cytometry after incubation of the sera of the two alpacas before immunization with CHO-S cells and CHO-S-CLDN6 cells was similar. The binding ability of the two alpacas to the two cells before immunization was comparable, indicating that the content of CLDN6-specific antibodies contained therein 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 was significantly increased after multiple immunizations, and the immunization was successful, which can be used for the construction of phage surface display libraries.

[0066] Table 1

[0067]

[0068] Table 2

[0069]

[0070] Table 3

[0071]

[0072] Table 4

[0073]

[0074] Example 2 Construction of single domain antibody phage display library

[0075] (1) VHH antibody fragment cloning

[0076] After confirming that the serum of the two alpacas contained CLDN6-specific antibodies, 100 mL of peripheral blood was collected, PBMCs were separated using lymphocyte separation solution, and RNA was extracted. TM II 1 st Strand cDNA Synthesis Kit was used for reverse transcription to obtain cDNA. Using PBMC cDNA as a template, specific primers (upstream primer binds to the signal peptide of VHH antibody ORF, downstream primer binds to CH2 region) were used to PCR amplify the alpaca heavy chain antibody sequence. 1% agarose was used for electrophoresis analysis of PCR products, and the target fragment with a molecular weight of about 750 bp was recovered and separated ( Figure 3A). The first round of PCR product was used as a template to amplify the heavy chain antibody VHH fragment using specific primers (the upstream primer binds to the antibody FR1 region, and the 5' end contains the SfiI restriction site GGCCCAGCCGGCC, SEQ ID NO.19; the downstream primer binds to the antibody Hinge and FR4 region, and the 5' end contains the SfiI restriction site GGCCACGAAGGCC, SEQ ID NO.20). The PCR product was analyzed by electrophoresis using 1% agarose, and the target fragment with a molecular weight of about 400 bp was recovered and separated ( Figure 3 (B). The target fragment was stored at -80°C (the storage solution contained 1 / 10 volume of 3M sodium acetate, 1 μg / μL glycogen, and 80% anhydrous ethanol).

[0077] (2) Electroporation of library vectors and detection of library capacity and diversity

[0078] The phage surface display vector pComf and the VHH fragment library obtained above were digested with SfiI endonuclease. The linearized pComf vector and the VHH fragment library were connected overnight at 16°C using T4 ligase. The ligation product was stored at -80°C (the storage solution contained 1 / 10 volume of 3M sodium acetate, 1μg / μL glycogen, and 80% anhydrous ethanol). 300μL of the mixture of the above ligation product and E. coli SS320 competent cells was added to the pre-cooled electroporation cup, and the ligation product was transformed into E. coli by electroporation (2500V, 5ms), and then 20mL of SOC medium was added to resuspend the bacteria, and the culture was revived on a shaking table at 37°C for 1h. 15mL of the bacterial solution was taken for subsequent phage production enrichment, and the remaining 5mL of the electroporation product was added with an equal volume of 50% glycerol, mixed evenly, and stored at -80°C. In addition, 20 μL of bacterial solution was diluted with 2YT medium and evenly spread on LB plate containing ampicillin, and cultured at 37°C overnight. The number of clones that can be produced by each ligation reaction was calculated to obtain the capacity of the single-domain antibody phage surface display library. The results showed that the library capacity of the single-domain antibody phage surface display library obtained from 72# alpaca PBMC was 1.33×10 9 The single-domain antibody phage display library obtained from 110# alpaca PBMC has a library capacity of 1.18×10 9 . Twenty single clones on the plate were picked and Sanger sequencing was performed with M13R primers. The results showed that the sequences of the phage library were highly different, with no repeated sequences and good diversity.

[0079] (3) Enrichment of phage display libraries

[0080] Take 15 mL of the bacterial solution after electroporation and recovery culture, and dilute it with 2YT to adjust the OD 600When the OD is about 0.25, add ampicillin with a final concentration of 100 μg / mL and place in a constant temperature shaker at 37°C and 225 rpm. 600 When the value was 0.6, M13KO7 helper phage was added (the volume of M13KO7 helper phage added = 10 × bacterial solution volume × OD 600 ×5×10 8 / M13KO7 titer), shake well and let stand at 37℃ for 30min, then culture at 37℃ shaker at 225rpm for 1h. After the helper phage completes the infection of the target strain, centrifuge at 6000rpm for 10min, discard the supernatant, resuspend with 2YT-AK medium, and culture overnight at 25℃ shaker at 200rpm. Then centrifuge the bacterial solution at 10000rpm for 15min, transfer the supernatant containing phage particles to a new centrifuge tube (add 1 / 5 of the volume of bacterial solution PEG / NaCl to the tube), mix well and let stand at 4℃. After standing for 2h, centrifuge at 10000rpm for 30min at 4℃, collect the phage precipitate, and resuspend with PBS 1 / 50 of the original volume. Transfer the resuspended phage to a 1.5mL EP tube and centrifuge at 12000g for 5min at 4℃ to remove insoluble impurities. The supernatant was then transferred to a new 1.5 mL EP tube, 250 μL of PEG / NaCl was added to each tube, mixed and allowed to stand at 4°C for 10 min, centrifuged at 12000 g for 10 min at 4°C, and the supernatant was discarded. After resuspending in 1 mL PBS, the supernatant was centrifuged at 12000 g for 5 min at 4°C, and the supernatant was transferred to a new 1.5 mL EP tube to obtain the original library of single domain antibody phage surface display.

[0081] Take 10 μL of the precipitate and perform 10-fold gradient dilutions. Add 200 μL of OD 600 ER2738 Escherichia coli was added at 0.5, mixed well, placed in a 37°C water bath, left to stand for 10 minutes, and then spread on an LB plate. After overnight culture at 37°C, the plaques were counted to obtain the titer of the phage surface display library.

[0082] Example 3 Target Antibody Library Selection

[0083] (1) First round of panning and product amplification

[0084] Seal the 1.5 mL centrifuge tube with 3% MPBS and incubate at 4°C overnight. Dilute the CLDN6-VLP antigen to 50 μg / mL with CBS solution and add to the 96-well solid phase plate. Coat at 4°C overnight. Take 1×10 7CHO-S-CLDN9 cells were washed 3 times with PBS, resuspended in 3% PBSA, and blocked at 37°C for 1h on a 360° low-speed rotating mixer. At the same time, 150 μL of the precipitate product of the original library displayed on the surface of the single-domain antibody phage was taken, 350 μL of 1% PBSA was added to the 360° low-speed rotating mixer, and blocked at 4°C for 1h as a premix. The blocked CHO-S-CLDN9 cells were centrifuged at 500g for 10min, the supernatant was removed, and the phage premix was added and incubated at 4°C for 1h on a 360° low-speed rotating mixer 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 allowed to stand at room temperature. After blocking for 1h, 3% MPBS buffer was used. CHO-S-CLDN9 cells were centrifuged at 500g for 10 minutes, and the supernatant was taken and added to the CLDN6-VLP protein wells respectively, and incubated at room temperature for 1 hour. The phage supernatant in the CLDN6-VLP protein wells was discarded, and 0.05% PBST was used to wash 6 times and PBS was washed 4 times. 100 μl of Gly-HCl elution solution at pH 2.2 was added to each well, and incubated at 37°C for 8 minutes. The specifically bound phages were eluted twice, and the eluted products were stored at 4°C in pre-sealed centrifuge tubes (washed twice with PBS after sealing).

[0085] 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 OD 600 The eluted phage product was added to the ER2738 bacterial solution, mixed and incubated at 37°C for 30 min, then 20 mL of 2YT medium was added and cultured at 37°C and 225 rpm for 30 min. 600 When the value was 0.5 again, M13KO7 helper phage was added (the added volume of M13KO7 = 10 × bacterial solution volume × OD 600 ×5×10 8 / M13KO7 titer), shake well and let stand at 37℃ for 30min. Add ampicillin at a final concentration of 100μg / mL to the bacterial solution, culture at 225rpm at 37℃ for 45min, centrifuge at 8000rpm for 20min, discard the supernatant and collect the bacteria. Resuspend with 40mL 2YT-AK medium and culture overnight at 210rpm at 30℃. Transfer the phage suspension infected and amplified overnight to a 50mL centrifuge tube, centrifuge at 8000rpm at 4℃ for 30min, and divide the supernatant into 40mL centrifuge tubes. Add 10mL PEG / NaCl to each tube, mix well and place on ice, let stand for 1h to precipitate the phage, and centrifuge at 8000rpm at 4℃ for 30min. Discard the supernatant and resuspend the phage with 1mL sterile PBS, centrifuge at 12000g at 4℃ for 5min to remove insoluble impurities. Transfer 1 mL of phage suspension to a new 1.5 mL centrifuge tube, add 250 μL of PEG / NaCl, mix well, and let stand at 4°C for 10 min to precipitate phage. Centrifuge at 12000g for 10 min at 4°C, and discard the supernatant. Resuspend the phage with 1 mL of PBS, and centrifuge at 12000g for 5 min at 4°C to remove insoluble impurities. This is the phage product obtained in the first round of panning amplification.

[0086] (2) Second to fourth rounds of panning and product amplification

[0087] The CLDN6-VLP antigen was diluted to 10 μg / mL using CBS solution, added to a 96-well solid phase plate, and coated overnight at 4°C. The phage products amplified in the first round of panning were used for 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 Tables 5 and 6. There was 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, and the enrichment index of the fourth round of the 110# alpaca single domain antibody phage surface display library was about 27 times lower than that of the first round, indicating that the 110# alpaca single domain antibody phage surface display library may contain more CLDN6-specific single domain antibody clones. 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.

[0088] Table 5

[0089]

[0090] Table 6

[0091]

[0092] (3) ELISA detection and sequencing of monoclonal phage

[0093] The single-domain antibody phage surface display library after multiple rounds of panning was used to infect ER2738 Escherichia coli, mixed and placed in a 37°C water bath for 10 minutes, then spread on an LB plate and incubated at 37°C overnight. 2YT-A medium was added to a 96-well deep-well plate at 200 μL per well, and a single clone on the plate was picked and incubated at 37°C and 225 rpm overnight. 2YT-A medium was added to a 96-well deep-well plate at 150 μL per well, and 20 μL of the overnight culture solution was added to each well, and incubated at 37°C and 225 rpm until OD 600 Add M13KO7 helper phage, mix well and let stand at 37℃ for 15min (M13KO7 volume = 10×bacterial solution volume×OD600×5×10 8 / M13KO7 titer), cultured at 37°C and 225 rpm for 45 min. Centrifuged at 3900 rpm for 10 min, discarded the supernatant, resuspended in 500 μL 2YT-AK medium per well, cultured at 30°C and 220 rpm overnight. Centrifuged at 3900 rpm for 10 min, the supernatant obtained was the monoclonal phage particles.

[0094] While amplifying the phage monoclonal, coat the CLDN6-VLP antigen protein with CBS at pH 9.6 to the ELISA plate (2 μg / mL, 100 μL / well). After overnight coating at 4°C, discard the antigen, wash three times with PBST, add 250 μL 3% MPBS to each well, and block overnight at 4°C. After discarding the blocking solution, add 200 μL 0.05% PBST to each well, wash four times, add 50 μL 0.1% PBST, and then add 50 μL of the above monoclonal phage supernatant one by one. After incubation at 4°C for 1 hour, wash five times with 0.05% PBST. Dilute the anti M13-HRP antibody (1:5000) with 0.05% PBST, add 100 μL to each well, and incubate at 4°C for 45 minutes. After washing five times with 0.05% PBST, 100 μL TMB was added to develop color at room temperature for 10 min, and then 50 μL 0.2 M hydrochloric acid was added to terminate the color development. The OD was read on the ELISA plate. 450 The sample / negative control value was calculated, and clones with a ratio significantly greater than that of the positive serum control group were selected for sequencing to obtain antibodies 72-2-C01, 72-2-E02, and 110-1-C12 (see sequence listing).

[0095] Example 4 Expression of recombinant single domain antibodies and detection of binding to target proteins

[0096] The candidate VHH sequences were amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.4, and the C-terminus was fused with the human IgG1 Fc fragment for expression. After transient transfection of the obtained expression plasmid into HEK293 cells, the harvested cell culture supernatant contained each recombinant single domain antibody. In the same manner, the CLDN6-specific antibody 89A (from CN111875703A, see sequence listing) was expressed as a positive control.

[0097] The binding specificity of each recombinant single domain antibody was tested. The culture supernatant containing each recombinant single domain antibody was mixed with 3×10 5 CHO-S, CHO-S-CLDN6 or CHO-S-CLDN9 cells were incubated at room temperature for 1 hour. After centrifugation at 800g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed three times with PBS. 100 μL of PE-labeled anti-human IgG antibody (1:500 dilution) was added and incubated at room temperature in the dark for 45 minutes. After centrifugation at 800g for 5 minutes at room temperature, the supernatant was discarded and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS and analyzed by flow cytometry.

[0098] The results are as follows Figure 4 As shown, the expression supernatant of the negative control group had no significant binding to CHO-S-CLDN6 or CHO-S-CLDN9; the positive control antibody 89A had significant binding to both CHO-S-CLDN6 and CHO-S-CLDN9, but the binding level to CHO-S-CLDN9 cells was slightly weaker than that to CHO-S-CLDN6 cells. Similar to the positive control, the binding level of 72-2-C01 and 72-2-E02 recombinant single domain antibodies to CHO-S-CLDN9 cells was slightly weaker than that to CHO-S-CLDN6 cells, indicating that these antibodies can bind to CLDN6, but with poor specificity. In contrast, the binding level of 110-1-C12 to CHO-S-CLDN9 cells was significantly weaker than that to CHO-S-CLDN6 cells, indicating that its specificity to CLDN6 is higher than that of other antibodies.

[0099] Example 5 Purification of recombinant antibodies and half effective concentration (EC 50 ) determination

[0100] To determine the EC of 110-1-C12 antibody 50, transiently transfect the relevant expression plasmid into 293F cells, and shake the flask to culture 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 30mL PBS buffer, 0.1M sodium hydroxide, and PBS buffer in sequence. Add the corresponding volume of Protein A magnetic beads (calculated as 20mg IgG / mL Protein A magnetic beads) to the 293F cell shake flask according to the required amount of the sample. Incubate at room temperature for 1 to 4 hours or overnight at 4°C at 120rpm in a shaking incubator. Collect the Protein A magnetic beads with a magnetic separation rack and transfer them to a 50mL centrifuge tube. After washing twice with 30mL PBS buffer and deionized water, resuspend them with 1mL elution buffer. After incubation at room temperature for 5min, collect the magnetic beads with a magnetic separation rack, and transfer the supernatant containing the target antibody to a 15mL centrifuge tube. After eluting the Protein A magnetic beads twice, the eluates were combined and the neutralization buffer was added to adjust the pH of the solution. The eluted sample was dialyzed with PBS at least 100 times the volume of the sample, first dialyzed at 18-25°C for 2 hours, and then dialyzed at 2-8°C for 14-16 hours after changing the solution once. Finally, the protein concentration was determined and the sample was filtered with a 0.22μm sterile filter membrane and packaged, and stored in a -80°C refrigerator for use.

[0101] Serial dilutions of the target antibody were added to 3×10 5 CHO-S-CLDN6 and CHO-S-CLDN9 were incubated at room temperature for 1 hour. Centrifuge at 800g for 5 minutes at room temperature, discard the supernatant containing antibodies, and wash the cells three times with PBS. Add 100μL PE-labeled Anti-human IgG antibody (1:500 dilution), mix well, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800g for 5 minutes at room temperature, discard the supernatant containing antibodies, wash the cells three times with PBS, and resuspend the cells with 500μL PBS for flow cytometry analysis.

[0102] The results are as follows Figure 5 , Figure 6 As shown in Table 7, Table 7 is the EC of recombinant single domain antibodies detected by flow cytometry. 50 Results: EC of positive control antibody 89A against CHO-S-CLDN6 50 The value was 1.26 μg / mL, and the EC value of 110-1-C12 for CHO-S-CLDN6 was 50 The value is 0.4045 μg / mL, indicating that 110-1-C12 has a higher affinity for CLDN6; the EC value of the positive control antibody 89A for CHO-S-CLDN9 50 The value was 14.49 μg / mL, and the EC value of 110-1-C12 for CHO-S-CLDN9 was50 The value is 16.12 μg / mL, indicating that 110-1-C12 has a lower affinity for CLDN9. The above results show that compared with the prior art, 110-1-C12 has both higher affinity and specificity for CLDN6.

[0103] Table 7

[0104]

[0105] Example 6 Detection of CLDN6 expression in tumor cells

[0106] (1) Expression detection in ovarian cancer cells

[0107] The positive control antibody A-4 is an antibody that binds to the intracellular domain of CLDN6 but not CLDN9, and can effectively distinguish CLDN6 from CLDN9. The A-4 antibody was used to detect the expression of CLDN6 in ovarian cancer cells (OVCAR-3, SK-OV-3, and CAOV-3 cells that overexpress firefly luciferase and red fluorescent protein, respectively) by immunohistochemistry (IHC). The results are shown in Figure 7 As shown, only OVCAR-3-Luc-mCherry cells are CLDN6 positive, while SK-OV-3-Luc-mCherry and CAOV-3-Luc-mCherry cells are CLDN6 negative. The three cells were incubated with 110-1-C12 antibody at room temperature for 1 hour. Centrifuge at 800g for 5 minutes at room temperature, discard the supernatant containing the antibody, and wash the cells three times with PBS. Add 100μL AF647-labeled Anti-human IgG antibody, mix thoroughly, and incubate at room temperature in the dark for 45 minutes. Centrifuge at 800g for 5 minutes at room temperature, discard the supernatant containing the antibody, wash the cells three times with PBS, resuspend the cells with 200μL PBS, and perform flow cytometric analysis. Figure 7 As shown, the 110-1-C12 antibody could only detect the significant expression of CLDN6 in OVCAR-3-Luc-mCherry cells, indicating that the 110-1-C12 antibody also has good affinity and specificity for CLDN6 in tumor cells.

[0108] (2) Expression detection in liver cancer cells

[0109] The anti-human CLDN6 antibody labeled with AF647 was incubated with the liver cancer cells HuH-7-Luc-mCherry overexpressing firefly luciferase and red fluorescent protein for 1 hour. Centrifuge at 800g for 5 minutes at room temperature, discard the supernatant containing the antibody, and wash the cells three times with PBS. Resuspend the cells with 200μL PBS and perform flow cytometric analysis. Figure 8 As shown, CLDN6 is significantly expressed in HuH-7-Luc-mCherry cells and can be used for subsequent functional evaluation experiments.

[0110] Example 7 ADCC Functional Detection

[0111] HuH7-Luc-Mcherry cells were used to detect the ADCC function of the target antibody. After washing the target cells three times with OptiVitro NK cell expansion medium, 1×10 4 The target cells were inoculated into a 96-well plate at a density of 1:1 / well and cultured overnight. NK cells were added at an effector-target ratio of 2:1, 4:1 or 8:1, and CLDN6 positive control antibody 89A or target antibody 110-1-C12 was added at a final concentration of 5 μg / mL.

[0112] The real-time quantitative live cell imaging and analysis platform IncuCyte was used for detection. The changes in the fluorescence signals of target cells in each group were calculated using the IncuCyte SX5 software. The lower the signal value, the fewer cells in the group and the better the NK cell killing effect. Fig. 9 As shown, compared with the NK cell group without antibody, both 110-1-C12 antibody and positive control antibody can significantly enhance the killing effect of NK cells on tumor cells, and the enhancement is greater under the condition of low efficiency target ratio.

[0113] Example 8 Construction of CAR-T cells based on target antibodies

[0114] (1) Design of CAR molecules

[0115] The target gene structure of the lentiviral vector involved in this example is as follows Fig.10 As shown in A.

[0116] BVHCN6-007 is composed of the following structures in series: human CD8 signal peptide (SP for short), anti-human CLDN6 single-domain antibody 110-1-C12 [VHH(110-1-C12) for short], human IgG4 hinge region (IgG4 hinge for short), human CD8 transmembrane domain (CD8 TM for short), human 4-1BB intracellular co-stimulatory domain (4-1BB ICD for short), and human CD3ζ intracellular signal transduction domain (CD3ζICD for short).

[0117] BN124 is composed of the following structures in series: human CD8 signal peptide (SP), anti-human CLDN6 single-chain antibody [scFv(67A)], human IgG4 hinge region (IgG4 hinge), human CD8 transmembrane domain (CD8 TM), human 4-1BB intracellular co-stimulatory domain (4-1BB ICD), and human CD3ζ intracellular signal transduction domain (CD3ζICD).

[0118] (2) Lentivirus preparation

[0119] The above CAR molecule expression sequence was fully synthesized and then connected to the lentiviral vector pCDH-EF1α-MCS plasmid by molecular cloning, so that it was expressed under the regulation of the human EF-1α promoter and Kozak sequence. 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 using the transfection reagent Lipofectamine 3000. The viral supernatant was collected 48 hours after transfection, centrifuged at 3000rpm for 10-15min at 4°C, filtered through a 0.45μm pore size filter membrane, and finally ultracentrifuged at 25,000rpm at 4°C for 2-3h. The obtained virus concentrate was stored at -80°C and named BVHCN6-007 and BN124 respectively. Finally, Jurkat cells were used as materials to detect the activity titer of the above lentivirus.

[0120] (3) CAR-T cell preparation

[0121] PBMCs from healthy donors were revived in AIM V medium, and 25 ng / mL anti-CD3 antibody, 25 ng / mL anti-CD28 antibody and 300 IU / mL recombinant hIL-2 were added, and cultured in a cell culture incubator for 24 h (culture temperature was 37 ° C, carbon dioxide concentration was 5%). The obtained T cells were washed, and lentivirus was added at an MOI of 5TU / mL for transduction. 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 cultured in a cell culture 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 supplemented with 300IU / mL of hIL-2. On the 4th day after transduction, the cells were washed to remove the residual lentiviral particles in the supernatant, and then cultured in a cell culture incubator for 5 days (culture temperature was 37°C, carbon dioxide concentration was 5%), during which the cell density was maintained at 1-2×10 6 / mL. The cells were collected on the 10th day after transduction and frozen in liquid nitrogen for future use. The obtained CAR-T cells were named after the corresponding CAR molecules, and the T cells not transduced with lentivirus were named Ctrl T.

[0122] (4) Detection of CAR molecule expression

[0123] The BVHCN6-007 CAR-T cells to be tested were washed twice with PBS and resuspended in FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). According to the antibody instructions, the FITC-labeled anti-VHH antibody was incubated with the CAR-T cells for 1 hour. The supernatant was then removed by centrifugation, and the cells were washed twice with FACS buffer and resuspended. Using Ctrl T cells as negative control, the CAR molecule expression rate of BVHCN6-007 cells was detected by flow cytometry. The results are shown in Fig.10 As shown in B, the expression rate of BVHCN6-007CAR was 80.1%.

[0124] The BN124 CAR-T cells to be tested were washed twice with PBS and resuspended with FACS buffer (PBS containing 0.1% sodium azide and 0.4% BSA). CAR-T cells were incubated with CLDN6-VLP for 1 hour. The supernatant was then removed by centrifugation and washed twice with FACS buffer. CAR-T cells were then incubated with AF647-labeled anti-human CLDN6 antibody for 1 hour. The supernatant was removed by centrifugation and washed twice with FACS buffer. Using Ctrl T cells as negative control, the CAR molecule expression rate of BN124 cells was detected by flow cytometry. The results are shown in Figure 2. Fig.10 As shown in center C, the expression rate of BN124 CAR was 97.8%.

[0125] Example 9 Study on the function of CAR-T cells in vivo and in vitro

[0126] (1) In vitro killing experiment of BVHCN6-007 cells

[0127] Use culture medium at 1×10 5 OVCAR-3-Luc-mCherry cells and SK-OV-3-Luc-mCherry cells were resuspended at a density of 100 μL / mL and inoculated into a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte SX5 live cell imaging analysis system, CAR-T cells were added for co-culture at an effect-target ratio of 4.5:1, and the killing effect of CAR-T cells on tumor cells was recorded in real time. After the co-culture, the changes in the mCherry fluorescence signal of the target cells in each group were calculated using the IncuCyte SX5 software. The lower the signal value, the fewer cells in the group and the better the killing effect of CAR-T cells. The results are shown in Fig.11As shown, for CLDN6-positive OVCAR-3-Luc-mCherry cells, the killing effect of the BN124 group (relative fluorescence area at the last time point was 0.68±0.02) was only slightly improved compared with the Ctrl T group (relative fluorescence area at the last time point was 0.79±0.02), while the killing effect of the BVHCN6-007 group (relative fluorescence area at the last time point was 0.03±0.01) was greatly improved (P<0.05). For CLDN6-negative SK-OV-3-Luc-mCherry cells, BVHCN6-007 cells had no significant killing effect. This result shows that compared with existing antibodies, CAR-T cells constructed based on the 110-1-C12 antibody have stronger specific killing function against CLDN6-positive tumor cells.

[0128] To study the specificity of BVHCN6-007 cell killing function, 293T cells, OVCAR-3-Luc-mCherry cells, SK-OV-3-Luc-mCherry cells and CAOV-3-Luc-mCherry cells were used as target cells, and Ctrl T cells and BVHCN6-007 cells were added respectively. After overnight co-culture, the culture supernatant was collected and the IFN-γ content was detected by ELISA. The results are shown in Fig.12 As shown in the figure, BVHCN6-007 was significantly activated only by CLDN6-positive OVCAR-3-Luc-mCherry cells and released high levels of IFN-γ (P<0.05), but was hardly activated by CLDN6-negative target cells. This result further shows that CAR-T cells constructed based on the 110-1-C12 antibody have good specificity in killing CLDN6-positive tumor cells.

[0129] (2) In vivo tumor inhibition experiment of BVHCN6-007 cells

[0130] OVCAR-3-Luc-mCherry target cells in the logarithmic growth phase and in good growth condition were collected by trypsin digestion, washed with physiological saline, and the cell density was adjusted to 2×10 7 / mL. 100 μL of cell suspension was injected subcutaneously on the right side of severely immunodeficient mice near the armpit, that is, 2×10 6 Target cells. On the 7th day after inoculation of target cells (or when the average tumor volume is about 100mm 3 CAR-T cells, Ctrl T cells and vehicle were injected through the tail vein, and the day of injection of the test substance was recorded as treatment day 0. The tumor size was measured 2 to 3 times a week. Fig.13As shown, the tumor volume of the vehicle control group and the Ctrl T control group gradually increased. The tumor in the BN124 treatment group also gradually increased, and there was no significant difference from the Ctrl T group (P>0.05). On the contrary, the tumor volume in the BVHCN6-007 treatment group was significantly smaller than that in the control group and the BN124 treatment group (P<0.05). This result shows that the CAR-T cells constructed based on 110-1-C12 of the present invention can also effectively inhibit the growth of solid tumors in vivo, and are more suitable for use in CAR-T treatment technology than existing antibodies.

[0131] In summary, the anti-human CLDN6 single-domain antibody of the present invention has a higher affinity for CLDN6 and a lower affinity for CLDN9, that is, it has a higher specificity for CLDN6, and at the same time, it has a better antibody-dependent cell-mediated cytotoxicity. The chimeric antigen receptor molecule constructed based on it can better mediate immune cell killing of tumors.

[0132] The amino acid sequences involved in the above specific embodiments are shown below.

[0133] 89A VL amino acid sequence (underlined is the CDR region sequence): SEQ ID NO.13:

[0134] QIVLTQSPAIMSASPGEKVTITCSAS SSVNY MHWFQLKPGTSPKLLIY STS NLASGVPARFSGRGSGTSYSLTISRMEAEDAATYYC QQRNNYPPWT FGGGTKLEIK.

[0135] 89A VH amino acid sequence (underlined is the CDR region sequence): SEQ ID NO.14:

[0136] EVQLQQSRPELVKPGASMKISCKAS GYSFTGYT LNWVKQSHGKNLEWIGL INPYNGGS SYNQKFKDKATLTVDKSSSTAYMELLSLTSEDSAVYYC ARDYGYVFDY WGQGTTLTVSS.

[0137] 67A VL amino acid sequence (underlined is the CDR region sequence): SEQ ID NO.15:

[0138] QIVLTQSPAIMSASPGEKVTITCSAS SSVSY MHWFQQKPGTSPKLWIY STSNLASGVPARFSGSGSGTSYSLTISRVAAEDAATYYC QQRSTYPPWT FGGGTKLEIK.

[0139] 67A VH amino acid sequence (underlined CDR region sequence): SEQ ID NO.16

[0140] EVQLQQSGPELVKPGASMKISCKAS GYSFTGYT MNWVKQSHGKNLEWIGL INPYNGGI IYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVFYC ARDFGYVLDY WGQGTTLTVSS.

[0141] 72-2-C01 amino acid sequence (underlined is the CDR region sequence): SEQ ID NO.17:

[0142] AVQLVDSGGGLVQPGGSMRLSCAA SGFTVSSYA MSWVRQAPGKGLEWV SHIYPGGI TSYADSVKGRFTISRDNAKNTLYLQMDSLKPEDTAVYYC ALSQSGY RGQGTLVTVSS.

[0143] 72-2-E02 amino acid sequence (underlined is the CDR region sequence): SEQ ID NO.18:

[0144] QLRLVESGGGLVQPGGSRRLDCAA SGFSFGNV AMSWVRQAPGKGLELV STIYSFGGT YYAKSVQGRFTISRDNAKHTVYLQMNNLKPEDTAVYYC AITGLEERP PGQGTQVTVSS.

[0145] 110-1-C12 amino acid sequence (underlined is the CDR region sequence): SEQ ID NO.4:

[0146] AVQLVESGGGLVQPGGSLRLSCAV PGSISSILL MAWYRQAPGKQRELVA HIIGGEHT NYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC TAGGTY WGQGTQVTVSS.

[0147] The amino acid sequence of CDR1 of 110-1-C12: SEQ ID NO.1: PGSISSILL.

[0148] The amino acid sequence of CDR2 of 110-1-C12: SEQ ID NO.2: HIIGGEHT.

[0149] The amino acid sequence of CDR3 of 110-1-C12: SEQ ID NO.3: TAGGTY.

[0150] Human IgG1 Fc amino acid sequence: SEQ ID NO.5:

[0151] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0152] The amino acid sequence of the heavy chain antibody is SEQ ID NO.6:

[0153] MAAVQLVESGGGLVQPGGSLRLSCAV PGSISSILL MAWYRQAPGKQRELVA HIIGGEHT NYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYC TAGGTY WGQGTQVTVSSAHHSEDPKATKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0154] The amino acid sequence of the anti-human CLDN6 chimeric antigen receptor is shown in SEQ ID NO.7:

[0155] MALPVTALLLPLALLLHAARPSAQPAMAAVQLVESGGGLVQPGGSLRLSCAVPGSISSILLMAWYRQAPGKQRELVAHIIGGEHTNYADSVKGRFTISRDNAKNTVYLQMNSLR PEDTAVYYCTAGGTYWGQGTQVTVSSAHHSEDPKATSGESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCDIYIWAPLAGTCGVLLLSLVITLYCNHRNRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0156] Human CD8 signal peptide amino acid sequence: SEQ ID NO.8: MALPVTALLLPLALLLHAARPS.

[0157] Human IgG4 hinge amino acid sequence: SEQ ID NO.9:

[0158] ESKYGPPCPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0159] Human CD8 transmembrane domain amino acid sequence: SEQ ID NO.10:

[0160] CDIYIWAPLAGTCGVLLLSLVITLYCNHRNR.

[0161] Human 4-1BB intracellular domain amino acid sequence: SEQ ID NO.11:

[0162] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0163] Human CD3ζ intracellular domain amino acid sequence: SEQ ID NO.12:

[0164] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0165] The applicant declares that the above is only a specific implementation mode 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 shall fall within the protection scope and disclosure scope of the present invention.

Claims

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

3.

2. The anti-human CLDN6 single domain antibody according to claim 1, characterized in that The heavy chain variable region sequence of the single-domain antibody includes SEQ ID NO.

4.

3. A heavy chain antibody, characterized in that The heavy chain antibody comprises the heavy chain variable region sequence of claim 2 and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin.

4. The heavy chain antibody 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 heavy chain antibody according to claim 4, characterized in that The IgG1 Fc amino acid sequence includes that shown in SEQ ID NO.

5.

6. The heavy chain antibody according to claim 5, characterized in that The amino acid sequence of the heavy chain antibody includes SEQ ID NO.

6.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-human CLDN6 single domain antibody according to claim 1 or 2 or the heavy chain antibody according to any one of claims 3 to 6.

8. An expression vector, characterized in that The expression vector contains the nucleic acid molecule of claim 7; and after transfection / transduction / transformation of a host cell, the expression vector causes the host cell to express the anti-human CLDN6 single domain antibody of claim 1 or 2 or the heavy chain antibody of 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 of claim 8, or at least one copy of the nucleic acid molecule of claim 7.

10. A composition for detecting human CLDN6, characterized in that: The composition comprises the anti-human CLDN6 single domain antibody according to claim 1 or 2 or the heavy chain antibody according to any one of claims 3-6.

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

12. The anti-human CLDN6 chimeric antigen receptor according to claim 11, characterized in that 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.

13. The anti-human CLDN6 chimeric antigen receptor according to claim 12, characterized in that: The amino acid sequence of the signal peptide is shown in SEQ ID NO.

8.

14. The anti-human CLDN6 chimeric antigen receptor according to claim 11, characterized in that The hinge region is selected from the hinge regions of the following proteins: CD8, CD28, CD137, IgG1, IgG4, TCRα, TCRβ, or a combination thereof.

15. The anti-human CLDN6 chimeric antigen receptor according to claim 14, characterized in that The amino acid sequence of the hinge region includes that shown in SEQ ID NO.

9.

16. The anti-human CLDN6 chimeric antigen receptor according to claim 11, characterized in that 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.

17. The anti-human CLDN6 chimeric antigen receptor according to claim 16, characterized in that The amino acid sequence of the transmembrane domain includes that shown in SEQ ID NO.

10.

18. The anti-human CLDN6 chimeric antigen receptor according to claim 11, characterized in that 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 (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, or a combination thereof.

19. The anti-human CLDN6 chimeric antigen receptor according to claim 18, characterized in that The amino acid sequence of the intracellular co-stimulatory domain includes that shown in SEQ ID NO.

11.

20. The anti-human CLDN6 chimeric antigen receptor according to claim 11, characterized in that The human CD3 intracellular signal transduction domain is selected from the following proteins: CD3ζ, CD3ε, CD3γ, CD3δ, or a combination thereof.

21. The anti-human CLDN6 chimeric antigen receptor according to claim 20, 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-human CLDN6 chimeric antigen receptor according to claim 11, characterized in that The amino acid sequence of the anti-human 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-human 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 of claim 23; and after transfection / transduction / transformation of a host cell, the expression vector enables the host cell to express the anti-human CLDN6 chimeric antigen receptor of any one of claims 11-22.

25. The expression vector according to claim 24, characterized in that The expression vector comprises a lentiviral expression vector.

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

27. The cell expressing anti-human CLDN6 chimeric antigen receptor according to claim 26, characterized in that The cells include T cells.

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

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

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