A tight junction protein antibody E02 and its application

Abstract

The present invention provides a tight junction protein antibody E02 and its applications. The antibody E02 is a single-domain antibody, and the variable region of the heavy chain 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 E02 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 Claudin 6 antibody E02 and its application. Background Art

[0002] Tight junctions are formed by the direct contact of specific proteins in the cell membranes of two adjacent cells, where the proteins of one cell membrane insert into the groove of the other cell membrane in the form of ridges to form bundles. Tight junctions are often located between sheets of epithelial cells and have the ability to seal or prevent leakage. At the same time, tight junctions can also prevent the lateral diffusion of membrane proteins, thus maintaining the polarity of the cell membrane.

[0003] 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 almost not 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 tumor 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.

[0004] 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 entered clinical research.

[0005] CN117534767A discloses a chimeric antigen receptor macrophage targeting CLDN6 and its preparation method and application. The invention provides a chimeric antigen receptor specifically targeting CLDN6, its gene and recombinant expression vector, an engineered immune response cell specifically targeting CLDN6 chimeric antigen receptor modified and its application, providing a new means for tumor treatment with application prospects.

[0006] CN116003622A discloses a chimeric antigen receptor for the treatment of ovarian cancer and its application. The chimeric antigen receptor includes a CD8 signal peptide, a single-chain antibody CLDN6 scFv specifically recognizing CLDN6, a CD8 hinge region, an NKG2D transmembrane region, a 2B4 intracellular co-stimulatory domain, a DAP10 intracellular co-stimulatory domain, and a CD3ζ signaling region. The third-generation CAR-NK cells based on this chimeric antigen receptor and targeting CLDN6 can mediate stronger NK cell signal transduction and killing ability, significantly improve the activation level of NK cells and the ability to secrete cytokines, have a faster killing effect and require fewer NK cells, endow NK cells with continuous killing ability, and the combined application with anti-PD-L1 antibody can further improve the persistence of CAR-NK cells in vivo and intratumoral infiltration, enhancing the killing effect.

[0007] However, the current number of antibodies and chimeric antigen receptors targeting CLDN6 that have been developed is insufficient, and most of them are only prepared based on traditional antibodies or single-chain antibodies (scFv). In contrast, nanobodies have advantages such as small molecular weight, low production cost, high stability, low immunogenicity, and strong tissue penetration ability. Therefore, providing more single-domain antibodies and chimeric antigen receptors targeting CLDN6 has important application value for the treatment of solid tumors.

[0008] In addition, 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 that many CLDN6 antibodies in the prior art cannot 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 weak affinity for CLDN6. Therefore, there is still room for improvement in the therapeutic effect of related drugs. In addition, all CLDN6 antibodies in the prior art are murine / humanized antibodies. When applied to antibody drugs, their large molecular weight is not conducive to improving the penetration rate of the drug into solid tumor lesions; when applied to cell drugs, they are usually expressed in the form of single-chain antibodies (scFv), and their 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 the 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

[0009] Aiming at the deficiencies of the existing technologies, the purpose of the present invention is to provide a Claudin antibody E02 and its applications. To solve the problem that the existing CLDN6 antibodies cannot have both high affinity and specificity, the present invention screens and obtains a CLDN6 antibody with higher affinity, better specificity and smaller molecular weight than the existing CLDN6 antibodies. At the same time, the antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by it 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 antibodies and cell drugs, and has important application value in the development of tumor treatment drugs.

[0010] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0011] In the first aspect, the present invention provides a Claudin antibody E02. The antibody E02 is a single-domain antibody, and the heavy-chain variable region of the single-domain antibody includes CDR1, CDR2 and CDR3;

[0012] The amino acid sequence of CDR1 includes that shown in SEQ ID NO.1;

[0013] The amino acid sequence of CDR2 includes that shown in SEQ ID NO.2;

[0014] The amino acid sequence of CDR3 includes that shown in SEQ ID NO.3.

[0015] Preferably, the heavy-chain variable region sequence of the single-domain antibody includes that shown in SEQ ID NO.4.

[0016] In the second aspect, the present invention provides a heavy-chain antibody, which includes the heavy-chain variable region sequence described in the first aspect and the full-length or partial amino acid sequence of the crystallizable fragment Ig Fc of human immunoglobulin.

[0017] Preferably, the Ig Fc includes the full-length or partial sequence of the Fc segment of IgG1, IgG2, IgG3 or IgG4, or a combination thereof.

[0018] Preferably, the amino acid sequence of IgG1 Fc includes that shown in SEQ ID NO.5.

[0019] Preferably, the amino acid sequence of the heavy-chain antibody includes that shown in SEQ ID NO.6.

[0020] In the present invention, alpacas were immunized 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) were isolated, RNA was extracted and reverse transcribed to obtain cDNA. Using specific primers for alpaca single-domain antibodies, the VHH sequences were amplified and cloned into a phage expression plasmid to construct a phage display library. CHO-S cells overexpressing CLDN9 were incubated with the phage display library to eliminate antibody clones that bind to CLDN9 cells. Then, the phage display library was subjected to 3-4 rounds of enrichment panning using virus-like particles (VLP) containing CLDN6. Monoclonal clones were picked from the enriched products, and the specific binding of the candidate antibodies to CLDN6-VLP was detected by ELISA. After the positive antibodies were expressed and purified, their affinities for CHO-S cells overexpressing CLDN6 or CLDN9 were detected respectively. Antibody clones with strong affinity for CLDN6 and weak affinity for CLDN9 were selected and co-cultured with NK cells and CLDN6-positive Huh-7 hepatoma cells to detect the antibody-dependent cell-mediated cytotoxicity (ADCC) of each clone. Meanwhile, the CLDN6 single-domain antibody sequence was cloned into a CAR expression vector to construct CAR-T cells with the CLDN6 single-domain antibody as the antigen-binding domain, and their killing abilities against CLDN6-positive OVCAR3 cells in vitro and in vivo were detected. The results showed that compared with the prior art, the CLDN6 single-domain antibody obtained in the present invention had 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 were better.

[0021] In a third aspect, the present invention provides a nucleic acid molecule encoding the tight junction protein antibody E02 described in the first aspect or the heavy chain antibody described in the second aspect.

[0022] In a fourth aspect, the present invention provides an expression vector containing the nucleic acid molecule described in the third aspect; and after transfection / transduction / transformation of a host cell, the host cell expresses the tight junction protein antibody E02 described in the first aspect or the heavy chain antibody described in the second aspect.

[0023] 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.

[0024] In a sixth aspect, the present invention provides a composition for detecting claudin, the composition comprising the claudin antibody E02 described in the first aspect or the heavy chain antibody described in the second aspect.

[0025] In a seventh aspect, the present invention provides an anti-claudin chimeric antigen receptor that recognizes claudin, the chimeric antigen receptor being composed of the following structures in series: a signal peptide, the claudin antibody E02 described in the first aspect, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain of human CD3.

[0026] 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.

[0027] Preferably, the amino acid sequence of the signal peptide comprises that shown in SEQ ID NO.8.

[0028] 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.

[0029] Preferably, the amino acid sequence of the hinge region comprises that shown in SEQ ID NO.9.

[0030] 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.

[0031] Preferably, the amino acid sequence of the transmembrane domain comprises that shown in SEQ ID NO.10.

[0032] 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.

[0033] Preferably, the amino acid sequence of the intracellular co-stimulatory domain comprises that shown in SEQ ID NO.11.

[0034] Preferably, the human CD3 intracellular signal transduction domain is selected from the following proteins: CD3ζ, CD3ε, CD3γ, CD3δ, or a combination thereof.

[0035] Preferably, the amino acid sequence of the human CD3 intracellular signal transduction domain comprises that shown in SEQ ID NO.12.

[0036] Preferably, the amino acid sequence of the anti-occludin chimeric antigen receptor comprises that shown in SEQ ID NO.7.

[0037] In an eighth aspect, the present invention provides a nucleic acid molecule encoding the anti-occludin chimeric antigen receptor described in the seventh aspect.

[0038] 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.

[0039] 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.

[0040] Preferably, the host cell includes a T cell.

[0041] 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.

[0042] In a twelfth aspect, the present invention provides the application of any one or at least two combinations of the occludin antibody E02 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.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 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 efficacy, stronger tissue penetration, better in vivo stability, and superior pharmacodynamic effects; based on it, the engineered immune cells developed have higher exogenous gene expression levels, better efficacy, superior pharmacodynamic effects, and lower modification difficulty; based on it, the detection reagents developed have higher sensitivity and lower false positives. Description of the Drawings

[0045] Figure 1 It is the detection result of the titer of alpaca serum No. 72.

[0046] Figure 2 It is the detection result of the titer of alpaca serum No. 110.

[0047] Figure 3 It is the amplification result of the VHH region of the alpaca antibody; among them, A is the first-round PCR amplification of the heavy-chain antibody fragment; B is the second-round PCR amplification of the VHH region fragment.

[0048] Figure 4 It is the result of detecting the specificity of the recombinant single-domain antibody by flow cytometry.

[0049] Figure 5 It is the result of detecting the EC of the recombinant single-domain antibody by flow cytometry. 50 of.

[0050] Figure 6 It is the result of detecting the EC of the recombinant single-domain antibody by flow cytometry. 50 of; among them, A is the concentration-effect curve detected with CHO-S-CLDN6 cells; B is the concentration-effect curve detected with CHO-S-CLDN9 cells.

[0051] 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.

[0052] Figure 8 It is the result of detecting the expression of CLDN6 in liver cancer cells by flow cytometry.

[0053] Figure 9 It is the IncuCyte real-time quantitative live cell imaging to analyze the ADCC function mediated by the recombinant single-domain antibody.

[0054] Figure 10 It is the CAR molecular structure targeting CLDN6 and its expression in T cells; A is the CAR molecular structure; B is the detection of the expression of BVHCN6-002 in T cells by flow cytometry; C is the detection of the expression of BN124 in T cells by flow cytometry.

[0055] Figure 11It is the result of analyzing the killing function and specificity of CAR-T cells targeting CLDN6 by IncuCyte real-time quantitative live cell imaging analysis.

[0056] Figure 12 It is the result of analyzing the function and specificity of CAR-T cells targeting CLDN6 to secrete IFN-γ by ELISA detection.

[0057] Figure 13 It is the antitumor effect of CAR-T cells targeting CLDN6 on ovarian cancer in vivo. Specific implementation manners

[0058] 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 described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0059] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0060] The sources of the following experimental materials are as follows:

[0061] 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).

[0062] The sequences involved in the present invention and their numbers are as follows:

[0063] The amino acid sequence of 89A VL SEQ ID NO.13.

[0064] The amino acid sequence of 89A VH SEQ ID NO.14.

[0065] The amino acid sequence of 67AVL, SEQ ID NO.15.

[0066] The amino acid sequence of 67AVH, SEQ ID NO.16.

[0067] The amino acid sequence of 72-2-C01, SEQ ID NO.17.

[0068] The amino acid sequence of 72-2-E02, SEQ ID NO.18.

[0069] The amino acid sequence of 110-1-E02, SEQ ID NO.4.

[0070] The CDR1 amino acid sequence of 110-1-E02, SEQ ID NO.1: GSIFSFYA.

[0071] The CDR2 amino acid sequence of 110-1-E02, SEQ ID NO.2: GINDFGMT.

[0072] The CDR3 amino acid sequence of 110-1-E02, SEQ ID NO.3: NAQRLGSPPY.

[0073] The amino acid sequence of human IgG1 Fc, SEQ ID NO.5.

[0074] The amino acid sequence of the heavy chain antibody described above, SEQ ID NO.6.

[0075] The amino acid sequence of human CD8 signal peptide, SEQ ID NO.8.

[0076] The amino acid sequence of human IgG4 hinge, SEQ ID NO.9.

[0077] The amino acid sequence of human CD8 transmembrane domain, SEQ ID NO.10.

[0078] The amino acid sequence of human 4-1BB intracellular domain, SEQ ID NO.11.

[0079] The amino acid sequence of human CD3ζ intracellular domain, SEQ ID NO.12.

[0080] The amino acid sequence of the anti-human CLDN6 chimeric antigen receptor described above, SEQ ID NO.7.

[0081] Example 1: Alpaca immunization and antibody titer determination

[0082] (1) Construction of CLDN6- and CLDN9-overexpressing cells

[0083] 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, obtaining cell lines 293T-CLDN6 and CHO-S-CLDN6 overexpressing CLDN6, which were used for alpaca immunization, serum titer detection, and antibody affinity verification respectively.

[0084] 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, obtaining the cell line CHO-S-CLDN9 overexpressing CLDN9, which was used for antibody panning and affinity verification.

[0085] (2) Alpaca immunization and serum titer detection

[0086] 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 serially diluted with PBS. 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 then detected using a flow cytometer.

[0087] 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.

[0088] The mean fluorescence intensity (MFI) detected by flow cytometry after incubating the sera of 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. In contrast, 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.

[0089] Table 1

[0090]

[0091] Table 2

[0092]

[0093] Table 3

[0094]

[0095] Table 4

[0096]

[0097] Example 2: Construction of a single-domain antibody phage surface display library

[0098] (1) Cloning of VHH antibody fragment

[0099] After confirming that the sera of two alpacas contained CLDN6-specific antibodies, 100 mL of peripheral blood was collected, and PBMC was 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 amplified by PCR 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 3In A), using the first-round PCR product as a template, specific primers (the upstream primer binds to the antibody FR1 region, with an SfiI restriction site GGCCCAGCCGGCC at the 5' end, SEQ ID NO. 19; the downstream primer binds to the antibody Hinge and FR4 regions, with an SfiI restriction site GGCCACGAAGGCC at the 5' end, SEQ ID NO. 20) are used to amplify the VHH fragment of the heavy-chain antibody. The PCR products are analyzed by electrophoresis using 1% agarose, and the target fragment with a molecular weight of about 400 bp is recovered and separated ( Figure 3 In B). The target fragment is stored at -80 °C (the storage solution contains 1 / 10 volume of 3 M sodium acetate, 1 μg / μL glycogen, and 80% absolute ethanol).

[0100] (2) Electroporation of the library vector and detection of library capacity and diversity

[0101] The phage surface display vector pComf and the above-obtained VHH fragment library are digested with SfiI endonuclease. The linearized pComf vector and the VHH fragment library are ligated overnight at 16 °C with T4 ligase. The ligation product is stored at -80 °C (the storage solution contains 1 / 10 volume of 3 M sodium acetate, 1 μg / μL glycogen, and 80% absolute ethanol). A mixture of 300 μL of the above ligation product and Escherichia coli SS320 competent cells is added to a pre-chilled electroporation cuvette, and the ligation product is transformed into Escherichia coli by electroporation (2500 V, 5 ms). Then, 20 mL of SOC medium is added to resuspend the cells, and the cells are recovered and cultured on a shaker at 37 °C for 1 h. 15 mL of the bacterial solution is taken for subsequent phage production and enrichment, and the remaining 5 mL of the electroporated product is added with an equal volume of 50% glycerol, mixed evenly, and stored at -80 °C. Additionally, 20 μL of the bacterial solution is serially diluted with 2YT medium and evenly spread on an LB plate containing ampicillin, and cultured overnight at 37 °C to 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 72# alpaca PBMC is 1.33×10 9 , and the library capacity of the single-domain antibody phage surface display library obtained from 110# alpaca PBMC is 1.18×10 9 . Twenty monoclonal colonies on the plate are picked and subjected to Sanger sequencing with the M13R primer. The results show that the phage library sequences have large differences, no repeated sequences, and good diversity.

[0102] (3) Enrichment of the phage surface display library

[0103] Take 15 mL of the bacterial solution after the above electroporation and recovery culture, and dilute it with 2YT to adjust the OD 600is about 0.25, add ampicillin with a final concentration of 100 μg / mL, place it in a constant temperature shaker, and culture at 225 rpm in a 37 °C shaker. When OD 600 is 0.6, add M13KO7 helper phage (volume of M13KO7 helper phage added = 10 × volume of bacterial solution × OD 600 × 5 × 10 8 / titer of M13KO7), shake well and let it stand at 37 °C for 30 min, then culture at 225 rpm in a 37 °C shaker for 1 h. After the helper phage infects the target strain, centrifuge at 6000 rpm for 10 min, discard the supernatant, resuspend with 2YT-AK medium, and culture overnight at 200 rpm in a 25 °C shaker. 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), mix well and place at 4 °C. After standing for 2 h, centrifuge at 10000 rpm for 30 min at 4 °C, collect the phage precipitate, and resuspend 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 let it stand at 4 °C for 10 min, then centrifuge at 12000 g for 10 min at 4 °C, discard the supernatant. Add 1 mL of PBS to resuspend, 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 display.

[0104] Take 10 μL of the precipitate for 10-fold serial dilution, add 200 μL of ER2738 Escherichia coli with OD 600 of 0.5 respectively, mix well and place in a 37 °C water bath, let it stand for 10 min, and then spread on an LB plate. After overnight culture at 37 °C, count the plaques to obtain the titer of the phage display library.

[0105] Example 3: Panning of the target antibody library

[0106] (1) First-round panning and product amplification

[0107] Block a 1.5 mL centrifuge tube with 3% MPBS, 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 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 to 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 allowed to stand at room temperature. After blocking for 1 h, 3% MPBS buffer was used. The CHO-S-CLDN9 cells were centrifuged at 500 g for 10 min, the supernatant was taken and added to the wells containing CLDN6-VLP protein, and the mixture was incubated with shaking at room temperature for 1 h. The phage supernatant in the wells containing CLDN6-VLP protein was discarded, and the wells were washed 6 times with 0.05% PBST and 4 times with PBS. 100 μL of Gly-HCl elution buffer at 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 twice, and the eluted product was stored at 4°C in a pre-blocked centrifuge tube (washed twice with PBS after blocking).

[0108] 20 mL of 2YT medium was taken, and tetracycline at a final concentration of 100 μg / mL and 20 μL of Escherichia coli ER2738 were added and cultured at 37°C and 225 rpm in an incubator until the OD 600 reached 0.5. The above-eluted phage product was added to the ER2738 bacterial solution. After mixing, the mixture was incubated statically at 37°C for 30 min, and 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 600 of the bacterial solution reached 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 let stand at 37 °C for 30 min. Add ampicillin with a final concentration of 100 μg / mL to the bacterial solution, culture at 37 °C at 225 rpm for 45 min, then centrifuge at 8000 rpm for 20 min, discard the supernatant and collect the bacterial cells. 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 aliquot the supernatant into 40 mL centrifuge tubes. Add 10 mL of PEG / NaCl to each tube, mix well and place on ice. Let stand for 1 h to precipitate the phages, then 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 let stand at 4 °C for 10 min to precipitate the phages. Centrifuge at 12000 g at 4 °C for 10 min, then 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.

[0109] (2) Second to fourth rounds of panning and product amplification

[0110] Dilute the CLDN6-VLP antigen with CBS solution to 10 μg / mL, add it to a 96-well solid-phase plate, and coat 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 Tables 5 and 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.

[0111] Table 5

[0112]

[0113]

[0114] Table 6

[0115]

[0116] (3) ELISA detection and sequencing of monoclonal phages

[0117] Infect Escherichia coli ER2738 with the single-domain antibody phage surface display library after the above-mentioned 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 on 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-mentioned overnight cultured bacterial solution to each well and culture at 37°C at 225 rpm until the 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, and 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.

[0118] While amplifying the phage monoclonal, coat the CLDN6-VLP antigen protein with CBS at pH 9.6 onto an ELISA plate (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-mentioned monoclonal phage supernatant in a 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, then 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 the OD 450 . 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 110-1-E02 (see the sequence listing).

[0119] Example 4: Expression of recombinant single-domain antibody and detection of its binding to the target protein

[0120] 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, used as a positive control, was expressed.

[0121] 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 the 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.

[0122] 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, 110-1-E02 strongly bound to CHO-S-CLDN6 cells and hardly bound to CHO-S-CLDN9 cells, indicating that its specificity for CLDN6 was higher than that of other antibodies.

[0123] Example 5: Purification of the recombinant antibody and determination of the half-maximal effective concentration (EC 50 )

[0124] To determine the EC of the 110-1-E02 antibody 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 requirement (calculated as 20 mg IgG / mL Protein A magnetic beads). Incubate at 120 rpm at room temperature for 1 - 4 hours or overnight at 4°C. 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.

[0125] After incubating 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 Protein A magnetic beads twice, combine the elution solutions, and add neutralization buffer to adjust the solution pH. 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 for later use.

[0126] Gradient-dilute the target antibody and incubate it with 3×10 5 CHO-S-CLDN6 and CHO-S-CLDN9 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. After discarding 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.

[0127] The results are as Figure 5 , Figure 6 and shown in Table 7. Table 7 is the EC 50 result of the recombinant single-domain antibody detected by flow cytometry. The EC 50 value of the positive control antibody 89A for CHO-S-CLDN6 is 1.26 μg / mL, and the EC 50 value of 110-1-E02 for CHO-S-CLDN6 is 0.5215 μg / mL, which is 58.6% lower than the EC 50 value of the positive antibody, indicating that 110-1-E02 has a higher affinity for CLDN6. The EC of the positive control antibody 89A for CHO-S-CLDN950 The value was 14.49 μg / mL, and the binding of 110-1-E02 to CHO-S-CLDN9 could not be detected at the corresponding dilution gradients, indicating that the affinity of 110-1-E02 for CLDN9 was extremely weak, significantly lower than that of the positive control antibody. The above results show that, compared with the prior art, 110-1-E02 has higher affinity and specificity for CLDN6.

[0128] Table 7

[0129]

[0130] Example 6: Detection of CLDN6 Expression in Tumor Cells

[0131] (1) Detection of Expression in Ovarian Cancer Cells

[0132] 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 using the A-4 antibody by immunohistochemistry (IHC). 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 110-1-E02 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 the cells were washed 3 times with PBS and then resuspended in 200 μL of PBS for flow cytometry analysis. As Figure 7 shown, the 110-1-E02 antibody could only detect significant expression of CLDN6 in OVCAR-3-Luc-mCherry cells, indicating that the 110-1-E02 antibody also has good affinity and specificity for CLDN6 in tumor cells.

[0133] (2) Detection of Expression in Hepatocellular Carcinoma Cells

[0134] 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 and subjected to 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.

[0135] Example 7: Detection of ADCC function

[0136] 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 at the same time, the CLDN6 positive control antibody 89A or the target antibody 110-1-E02 at a final concentration of 5 μg / mL was added.

[0137] 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 number of cells in that group, indicating a better killing effect of NK cells. The results are as Figure 9 shown. Compared with the NK cell group without antibody addition, both the 110-1-E02 antibody and the positive control antibody could significantly improve the killing effect of NK cells on tumor cells, indicating that 110-1-E02 has a good function of mediating ADCC.

[0138] Example 8: Construction of CAR-T cells based on the target antibody

[0139] (1) Design of the CAR molecule

[0140] The target gene structure of the lentiviral vector involved in this example is as Figure 10 shown in A.

[0141] BVHCN6-002 is composed of the following structures in series: human CD8 signal peptide (abbreviated as SP), the CLDN6 antibody E02 [110-1-E02, abbreviated as VHH(110-1-E02)], 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).

[0142] BN124 is composed of the following structures connected in series in sequence: human CD8 signal peptide (abbreviated as SP), anti-human CLDN6 single-chain antibody [abbreviated as scFv(67A)], 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).

[0143] (2) Lentivirus preparation

[0144] The above-mentioned CAR molecule expression sequence was synthesized by total gene synthesis, and then ligated into 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. Using the transfection reagent Lipofectamine 3000, the above-mentioned 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 virus supernatant was collected 48 h after transfection, centrifuged at 3000 rpm for 10 - 15 min at 4°C, filtered through a filter membrane with a pore size of 0.45 μm, and finally ultracentrifuged at 25000 rpm for 2 - 3 h at 4°C. The obtained virus concentrate was stored at -80°C and named BVHCN6-002 and BN124 respectively. Finally, using Jurkat cells as materials, the above-mentioned lentivirus was detected for its active titer.

[0145] (3) CAR-T cell preparation

[0146] 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 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 for transduction at a dosage of MOI = 5 TU / mL, and 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 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 culture 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. Cells were harvested on the 10th day after transduction and cryopreserved 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.

[0147] (4) Detection of CAR molecule expression

[0148] The BVHCN6-002 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 the negative control, the CAR molecule expression rate of BVHCN6-002 cells was detected by flow cytometry. The results are as Figure 10 shown in B, and the expression rate of BVHCN6-002 CAR was 42.2%.

[0149] The BN124 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 CAR-T cells were incubated with CLDN6-VLP for 1 h. Subsequently, the supernatant was removed by centrifugation and the cells were washed twice with FACS buffer. Then the CAR-T cells were incubated with the AF647-labeled anti-human CLDN6 antibody for 1 h. The supernatant was removed by centrifugation and the cells were washed twice with FACS buffer. Using Ctrl T cells as the negative control, the CAR molecule expression rate of BN124 cells was detected by flow cytometry. The results are as Figure 10 shown in C, and the expression rate of BN124 CAR was 97.8%.

[0150] Example 9: In vitro and in vivo functional study of CAR-T cells

[0151] (1) In vitro killing experiment of BVHCN6-002 cells

[0152] OVCAR-3-Luc-mCherry cells and SK-OV-3-Luc-mCherry cells were resuspended with medium at a density of 1×10 5 cells / mL and inoculated into 96-well plates at a volume of 100 μL per well. After culturing overnight in the IncuCyte SX5 live cell imaging analysis system, CAR-T cells were added for co-culture at an effector-to-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 ended, the change in the mCherry fluorescence signal of the target cells in each group was calculated by the IncuCyte SX5 software. The lower the signal value, the fewer the cell number in that group, and the better the killing effect of CAR-T cells. The results are asFigure 11 As 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 0.714 ± 0.016) was only slightly improved compared to the Ctrl T group (relative fluorescence area at the last time point was 0.832 ± 0.018), while the killing effect of the BVHCN6-002 group (relative fluorescence area at the last time point was 0.191 ± 0.021) was significantly improved (P < 0.05). For CLDN6-negative SK-OV-3-Luc-mCherry cells, the killing effects of BVHCN6-002 cells were consistent with those of the BN124 group and the Ctrl T group. This result indicates that, compared with existing antibodies, the CAR-T cells constructed based on the 110-1-E02 antibody have a stronger specific killing function against CLDN6-positive tumor cells.

[0153] To study the specificity of the killing function of BVHCN6-002 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 BVHCN6-002 cells were added respectively. After overnight co-culture, the culture supernatants were collected, and the content of IFN-γ was detected by ELISA. The results are as Figure 12 shown. BVHCN6-002 was significantly activated only by CLDN6-positive OVCAR-3-Luc-mCherry cells and released high levels of IFN-γ, while it was hardly activated by CLDN6-negative target cells. This result further demonstrates that the CAR-T cells constructed based on the 110-1-E02 antibody have good specificity in the killing function against CLDN6-positive tumor cells.

[0154] (2) In vivo tumor suppression experiment of BVHCN6-002 cells

[0155] The OVCAR-3-Luc-mCherry target cells in the logarithmic growth phase with good growth state were collected by trypsin digestion method. After washing with physiological saline, the cell density was adjusted to 2×10 7 / mL. 100 μL of cell suspension was subcutaneously injected into the right axilla area of severely immunodeficient mice, that is, each mouse was inoculated with 2×10 6 target cells. On the 7th day after inoculating the target cells (or when the average tumor volume was about 100 mm 3 ), CAR-T cells, Ctrl T cells, and vehicle were injected through the tail vein respectively. The day of injecting the test substance was recorded as day 0 of treatment. The tumor size was measured 2 - 3 times a week, and the results are as Figure 13As shown, the tumor volumes of both the solvent control group and the CtrlT control group gradually increased. The tumors in the BN124 treatment group also gradually increased and there was no significant difference compared with the Ctrl T group (P>0.05). In contrast, the tumor volume in the BVHCN6-002 treatment group did not increase significantly over time and there were significant differences compared with the control group and the BN124 treatment group (P<0.05). This result indicates that the CAR-T cells constructed based on 110-1-E02 in 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.

[0156] In summary, the tight junction protein antibody E02 of 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 antibody-dependent cell-mediated cytotoxicity mediated by it is better, and the chimeric antigen receptor molecule constructed based on it can better mediate the killing of tumors by immune cells.

[0157] 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 any person 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. A tight junction protein antibody, characterized in that The antibody 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 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; The heavy chain variable region sequence of the single-domain antibody is shown in SEQ ID NO.

4.

2. A heavy chain antibody, characterized in that The heavy chain antibody comprises the heavy chain variable region sequence described in claim 1 and the full-length or partial amino acid sequence of the crystallizable segment Ig Fc of human immunoglobulin.

3. The heavy chain antibody according to claim 2, 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.

4. The heavy chain antibody according to claim 3, characterized in that The IgG1 Fc amino acid sequence is shown in SEQ ID NO.

5.

5. The heavy chain antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain antibody is shown in SEQ ID NO.

6.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the claudin antibody according to claim 1 or the heavy chain antibody according to any one of claims 2 to 5.

7. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule of claim 6; and after transfection / transduction / transformation of a host cell, the expression vector enables the host cell to express the tight junction protein antibody of claim 1 or the heavy chain antibody of any one of claims 2-5.

8. A host cell, characterized in that The host cell contains at least one copy of the expression vector of claim 7, or at least one copy of the nucleic acid molecule of claim 6.

9. A composition for detecting tight junction proteins, characterized in that: The composition comprises the claudin antibody according to claim 1 or the heavy chain antibody according to any one of claims 2 to 5.

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

11. The anti-claudin chimeric antigen receptor according to claim 10, 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.

12. The anti-claudin chimeric antigen receptor according to claim 11, characterized in that The amino acid sequence of the signal peptide is shown in SEQ ID NO.

8.

13. The anti-claudin chimeric antigen receptor according to claim 10, 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.

14. The anti-claudin chimeric antigen receptor according to claim 13, characterized in that: The amino acid sequence of the hinge region is shown in SEQ ID NO.

9.

15. The anti-claudin chimeric antigen receptor according to claim 10, 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.

16. The anti-claudin chimeric antigen receptor according to claim 15, characterized in that: The amino acid sequence of the transmembrane domain is shown in SEQ ID NO.

10.

17. The anti-claudin chimeric antigen receptor according to claim 10, 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, PD1, Dap10, CDS, ICAM-1, LFA-1 / CD11a / CD18, ICOS, NKG2D, GITR, OX40L, or a combination thereof.

18. The anti-claudin chimeric antigen receptor according to claim 17, characterized in that: The amino acid sequence of the intracellular co-stimulatory domain is shown in SEQ ID NO.

11.

19. The anti-claudin chimeric antigen receptor according to claim 10, characterized in that: The human CD3 intracellular signal transduction domain is selected from the following proteins: CD3ζ, CD3ε, CD3γ, CD3δ, or a combination thereof.

20. The anti-claudin chimeric antigen receptor according to claim 19, characterized in that: The amino acid sequence of the human CD3 intracellular signal transduction domain is shown in SEQ ID NO.

12.

21. The anti-claudin chimeric antigen receptor according to claim 10, characterized in that: The amino acid sequence of the anti-tight junction protein chimeric antigen receptor is shown in SEQ ID NO.

7.

22. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-claudin chimeric antigen receptor according to any one of claims 10-21.

23. An expression vector, characterized in that The expression vector contains the nucleic acid molecule of claim 22; and after transfection / transduction / transformation of a host cell, the expression vector enables the host cell to express the anti-claudin chimeric antigen receptor of any one of claims 10-21.

24. A cell expressing an anti-tight junction protein chimeric antigen receptor, characterized in that: The cell is obtained by transfecting / transducing / transforming a host cell with the expression vector according to claim 23 or the nucleic acid molecule according to claim 22, and expresses the anti-claudin chimeric antigen receptor according to any one of claims 10-21.

25. The cell expressing anti-claudin chimeric antigen receptor according to claim 24, characterized in that: The host cells include T cells.

26. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cell expressing the anti-claudin chimeric antigen receptor according to claim 24 or 25.

27. Use of the tight junction protein antibody according to claim 1, the heavy chain antibody according to any one of claims 2 to 5, or the host cell according to claim 8, or a combination of any one or at least two thereof, in the preparation of a drug for detecting ovarian cancer or liver cancer.

28. Use of any one or a combination of at least two of the claudin antibody of claim 1, the heavy chain antibody of any one of claims 2 to 5, the host cell of claim 8, the anti-claudin chimeric antigen receptor of any one of claims 10 to 21, or the cell expressing the anti-claudin chimeric antigen receptor of claim 24 or 25 in the preparation of a drug for treating ovarian cancer or liver cancer.

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