CAR-T cell targeting Nectin4 and application of CAR-T cell in cancer treatment

Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to a CAR-T cell targeting Nectin4 and application of the CAR-T cell in cancer treatment. The preparation method of the Nectin4-targeted CAR-T cell comprises the following step: introducing the Nectin4-targeted CAR gene into a T cell through a lentiviral vector, so that the CAR gene is integrated onto a T cell genome. In order to overcome the technical problems that in the prior art, the Nectin4-targeted CAR-T cells are few in variety and poor in treatment effect, a new antibody sequence of a target spot is found, and the Nectin4-targeted CAR-T cell is provided and can be applied to treatment of cancers such as ovarian cancer and breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy, and particularly relates to a Nectin4-targeted CAR-T cell and its application in cancer treatment. Background Art

[0002] Adoptive T cell immunotherapy is a promising method for cancer treatment. The immunotherapy method enhances the specificity of isolated genetically modified human T cells against specific tumor-associated antigens. Genetic modification may involve the expression of chimeric antigen receptors or exogenous T cell receptors to transplant antigen specificity onto T cells. Compared with exogenous T cell receptors, the specificity of chimeric antigen receptors is derived from the variable domains of monoclonal antibodies. Therefore, T cells expressing chimeric antigen receptors (CAR-T cells) induce tumor immune responses in a major histocompatibility complex-unrestricted manner. Adoptive T cell immunotherapy has been used as a clinical therapy for many cancers, including B cell malignancies, multiple myeloma, neuroblastoma, glioblastoma, advanced glioma, ovarian cancer, mesothelioma, melanoma, prostate cancer, and pancreatic cancer, etc.

[0003] Lectins Nectin1, Nectin2, and Nectin3 are widely expressed in normal adult tissues, but Nectin4, also known as PVRL4 (poliovirus receptor-like 4), is encoded by the NECTIN4 gene and has a molecular mass of about 66 kDa. Nectin4 exists only during embryogenesis and its expression level decreases in normal tissues after adulthood. However, Nectin4 is overexpressed in a variety of tumor cells, such as lung cancer, breast cancer, pancreatic cancer, ovarian cancer, urothelial cancer, and bladder cancer, etc. Nectin4 is a type I single-pass transmembrane protein and belongs to the Ca 2+ -dependent immunoglobulin-like molecule, which consists of an extracellular domain, a transmembrane region, and a cytoplasmic tail. The extracellular domain contains a membrane-distal IgV domain and two IgC domains, which can bind to a variety of growth factor receptors, including EGFR, HER2, etc., and then affect multiple signal transduction pathways, having a significant impact on cell growth, migration, and apoptosis, and being closely related to the occurrence and metastasis of tumors. The cytoplasmic tail of Nectin4 can bind to the adaptor molecule afadin through its C-terminal amino acid sequence, and then afadin will bind to and recruit filamentous actin components (F-actin), thereby promoting cell-cell adhesion. Nectin4 promotes tumor cell proliferation, differentiation, migration, invasion, etc. by activating the PI3K / Akt pathway. Targeting Nectin4 can be an effective strategy for treating cancers with high Nectin4 expression.

[0004] As disclosed in CN116589584A, the existing technology has monoclonal antibodies or fragments thereof designed to target human Nectin4 protein, which include heavy chains and light chains. The heavy chain includes three heavy chain complementarity-determining regions 1 to 3 (HCDR1, HCDR2, and HCDR3), the light chain includes three light chain complementarity-determining regions 1 to 3 (LCDR1, LCDR2, and LCDR3), and the heavy chain also includes the heavy chain variable region VH, and the light chain also includes the light chain variable region VL. Any amino acid sequence in the three heavy chain complementarity-determining regions can be paired and used in a double-stranded combination with any amino acid sequence in the three light chain complementarity-determining regions, or any amino acid sequence in the three heavy chain complementarity-determining regions can be used alone, or any amino acid sequence in the three light chain complementarity-determining regions can be used alone. The amino acid sequences of the corresponding complementarity-determining regions in the heavy and light chain variable regions of the monoclonal antibody are paired and used to express a humanized antibody. After the gene construction plasmid expression cassette for the humanized antibody, the expression cassette is transferred into 293T cells through a delivery system for antibody expression; among them, the delivery system can be one of lentivirus, retrovirus, ordinary plasmid vector, episomal vector, nano-delivery system, electroporation, and transposon. The recombinant cell line is an immune cell, which can be any one of T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, TCR-T cells. When the immune cell expresses the chimeric antigen receptor CAR, NK cells, NKT cells, TIL cells, gamma-delta T cells are equivalent to T cells (or T cells can replace NK cells). The monoclonal antibody targeting human Nectin4 protein of any of the above can be made into a pharmaceutically acceptable carrier, diluent, or excipient and applied to biomaterials and / or biopharmaceuticals, all of which have good shaping effects and maintain good drug efficacy at the same time. When the monoclonal antibody is applied to a biopharmaceutical, the biopharmaceutical contains an expression cassette (such as a gene expression cassette), a recombinant vector (such as a plasmid), a recombinant protein (such as a fusion protein, antibody protein, etc.), a recombinant microorganism (such as Escherichia coli, phage, etc.), or a recombinant cell line (such as an immune cell, CHO cell, etc.) constructed from the above nucleic acid sequence or amino acid sequence, and these nucleic acid sequences or amino acid sequences are derived from the above monoclonal antibody; among them, the recombinant vector includes a gene recombinant expression vector and a chimeric antigen receptor. When the monoclonal antibody is applied to a biopharmaceutical, it exists as a component of the biopharmaceutical configuration, and the biopharmaceutical component also contains a reagent for detecting the concentration of human Nectin4 protein, a reagent for detecting the expression level of human Nectin4 protein on the surface of tumor cells, an antibody-conjugated toxin for killing human Nectin4-positive cells, an antibody-conjugated with other antibodies to make a polyclonal antibody targeting human Nectin4 and other antigens, and an antibody-conjugated with other proteins to make a functional recombinant protein targeting human Nectin4. The chimeric antigen receptor (CAR) of this technology includes an extracellular domain, a transmembrane domain, and an intracellular domain.Among them, the extracellular domain includes an antigen-binding domain, and the intracellular domain includes a co-stimulatory signaling region, an intracellular region of a cytokine receptor, and a portion of the CD3ζ chain. The co-stimulatory signaling region refers to a part of the intracellular domain including a co-stimulatory molecule, and the co-stimulatory molecule is a cell surface molecule required for the effective response of lymphocytes to antigens. The extracellular domain of the CAR provided by this technology includes a humanized antibody heavy and light chain variable region antigen-binding domain targeting Nectin4. When the CAR is expressed in T cells, antigen recognition can be carried out based on antigen-binding specificity or protein receptor binding. The antigen-binding domain is fused with the intracellular domains from co-stimulatory molecules and the CD3ζ chain. The antigen-binding domain is respectively fused with the intracellular domains combined with the CD28, 4-1BB, ICOS signaling domains and the CD3ζ signaling domain. Therapeutic applications are carried out using cells transduced with a lentiviral vector of a nucleic acid construct encoding the antibody of the present invention. The transduced T cells can induce CAR-mediated T cell responses. The injected cells can kill the recipient's tumor cells, and the CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control. Moreover, the CAR-T cell membrane can express a chimeric antigen receptor of a humanized antibody scFv structure targeting Nectin4, specifically killing Nectin4-positive target cells without the risk of off-target effects.

[0005] In order to overcome the technical problems in the prior art that there are few types of CAR-T cells targeting Nectin4 and the treatment effect is not good, developing new antibody sequences for this target in order to provide a more efficient treatment plan has become a research hotspot. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a CAR-T cell targeting Nectin4 and its application in cancer treatment. The present invention has discovered new antibody sequences for this target, and the designed CAR-T cells can be applied to the treatment of cancers such as ovarian cancer and breast cancer.

[0007] The technical solution adopted by the present invention to achieve the above object is:

[0008] A method for preparing a CAR-T cell targeting Nectin4, comprising the following steps:

[0009] Introduce the CAR gene targeting Nectin4 into T cells through a lentiviral vector, so that the CAR gene is integrated into the T cell genome;

[0010] The CAR gene targeting Nectin4 has the following structure and sequence: CD8 signal peptide - anti-Nectin4 scFv - CD8α transmembrane domain - CD28 co-stimulatory domain - 4-1BB co-stimulatory domain - CD3ζ signaling domain; the nucleotide sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:1; the protein sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:2.

[0011] The specific steps are as follows:

[0012] (1) Select cells with passage numbers within 3 generations, adjust the cell density according to the cell growth density and status, and plate 293T cells with a growth density reaching 80%.

[0013] (2) When the growth density reaches 60 - 90% and the cell status is good, virus packaging can be carried out.

[0014] (3) Use the lentiviral packaging plasmids pPACKH1 - GAG, pPACKH1 - REV, and pVSV - G, as well as the constructed lentiviral plasmid containing the CAR structure, and perform the lentiviral plasmid ratio according to the plasmid instruction manual.

[0015] (4) Mix the plasmid mixture obtained in step (3) evenly with the transfection reagent, let it stand at room temperature, then add it to the 293T cells after replacing the fresh medium, mix well, and continue culturing.

[0016] (5) Collect the culture supernatant and filter it through a filter membrane.

[0017] (6) Concentrate the collected virus solution, and determine the virus titer by infecting 293T cells and detecting the CAR positivity of the 293T cells after infection by flow cytometry, and set aside for use.

[0018] The nucleotide and amino acid sequences involved in the present invention are as follows:

[0019] (1) The nucleotide sequence of the CAR gene targeting Nectin4 is as follows:

[0020] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGATGGCCCAGG

[0021] TGAAGCTGCAGGAGAGCGGCGCCGAGCTGGTGCGCAGCGGCGCCAGCGTGAAGCTGAGCTGCACCGCCAGCGGCTTC

[0022] AACATCAAGGACTACTACATGCACTGGGTGAAGCAGCGCCCCGAGCAGAGCCTGGAGTGGATCGGCAACTTCCACCC

[0023] CTACAACGACGACACCAAGTACAACGAGAAGTTCAAGGGCAAGGCCAAGCTGACCGCCGACAAGAGCAGCAGCACCG

[0024] CCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCAGCTACGGCAACTACCCC

[0025] TGGTTCGCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG

[0026] CGGCGGCGGCAGCGACATCGAGCTGACCCAGAGCCCCAGCAGCATGTACGCCAGCCTGGGCGAGCGCGTGACCATCA

[0027] CCTGCAAGGCCAGCCAGGACATCAAGAGCTACCTGAGCTGGTACCAGCAGAAGCCCTGGAAGAGCCCCAAGACCCTG

[0028] ATCTACTACGCCACCAGCCTGGCCGACGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGCCAGGACTACAGCCT

[0029] GACCATCAGCAGCCTGGAGAGCGACGACACCGCCACCTACTACTGCCAGCAGTTCACCAGCAGCCCCTTCACCTTCG

[0030] GCAGCGGCACCAAGCTGGAGCTGAAGCGCACCGCCGCCGGCGCCCCCGTGCCCTACCCCGACCCCCTGGAGCCCCGC

[0031] GCCGCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCC

[0032] AGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGG

[0033] CGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACAGGAGTAAGAGGAGCAGGCTC

[0034] CTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACC

[0035] ACGCGACTTCGCAGCCTATCGCTCCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC

[0036] CAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGA

[0037] GTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGG

[0038] ACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA

[0039] ACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC

[0040] GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT

[0041] TCACATGCAGGCCCTGCCCCCTCGC

[0042] (2) The protein sequence of the CAR gene targeting Nectin4 is as follows:

[0043] MALPVTALLLPLALLLHAARPMAQVKLQESGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQSLEWIGNFHPYNDDTKYNEKFKGKAKLTADKSSSTAYMQLSSLTSEDSAVYYCARSYGNYPWFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPWKSPKTLIYYATSLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCQQFTSSPFTFGSGTKLELKRTAAGAPVPYPDPLEPRAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0044] (3) The nucleotide sequence of the CD8 signal peptide is as follows:

[0045] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0046] (4) The protein sequence of the CD8 signal peptide is as follows:

[0047] MALPVTALLLPLALLLHAARP

[0048] (5) The nucleotide sequence of anti-Nectin4 scFv is as follows:

[0049] ATGGCCCAGGTGAAGCTGCAGGAGAGCGGCGCCGAGCTGGTGCGCAGCGGCGCCAGCGTGAAGCTGAGCTGCA

[0050] CCGCCAGCGGCTTCAACATCAAGGACTACTACATGCACTGGGTGAAGCAGCGCCCCGAGCAGAGCCTGGAGTGGATC

[0051] GGCAACTTCCACCCCTACAACGACGACACCAAGTACAACGAGAAGTTCAAGGGCAAGGCCAAGCTGACCGCCGACAA

[0052] GAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCCGCAGCT

[0053] ACGGCAACTACCCCTGGTTCGCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGC

[0054] GGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGAGCTGACCCAGAGCCCCAGCAGCATGTACGCCAGCCTGGGCGA

[0055] GCGCGTGACCATCACCTGCAAGGCCAGCCAGGACATCAAGAGCTACCTGAGCTGGTACCAGCAGAAGCCCTGGAAGA

[0056] GCCCCAAGACCCTGATCTACTACGCCACCAGCCTGGCCGACGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGGC

[0057] CAGGACTACAGCCTGACCATCAGCAGCCTGGAGAGCGACGACACCGCCACCTACTACTGCCAGCAGTTCACCAGCAG

[0058] CCCCTTCACCTTCGGCAGCGGCACCAAGCTGGAGCTGAAGCGCACCGCCGCCGGCGCCCCCGTGCCCTACCCCGACC

[0059] CCCTGGAGCCCCGCGCCGCC

[0060] (6) The protein sequence of anti-Nectin4 scFv is as follows:

[0061] MAQVKLQESGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQSLEWIGNFHPYNDDTKYNEKFKGKAKLTADKSSSTAYMQLSSLTSEDSAVYYCARSYGNYPWFAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPWKSPKTLIYYATSLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCQQFTSSPFTFGSGTKLELKRTAAGAPVPYPDPLEPRAA

[0062] (7) The nucleotide sequence of the CD8α transmembrane domain is as follows:

[0063] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAG

[0064] AGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCG

[0065] CCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTAC

[0066] (8) The protein sequence of the CD8α transmembrane domain is as follows:

[0067] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY

[0068] (9) The nucleotide sequence of the CD28 co-stimulatory domain is as follows:

[0069] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCA AGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[0070] (10) The protein sequence of the CD28 co-stimulatory domain is as follows:

[0071] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0072] (11) The nucleotide sequence of the 4-1BB co-stimulatory domain is as follows:

[0073] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGG AAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0074] (12) The protein sequence of the 4-1BB co-stimulatory domain is as follows:

[0075] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0076] (13) The nucleotide sequence of the CD3ζ signaling domain is as follows:

[0077] AGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0078] (14) The protein sequence of the CD3ζ signal transduction domain is as follows:

[0079] RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0080] Furthermore, the preparation method of the lentiviral plasmid containing the CAR structure is as follows:

[0081] S1. Insert the synthesized sequence into the vector plasmid, transform competent cells DH5α, spread the bacterial solution on an agar plate containing ampicillin for culture; pick multiple colonies on the agar plate and inoculate them into 5 mL of liquid LB medium containing ampicillin respectively, and culture them in a constant temperature shaker at 37 °C and 250 rmp for 12 - 16 h;

[0082] S2. Extract the plasmid according to the instructions, perform Sanger sequencing on each cloned plasmid to verify the accuracy of the inserted sequence, and select the bacterial solution with the correct clone number according to the sequencing data for inoculation and shake flask culture;

[0083] S3. Extract the expression vector plasmid, measure the concentration and purity using a spectrophotometer, then perform double digestion and agarose gel electrophoresis verification on the extracted expression vector plasmid, and perform Sanger sequencing to verify the accuracy of the inserted sequence.

[0084] Furthermore, in step (4), the transfection reagent is lipofectamine 2000, the addition amount is 2 μL / μg plasmid, and the standing time at room temperature is 20 min; in step (5), collect the culture supernatants after 48 h and 72 h respectively for filtration.

[0085] Application of the Nectin4 - targeted CAR - T cells prepared by the above method in cancer treatment.

[0086] The present invention has the following beneficial effects:

[0087] The present invention provides a preparation process of CAR-T cells targeting Nectin4, which realizes the stable integration of the CAR gene through a lentiviral vector and enhances the targeted killing ability of T cells. By measuring the virus titer, the virus infection efficiency can be precisely controlled, and the production process of CAR-T cells is optimized. The Nectin4-targeted CAR-T cells of the present invention can specifically recognize the Nectin4 antigen, only bind to Nectin4-positive tissues, do not recognize other tissues and organs, have no off-target risk, and have good targeting. In addition, the Nectin4-targeted CAR-T cells of the present invention have excellent tumor killing function and can effectively kill a variety of Nectin4-positive tumor cells. Brief Description of the Drawings

[0088] Figure 1 It is the verification of the expression vector plasmid by agarose gel electrophoresis.

[0089] Figure 2 It is the detection of the positive rate of CAR-T cells by flow cytometry using Nectin4 antibody.

[0090] Figure 3 It is the detection result of the specific killing of human ovarian cancer cell SK-OV-3 by CAR-T cells.

[0091] Figure 4 It is the detection result of the specific killing of non-small cell lung cancer cell A549 by CAR-T cells.

[0092] Figure 5 It is the detection result of the specific killing of human brain astrocytoma cell U-87MG by CAR-T cells. Detailed Embodiments

[0093] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0094] Use the endotoxin-free plasmid large-scale extraction kit of MN company to extract three packaging plasmids (pPACKH1-GAG, pPACKH1-REV, and pVSV-G (these three plasmids are purchased from SBI company, product number: LV550A-1)), and use a spectrophotometer to measure the concentration and purity; adopt a four-plasmid packaging system for lentiviral packaging. The four plasmids are a lentiviral expression plasmid containing the CAR structure, and the lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV, and pVSV-G (purchased from SBI company, product number: LV550A-1).

[0095] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0096] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained through commercial channels.

[0097] Example 1

[0098] Construction of lentiviral plasmid containing CAR structure

[0099] S1. Insert the synthesized sequence into the vector plasmid, transform competent cells DH5α, spread the bacterial solution on an agar plate containing ampicillin for culture; pick multiple clones on the agar plate and inoculate them into 5 mL of liquid LB medium (containing ampicillin) respectively, and culture them with shaking in a constant temperature shaker for 12 - 16 h under the conditions of 37 °C and 250 rmp;

[0100] S2. Extract the plasmid according to the instruction manual of the plasmid mini - kit (product number: DP103 - 03) purchased from Tiangen Biochemical Technology Co., Ltd., send the plasmid of each clone to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing to verify the accuracy of the inserted sequence, and select the bacterial solution of the correct clone number according to the sequencing data feedback by Sangon Biotech (Shanghai) Co., Ltd. for large - scale inoculation and shake - flask culture;

[0101] S3. Use the endotoxin - free plasmid large - scale extraction kit from MN company to extract the expression vector plasmid, measure the concentration and purity using a spectrophotometer, and then perform double - enzyme digestion of the extracted expression vector plasmid with AgeI - HF (purchased from NEB, product number: R3552S) and BsrGI (purchased from NEB, product number: R3575S) and verify it by agarose gel electrophoresis ( Figure 1 ), and finally send the expression vector plasmid to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing to verify the accuracy of the inserted sequence.

[0102] Example 2

[0103] Preparation of CAR - T cells targeting Nectin4

[0104] (1) Plate the cells within 24 h before transfection: Select cells with passage number within 3 generations, adjust the cell density according to the cell growth density and status, and plate the 293T cells with a growth density reaching 80%;

[0105] (2) When the growth density reaches 60 - 90% and the cell state is good, virus packaging can be carried out;

[0106] (3) Use the lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV, and pVSV-G, as well as the constructed lentiviral plasmid containing the CAR structure, and perform the lentiviral plasmid ratio according to the plasmid instruction manual;

[0107] (4) Mix the plasmid mixture obtained in step (3) evenly with the transfection reagent. After standing at room temperature for 20 min, add it to the 293T cells after replacing the fresh medium and mix well, then continue culturing; among them, the transfection reagent is lipofectamine 2000 (stored at 4 °C), and the addition amount is 2 μL / μg plasmid;

[0108] (5) Collect the culture supernatants after 48 h and 72 h respectively, and filter through a 0.45 μm filter membrane;

[0109] (6) Concentrate the collected virus solution by the PEG8000 concentration method, and determine the virus titer by infecting 293T cells and subsequent flow cytometry to detect the CAR positivity of the infected 293T cells, and store it at -80 °C for standby.

[0110] Example 3

[0111] PBMC Isolation, T Cell Activation, Lentiviral Infection and Flow Cytometry Detection

[0112] 1. PBMC Isolation

[0113] 1) Take 6 mL of human peripheral blood (for scientific research);

[0114] 2) Dilution: Add an equal volume of PBS at room temperature and gently pipette to mix well;

[0115] 3) Sample addition: Take a 50 mL centrifuge tube, pipette 6 mL of Ficoll (lymphocyte separation solution) into the centrifuge tube (the volume ratio of Ficoll to the blood before dilution is 1:1), tilt the centrifuge tube at 45°, and slowly add the diluted blood along the tube wall about 1 cm above the Ficoll liquid surface to the Ficoll;

[0116] 4) Centrifugation: Centrifuge at 18 - 20 °C and 2000 rpm for 30 min, with the acceleration and deceleration rate of 4 / 4. After centrifugation, there are four layers from the bottom to the liquid surface of the tube, namely the red blood cell and granulocyte layer, the separation liquid layer, the mononuclear cell layer, and the plasma layer;

[0117] 5) Recovery: Insert the pipette directly into the cloudy layer (or first aspirate the upper plasma), gently aspirate the cloudy layer, and place it in a new centrifuge tube;

[0118] 6) Washing: Add at least 3 times the volume of PBS of PBMC (peripheral blood mononuclear cells), centrifuge at 18 - 20 °C and 1400 rpm for 10 min, and repeat twice;

[0119] 7) Cell counting: Discard the supernatant, add 1 mL of lymphocyte medium, pipette and mix well to prepare a PBMC cell suspension. Use a hemocytometer for counting: Take a drop of PBMC suspension and mix it with a drop of 2% trypan blue staining solution, then add the mixture to the hemocytometer. Count the total number of cells in 4 large grids under the microscope. Cells / mL = Total number of cells in 4 large grids / 4 × 10 4 × 2 (dilution factor).

[0120] 2. Activation, lentiviral infection and cell expansion of T cells

[0121] Day 1: Cell recovery: Take PBMC cells from liquid nitrogen and recover them;

[0122] PBMC plating: Collect PBMC cells, count them and finally adjust the concentration to 2 × 10 6 cells / mL. Activate PBMC cells with magnetic beads (purchased from Miltenyi Biotec, catalog number: 130 - 128 - 758). Add 5 μL of magnetic beads to every 2 × 10 6 cells. After mixing the magnetic beads and PBMC cells, add 500 μL of cell suspension to each well in a 24 - well plate, that is, add 1 × 10 6 cells to each well;

[0123] Day 2: Virus infection: Infect at MOI = 5. Prepare 1 mL of virus medium suspension and add it to the 24 - well plate;

[0124] Day 4: Transfer all the cells in the 24 - well plate to a 75 cm 2 culture flask containing 20 mL of medium and observe the cell status;

[0125] Day 8: Observe the cell status and cell number, centrifuge and resuspend. Use the Nectin4 CAR detection antibody to detect the positive rate of Nectin4 CAR - positive CAR - T cells by flow cytometry. The detection results are as Figure 2 shown. The positive rate of Nectin4 CAR - T is 61.88%.

[0126] Example 4

[0127] SCRTM Real - Time Cell Killing Assay

[0128] 1) Take human ovarian cancer cells SK - OV - 3, non - small cell lung cancer cells A549, and human brain astrocytoma cells U - 87MG as examples (SK - OV - 3 and A549 are Nectin4 - target - positive cells, and U - 87MG is a Nectin4 - target - negative cell). Digest them to prepare a cell suspension, pipette and mix well, and then perform cell counting;

[0129] 2) Dilute the cell suspension to a concentration of 4×10 4 cells / mL, and place it on ice for later use;

[0130] 3) Take out the SCRTM detection plate and add 50 μL of culture medium;

[0131] 4) Select the SCRTM detector's built-in test program for this experiment in the SCRTM detector program;

[0132] 5) Place the SCRTM detection plate into the detector (purchased from Six Broad Beans, model: CM100-α), observe whether the Messege item in the program is normal, and start the experimental program after it is normal;

[0133] 6) After Program 1 finishes running, take out the detection plate, add 50 μL of tumor cell suspension to the corresponding wells, and mix each tube of cell suspension before adding;

[0134] 7) After adding the cell suspension, place the detection plate in the incubator and let it stand for 30 min to allow the cells to settle naturally;

[0135] 8) After 30 min, put the detection plate into the detector and run Program 2;

[0136] 9) Observe the cell growth curve after 24 h. When the cells are in the logarithmic growth phase, prepare to add effector T cells;

[0137] 10) Take out the effector T cells from the culture flask, centrifuge, wash, count, and prepare the effector cell concentration according to different effector-to-target ratios;

[0138] 11) Pause the program, take out the detection plate, add 100 μL of effector cells to the corresponding position, put it back into the detector, continue the program, and observe every day.

[0139] Figures 3 - 5 It is the result after the SCRTM program finishes running, showing that CAR-T can effectively kill tumor cells (SK-OV-3, A549) positive for the Nectin4 target, but has no killing effect on tumor cells (U-87MG) negative for the Nectin4 target, indicating that the Nectin4-targeted CAR-T cells of the present invention have excellent anti-tumor functions, good specificity, and high expected safety. Figures 3 - 5 The 2.5:1 in it means effector cell: target cell = 2.5:1; "culture medium" means: without adding effector cells, only tumor target cells; uninfected T cells mean: the effector cells are uninfected T cells.

[0140] Although embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing CAR-T cells targeting Nectin4, characterized in that: The following steps are involved: The CAR gene targeting Nectin4 is introduced into T cells via a lentiviral vector, so that the CAR gene is integrated into the T cell genome; The nucleotide sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:

1.

2. The method for preparing CAR-T cells targeting Nectin4 according to claim 1, characterized in that: The protein sequence of the CAR gene targeting Nectin4 is shown in SEQ ID NO:

2.

3. The method for preparing CAR-T cells targeting Nectin4 according to claim 1, characterized in that: The specific steps are: (1) Select cells with a passage number of less than 3 generations, adjust the cell density according to the cell growth density and status, and plate 293T cells with a growth density of 80%; (2) When the growth density reaches 60-90% and the cells are in good condition, virus packaging can be performed; (3) Using the lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV and pVSV-G, as well as the constructed lentiviral plasmid containing the CAR structure, the lentiviral plasmid ratio was prepared according to the plasmid instructions; (4) mixing the plasmid mixture obtained in step (3) with the transfection reagent, letting it stand at room temperature, adding it to the 293T cells after replacing the fresh culture medium, mixing it, and continuing to culture; (5) collecting the culture supernatant and filtering it through a filter membrane; (6) The collected virus solution is concentrated, and the virus titer is determined by infecting 293T cells and detecting the CAR positivity of the infected 293T cells by flow cytometry for later use.

4. The method for preparing CAR-T cells targeting Nectin4 according to claim 1, characterized in that: The preparation method of the lentiviral plasmid containing the CAR structure in step (3) is: S1. Insert the synthetic sequence into the vector plasmid, transform the competent cell DH5a, plate the bacterial solution on an agar plate containing ampicillin for culture; pick multiple clones on the agar plate, inoculate them into liquid LB medium containing ampicillin, and culture them on a constant temperature shaker; S2. Extract plasmids according to the instructions, perform Sanger sequencing on each cloned plasmid, and select the cloned bacterial solution with the correct sequence according to the sequencing data for inoculation into shake flask culture; S3. Extract the expression vector plasmid, measure the concentration and purity using a spectrophotometer, then perform double enzyme digestion and agarose gel electrophoresis verification on the extracted expression vector plasmid, and perform Sanger sequencing.

5. The method for preparing CAR-T cells targeting Nectin4 according to claim 1, characterized in that: The transfection reagent in step (4) is lipofectamine 2000, and the added amount is 2 μL / μg plasmid.

6. The method for preparing CAR-T cells targeting Nectin4 according to claim 1, characterized in that: The standing time at room temperature in step (4) is 20 minutes.

7. The method for preparing CAR-T cells targeting Nectin4 according to claim 1, characterized in that: In step (5), the culture supernatants were collected after 48 h and 72 h respectively and filtered.

8. CAR-T cells targeting Nectin4 prepared by the method according to any one of claims 1 to 7.

9. Use of the CAR-T cells targeting Nectin4 according to claim 8 in cancer treatment.

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