A CAR-T cell targeting Nectin4 and its application in cancer treatment

Abstract

The present invention belongs to the field of immunotherapy technology, and specifically relates to a CAR-T cell targeting Nectin4 and its application in cancer treatment. The method for preparing CAR-T cells targeting Nectin4 is: introducing a CAR gene targeting Nectin4 into T cells via a lentiviral vector, so that the CAR gene is integrated into the T cell genome. In order to overcome the technical difficulties in the prior art that there are few types of CAR-T cells targeting Nectin4 and the therapeutic effect is poor, the present invention discovers a new antibody sequence for this target and provides a CAR-T cell targeting Nectin4, which can be used in the 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 specifically relates to a CAR-T cell targeting Nectin4 and its application in cancer treatment. Background Art

[0002] T cell adoptive immunotherapy is a promising method for the treatment of cancer. Immunotherapy enhances its specificity to specific tumor-associated antigens by separating genetically modified human T cells. Genetic modification can 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 non-restricted manner of the major histocompatibility complex. T cell adoptive 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.

[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 66kDa. Nectin4 only exists 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. Nectin4 is a type I single-pass transmembrane protein belonging to the Ca 2+ Nectin4 is a cytoplasmic immunoglobulin-like molecule consisting 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 multiple growth factor receptors, including EGFR and HER2, thereby influencing multiple signal transduction pathways, significantly affecting cell growth, migration, and apoptosis, and is closely related to tumor occurrence and metastasis. The cytoplasmic tail of nectin4 can bind to the adaptor molecule afadin through its C-terminal amino acid sequence. Afadin then binds to and recruits filamentous actin components (F-actin), thereby promoting cell-cell adhesion. Nectin4 promotes tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K / Akt pathway. Using nectin4 as a therapeutic target could be an effective strategy for treating cancers with high Nectin4 expression.

[0004] For example, CN116589584A discloses existing technologies for designing monoclonal antibodies or fragments thereof targeting human Nectin 4 protein, which include heavy and light chains. The heavy chain includes three heavy chain complementary determining regions 1 to 3 (HCDR1, HCDR2, and HCDR3), and the light chain includes three light chain complementary determining regions 1 to 3 (HCDR1, HCDR2, and HCDR3). The heavy chain also includes a heavy chain variable region VH, and the light chain also includes a light chain variable region VL. Any amino acid sequence in the three heavy chain complementary determining regions can be paired with any amino acid sequence in the three light chain complementary determining regions for use in a double-chain combination, or any amino acid sequence in the three heavy chain complementary determining regions can be used as a single chain, or any amino acid sequence in the three light chain complementary determining regions can be used as a single chain. The amino acid sequences of the corresponding complementary determining regions in the heavy and light chain variable regions of the monoclonal antibody are paired and expressed as humanized antibodies. After the gene of the humanized antibody is constructed into a plasmid expression cassette, the expression cassette is transferred into 293T cells through a delivery system for antibody expression; wherein, the delivery system can be one of lentivirus, retrovirus, ordinary plasmid vector, episomal vector, nano-delivery system, electrotransduction and transposon. The recombinant cell line is an immune cell, which can be any immune cell including T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, and TCR-T cells. When the immune cells express the chimeric antigen receptor CAR, NK cells, NKT cells, TIL cells, and gamma-delta T cells are equivalent to T cells (or T cells can replace NK cells). Any of the above-mentioned monoclonal antibodies targeting human Nectin4 protein can be made into pharmaceutically acceptable carriers, diluents or excipients, and applied to biomaterials and / or biological preparations, all of which have good forming effects while maintaining good efficacy. When monoclonal antibodies are used in biological preparations, the biological preparations contain an expression cassette (such as a gene expression cassette) constructed from the above-mentioned nucleic acid sequence or amino acid sequence, 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.), and these nucleic acid sequences or amino acid sequences are derived from the above-mentioned monoclonal antibodies; wherein the recombinant vector includes a gene recombinant expression vector and a chimeric antigen receptor. When monoclonal antibodies are used in biological preparations, they exist as components of the biological preparation configuration, and the biological preparation components also include reagents for detecting the concentration of human Nectin 4 protein, reagents for detecting the expression level of human Nectin 4 protein on the surface of tumor cells, antibodies coupled to toxins to kill human Nectin 4-positive cells, antibodies coupled to other antibodies to produce polyclonal antibodies targeting human Nectin 4 and other antigens, and antibodies coupled to other proteins to produce functional recombinant proteins targeting human Nectin 4. The chimeric antigen receptor (CAR) of this technology includes an extracellular domain, a transmembrane domain, and an intracellular domain.The extracellular domain includes an antigen-binding domain, and the intracellular domain includes a co-stimulatory signaling region, a cytokine receptor intracellular region, and a portion of the CD3ζ chain. The co-stimulatory signaling region refers to a portion of the intracellular domain that includes a co-stimulatory molecule, which is a cell surface molecule required for lymphocytes to effectively respond to antigens. The extracellular domain of the CAR provided by this technology includes the antigen-binding domains of the heavy and light chain variable regions of a humanized antibody targeting Nectin4. When expressed in T cells, the CAR can recognize antigens based on antigen binding specificity or protein receptor binding. The antigen-binding domain is fused to the intracellular domains from the co-stimulatory molecule and the CD3ζ chain. The antigen-binding domain is fused to the intracellular domain of a combination of CD28, 4-1BB, ICOS signaling domains, and the CD3ζ signaling domain. The therapeutic application is performed using cells transduced with a lentiviral vector containing a nucleic acid construct encoding the nucleic acid of the antibody of the invention. The transduced T cells can elicit a CAR-mediated T cell response. The injected cells are able to kill the recipient's tumor cells, and the CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained tumor control. Furthermore, the CAR-T cell membrane can express a humanized antibody scFv structure antigen chimeric receptor targeting Nectin4, specifically killing Nectin4-positive target cells without the risk of off-target effects.

[0005] In order to overcome the technical difficulties of the existing technology of limited types of CAR-T cells targeting Nectin4 and poor therapeutic effects, developing new antibody sequences for this target in order to provide a more efficient treatment solution has become a research hotspot. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides a CAR-T cell targeting Nectin4 and its application in cancer treatment. The present invention discovered a new antibody sequence for this target, and the designed CAR-T cell can be used in the treatment of cancers such as ovarian cancer and breast cancer.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0008] A method for preparing CAR-T cells targeting Nectin4 comprises the following steps:

[0009] The CAR gene targeting Nectin4 is introduced into T cells via a lentiviral vector, allowing the CAR gene to integrate into the T cell genome;

[0010] The structure and order of the CAR gene targeting Nectin4 are: 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:

[0012] (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 when the growth density reaches 80%;

[0013] (2) When the growth density reaches 60-90% and the cells are in good condition, virus packaging can be carried out;

[0014] (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;

[0015] (4) The plasmid mixture obtained in step (3) was mixed evenly with the transfection reagent, allowed to stand at room temperature, and then added to the 293T cells after replacement of the fresh culture medium, mixed evenly, and continued to culture;

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

[0017] (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.

[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] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCAGGCCGATGGCCCAGG

[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] CCCCTTCACCTTCGGCAGCGGCACCAAGCTGGAGCTGAAGCGCACCGCCGCCGGCGCCCCGTGCCCTACCCGACC

[0059] CCCTGGAGCCCCGCGCCGCC

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

[0061] MAQVKLQESGAELVRSGASVKLSCTASGFNIKDYYMHWVKQRPEQSLEWIGNFHPYNDDTKYNEKFKGKAKLTADKSSSTAYMQLSSLTSEDSAVYYCARSYGNYPWFAYWGQGTTVTVSSGGGGSGGGGS GGGGSDIELTQSPSSMYASLGERVTITCKASQDIKSYLSWYQQKPWKSPKTLIYYATSLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCQQFTSSPFTFGSGTKLELKRTAAGAPVPYPDPLEPRAA

[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 costimulatory domain is as follows:

[0069] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCA AGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

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

[0071] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

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

[0073] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGG AAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

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

[0075] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

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

[0077] AGAGTGAAGTTCAGCAGGAGCGCAGAGCCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

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

[0079] RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

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

[0081] S1. Insert the synthesized sequence into the vector plasmid, transform competent DH5a cells, and plate the bacterial solution onto an agar plate containing ampicillin for culture. Pick multiple clones from the agar plate and inoculate them into 5 mL of liquid LB medium containing ampicillin. Incubate the culture in a constant temperature shaker at 37°C and 250 rpm for 12-16 hours.

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

[0083] S3. Extract the expression vector plasmid and measure its concentration and purity using a spectrophotometer. Then, perform double enzyme digestion and agarose gel electrophoresis 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), the culture supernatants after 48 h and 72 h are collected and filtered respectively.

[0085] Application of Nectin4-targeting 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 process for preparing CAR-T cells targeting Nectin4, which achieves stable integration of the CAR gene through a lentiviral vector and enhances the targeted killing ability of T cells. By measuring the viral titer, the viral infection efficiency can be precisely controlled, thereby optimizing the production process of CAR-T cells. The Nectin4-targeted CAR-T cells of the present invention can specifically recognize the Nectin4 antigen, bind only to Nectin4-positive tissues, and do not recognize other tissues and organs, without the risk of off-target effects, 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 The expression vector plasmid was verified by agarose gel electrophoresis.

[0089] Figure 2 The positive rate of CAR-T cells was detected by flow cytometry using Nectin4 antibody.

[0090] Figure 3 This is the detection result of CAR-T cells specifically killing human ovarian cancer cells SK-OV-3.

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

[0092] Figure 5 This is the test result of CAR-T cells specifically killing human brain glioblastoma cells U-87MG. DETAILED DESCRIPTION

[0093] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0094] Three packaging plasmids (pPACKH1-GAG, pPACKH1-REV, and pVSV-G (these three plasmids were purchased from SBI, catalog number: LV550A-1)) were extracted using the MN company's endotoxin-free plasmid extraction kit, and the concentration and purity were measured using a spectrophotometer; a four-plasmid packaging system was used for lentiviral packaging. The four plasmids are the lentiviral expression plasmid containing the CAR structure, and the lentiviral packaging plasmids pPACKH1-GAG, pPACKH1-REV, and pVSV-G (purchased from SBI, catalog number: LV550A-1).

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

[0096] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0097] Example 1

[0098] Construction of lentiviral plasmid containing CAR construct

[0099] S1. Insert the synthesized sequence into the vector plasmid, transform competent DH5a cells, and plate the bacterial solution onto an agar plate containing ampicillin for culture. Pick multiple clones from the agar plate and inoculate them into 5 mL of liquid LB medium (containing ampicillin). Incubate the culture in a constant temperature shaker at 37°C and 250 rpm for 12-16 hours.

[0100] S2. Plasmids were extracted according to the instructions of the plasmid extraction kit (catalog number: DP103-03) purchased from Tiangen Biochemical Technology Co., Ltd. Each cloned plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for Sanger sequencing to verify the accuracy of the inserted sequence. Based on the sequencing data provided by the sequencing company Shanghai Sangon Biotechnology Co., Ltd., the bacterial solution of the clone with the correct sequence was selected for large-scale inoculation in shake flask culture;

[0101] S3. The expression vector plasmid was extracted using the MN company's endotoxin-free plasmid extraction kit, and the concentration and purity were measured using a spectrophotometer. The extracted expression vector plasmid was then double-digested with AgeI-HF (purchased from NEB, catalog number: R3552S) and BsrGI (purchased from NEB, catalog number: R3575S) and verified by agarose gel electrophoresis ( Figure 1 ), and finally the expression vector plasmid was sent to Shanghai Sangon Biotechnology 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) Plating within 24 hours before transfection: 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 when the growth density reaches 80%;

[0105] (2) When the growth density reaches 60-90% and the cells are in good condition, 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 prepare the lentiviral plasmid ratio according to the plasmid instructions;

[0107] (4) The plasmid mixture obtained in step (3) was mixed evenly with a transfection reagent, allowed to stand at room temperature for 20 minutes, and then added to the 293T cells after replacement of fresh culture medium, mixed evenly, and continued to culture; wherein the transfection reagent was lipofectamine 2000 (stored at 4°C), and the addition amount was 2 μL / μg plasmid;

[0108] (5) The culture supernatants were collected after 48 h and 72 h, respectively, and filtered through a 0.45 μm filter membrane;

[0109] (6) The collected virus solution was concentrated using the PEG8000 concentration method, and the virus titer was determined by infecting 293T cells and subsequently detecting the CAR positivity of the infected 293T cells by flow cytometry, and stored at -80°C for future use.

[0110] Example 3

[0111] PBMC isolation, T cell activation, lentiviral infection, and flow cytometry

[0112] 1. PBMC Isolation

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

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

[0115] 3) Sample Addition: Take a 50 mL centrifuge tube and pipette 6 mL of Ficoll (lymphocyte separation medium) into the tube (the volume ratio of Ficoll to pre-dilution blood is 1:1). Tilt the tube at 45° and slowly add the diluted blood along the tube wall to the Ficoll approximately 1 cm above the liquid level.

[0116] 4) Centrifugation: Centrifuge at 18-20°C, 2000 rpm for 30 min, with the speed set at 4 / 4. After centrifugation, four layers are separated from the bottom of the tube to the liquid surface: red blood cells and granulocytes layer, stratified liquid layer, mononuclear cell layer, and plasma layer.

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

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

[0119] 7) Cell Count: Discard the supernatant, add 1 mL of lymphocyte culture medium, and mix thoroughly by pipetting to prepare a PBMC cell suspension. Count using a hemocytometer: Mix one drop of PBMC suspension with one drop of 2% trypan blue dye and add the mixture to a hemocytometer. Count the total number of cells within each of the four large squares under a microscope. Cells / mL = Total number of cells in each of the four large squares / 4 × 10 4 ×2 (dilution factor).

[0120] 2. T cell activation, lentiviral infection, and cell expansion

[0121] Day 1: Cell recovery: PBMC cells were collected from liquid nitrogen and recovered;

[0122] PBMC plating: Collect PBMC cells, count them and adjust the final concentration to 2 × 10 6 cells / mL, magnetic beads (purchased from Miltenyi Biotec, catalog number: 130-128-758) were used to activate PBMC cells, and 2×10 6 5 μL of magnetic beads were added to each cell, and after the magnetic beads and PBMC cells were mixed, 500 μL of cell suspension was added to each well of a 24-well plate, that is, 1×10 6 cells;

[0123] Day 2: Virus infection: Infect at an MOI of 5, prepare 1 mL of virus culture medium suspension, and add it to a 24-well plate;

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

[0125] Day 8: Observe the cell status and cell number, centrifuge and resuspend, and use Nectin4 CAR detection antibody to detect the positive rate of CAR-T cells targeting Nectin4 CAR by flow cytometry. Figure 2 As shown in the figure, the positive rate of nectin4-targeted CAR-T was 61.88%.

[0126] Example 4

[0127] SCRTM Real-Time Cell Killing Assay

[0128] 1) Using human ovarian cancer cells SK-OV-3, non-small cell lung cancer cells A549, and human glioblastoma cells U-87MG as examples (SK-OV-3 and A549 are Nectin 4-positive cells, while U-87MG is Nectin 4-negative), prepare a cell suspension after digestion, mix thoroughly by pipetting, and then count the cells.

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

[0130] 3) Remove the SCRTM assay plate and add 50 μL of culture medium;

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

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

[0133] 6) After Program 1 is complete, remove the assay plate and add 50 μL of tumor cell suspension to the corresponding wells. Mix each tube of cell suspension thoroughly before adding.

[0134] 7) After adding the cell suspension, place the test plate in an incubator and let it sit for 30 minutes to allow the cells to settle naturally.

[0135] 8) After 30 minutes, place the test plate in the tester and run Program 2;

[0136] 9) After 24 hours, observe the cell growth curve and prepare to add effector T cells when the cells are in the logarithmic growth phase;

[0137] 10) Remove effector T cells from the culture flask, centrifuge, wash, count, and adjust the concentration of effector group cells according to different effector-target ratios;

[0138] 11) Pause the procedure, remove the assay plate, add 100 μL of effector cells to the corresponding position, return the plate to the assay, continue the procedure, and observe daily.

[0139] Figure 3-5 This is the result after the SCRTM program is completed, showing that CAR-T can effectively kill Nectin4 target-positive tumor cells (SK-OV-3, A549), but has no killing effect on Nectin4 target-negative tumor cells (U-87MG), indicating that the targeted Nectin4 CAR-T cells of the present invention have excellent anti-tumor function, good specificity, and expected high safety. Figure 3-5 The 2.5:1 in the figure indicates that the ratio of effector cells to target cells is 2.5:1; “medium” indicates that only tumor target cells are present without effector cells; and uninfected T cells indicates that the effector cells are uninfected T cells.

[0140] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may 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, allowing the CAR gene to integrate 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 when the growth density reaches 80%; (2) When the growth density reaches 60-90% and the cells are in good condition, virus packaging can be carried out; (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) The plasmid mixture obtained in step (3) was mixed evenly with the transfection reagent, allowed to stand at room temperature, and then added to the 293T cells after replacement of the fresh culture medium, mixed evenly, and continued to culture; (5) Collect the culture supernatant and filter it through a 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 3, 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 DH5a cells, and 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 clone with the correct sequence according to the sequencing data for inoculation into shake flask culture; S3. Extract the expression vector plasmid, measure its 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 3, 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 3, 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. Nectin4-targeting CAR-T cells prepared by the preparation method according to any one of claims 1 to 7.

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