A method for preparing CAR-T cells

Abstract

The present invention belongs to the field of immunology, and particularly relates to a method for preparing CAR-T cells. The steps include: contacting and culturing PBMC cells or T cells with tumor antigens, anti-CD28 antibodies, and anti-CD6 antibodies, wherein the PBMC cells or T cells contain nucleic acids with coding sequences of chimeric antigen receptors targeting antigens in the cells. The CAR-T cells prepared by this method have uniform quality and similar activities, solving the problems of low transfection efficiency, less activation, poor proliferation, and difficult quality control of CAR-T cells caused by low expression of ZAP70, and meeting the requirements of cGMP production management and large-scale production.

Description

Technical Field

[0001] The present invention belongs to the field of immunology, and particularly relates to a method for preparing CAR-T cells. Background Art

[0002] Adoptive Cell Transfer Therapy (ACT) is an immunotherapy that separates tumor-specific or non-specific immune cells, such as TIL, NK cells, T cells, and DC cells, from donors, activates, expands, and functionally identifies them in vitro, and then transfuses them into patients to directly produce or activate an in vivo immune response to produce anti-tumor activity. Currently, more research is focused on adoptive T cell therapy - chimeric antigen receptor T cell (CAR-T) therapy. This therapy has a significant curative effect on the treatment of some B cell malignant hematological tumors clinically. CAR mainly consists of an extracellular single-chain variable fragment (scFv) domain that can specifically recognize tumor surface antigens and an intracellular signal transduction structure. After the CAR molecule is transfected into T cells, it can directly recognize tumor cells, activate T cells through intracellular co-stimulatory signal transduction, promote T cells to produce a large number of cell-killing and related factors to induce apoptosis of tumor cells, thereby achieving the effect of killing tumors. This CAR-T cell therapy has MHC-independent anti-tumor effects and simultaneously has the specific recognition of tumor-associated antigens and the cytotoxic effects of T cells. Compared with other traditional tumor treatment methods, CAR-T cell therapy is one of the most promising tumor treatment therapies.

[0003] At present, the global research and development of CAR-T is progressing rapidly. Two CAR-T cell therapy products have been launched on the market, and two CAR-T cell therapies submitted marketing applications to the US FDA at the end of 2019. The application prospect of CAR-T cell therapy is very promising. However, not all cancer patients can benefit from it. Especially after radiotherapy and chemotherapy, the quality of T cells drops sharply, which brings great difficulties to the preparation of CAR-T cells that meet the quality standards. In addition, there are many problems in the preparation of CAR-T cell products, such as poor raw material quality, long preparation cycle, immature preparation process, and difficult evaluation of biological function and safety. Therefore, the CAR-T cell therapy plan is constantly being optimized. Among them, it is worth noting that the quality of PBMC in cancer patients is uncontrollable. When preparing CAR-T cells, there are often situations such as low transfection efficiency, less activation, poor proliferation, and poor quality control data, which makes it difficult to carry out cGMP production management and large-scale production, resulting in difficulties in clinical transformation. Therefore, there is an urgent need to find a method to solve these difficulties. This study aims to optimize the problem that CAR-T cells cannot be well amplified in vitro due to poor quality of T cells in cancer patients.

[0004] To enhance the proliferation of CAR-T cells during in vitro preparation, the current optimization methods are mainly divided into two levels: (1) CAR structure optimization, introducing co-stimulatory molecules (such as third-generation CARs) (An immunoproteomic approach to characterize the CAR interactome and signalosome. Sciencesignaling, MC, R., BI, KB, L.-M. M, KW, SD, P.-S. M, DL, FB, RU, YS, BA, KJM, HEB&A.-D. D, 2019, 12, eaap9777.) or fragments of secreted cytokines (such as fourth-generation CARs) (the fourth generation of CARs. Expert opinion on biological therapy, M, C. & A. H, 2015, 15, 1145-54) into the intracellular domain of CAR; (2) CAR culture process optimization, adding factors that promote cell function to the culture environment, such as IL-2 (Interleukin-2(IL-2) in flounder(Paralichthys olivaceus): Molecular cloning, characterization and bioactivity analysis. Fish&shellfish immunology, X, T., GM, DY, XJ, SX&ZW, 2019, 93, 55-65.), IL-15 (IL15 Enhances CAR-T Cell Antitumor Activity by Reducing mTORC1 Activity and Preserving Their Stem Cell Memory Phenotype. Cancer immunology research, D, A., WR, A, YX, WD, PJ, R, KC, F, AB, QY, AD, K, SR, UR, WX, FS, J&B, CE, 2019, 7, 759-772), IL-12 (Anti-T12: an anti-CD6 monoclonal antibody, can activate human T lymphocytes.Journal of immunology (Baltimore, Md.: 1950), RM, G., SJ, A, GD, M & RP, L, 1989, 143, 2439 - 47.) and IL-21 (IL-21 Selectively Protects CD62L NKT Cells and Enhances Their Effector Functions for Adoptive Immunotherapy. Journal of immunology (Baltimore, Md.: 1950), H, N., TG, C, AN, RS, B, GL, LB, JJ, SE, T, DP, EJ & ML, S, 2018, 201, 2141 - 2153), etc. IL-2 is a bioactive cytokine widely used in the in vitro culture of CAR-T cells, which can promote the proliferation and activation of T cells. IL-15, IL-12 and IL-21 are also cytokines that can promote lymphocyte proliferation, differentiation and maintain the normal anti-tumor effect of the body in vivo, but it is difficult to act on T cells with weak ZAP70 expression. Because ZAP70 is a cytoplasmic protein tyrosine kinase, belonging to the Syk protein family, and is only expressed in T cells and natural killer cells. As part of the TCR, ZAP70 can regulate the opening and closing of T cell activation by regulating the expression of TCR on the T cell surface, and is an essential factor for TCR activation. The T cell activation pathway can be summarized as follows: after the activation of the TCR / CD3 complex, ZAP70 is recruited and activated, thereby phosphorylating Lat and CD6, and finally activating the SLP-76-dependent recruitment and other downstream effectors to regulate the T cell response (Malissen et al. 2014). Therefore, when the expression of ZAP70 is weak, the proximal TCR signal transduction ability of T cells is damaged, and the downstream activation signal cannot be initiated (AY et al. 2016). Summary of the Invention

[0005] The present invention provides a method for preparing CAR-T cells against abnormally immunodeficient cells (T cells) with weak ZAP70 expression. Without the need for other special modifications to the immunocytes (T cells), this preparation method can improve the transfection rate, activation rate, and proliferation rate of CAR-T cells, and does not affect the CAR-T cell preparation activity of normal immunocytes (T cells). When the quality of the immunocytes (T cells) used for preparing CAR-T cells is inconsistent, especially the autologous isolated immunocytes (T cells) in patients, the CAR-T preparation activity of abnormally immunodeficient cells can be enhanced without other special treatments, and the CAR-T cell preparation activity equivalent to or similar to that of normal immunocytes can be achieved. The quality is stable and controllable, the method is simple, safe, and controllable, and is suitable for large-scale production.

[0006] The technical solution of the present invention is: a method for preparing CAR-T cells, the steps of which include: contacting and culturing PBMC cells or T cells with a tumor antigen, an anti-CD28 antibody, and an anti-CD6 antibody, and the nucleic acid containing the coding sequence of the chimeric antigen receptor targeting the tumor antigen is present in the PBMC cells or T cells.

[0007] The conditions for the contact culture of the present invention are: the culture temperature is 37 °C, the CO2 concentration for culture is 5%, the culture medium is AIM-V + 2% FBS medium, the culture days are 5 - 10 days, and preferably 5 - 6 days; the culture medium contains cytokines, and the final concentration of the cytokines is 100 - 1000 U / ml, preferably 450 - 600 U / ml; the cytokines are selected from any one or more of IL-2, IL-15, IL-12, or IL-21, and preferably IL-2.

[0008] During the contact culture process of the present invention, the tumor antigen, the anti-CD28 antibody, and the anti-CD6 antibody are in a non-immobilized state or an immobilized state, preferably in an immobilized state. The tumor antigen, the anti-CD28 antibody, and the anti-CD6 antibody are connected to the matrix in a covalent or non-covalent manner. The matrix can be a fixed matrix, such as a solid well plate, culture dish, glass slide, etc., or a suspended matrix, such as magnetic beads, nanoparticles, etc.

[0009] When in the immobilized state, the coating concentration of the tumor antigen is 1 - 10 μg / ml, preferably 4.5 - 5 μg / ml; the coating concentration of the anti-CD28 antibody is 1 - 10 μg / ml, preferably 4.5 - 5 μg / ml; the coating concentration of the anti-CD6 antibody is 1 - 10 μg / ml, preferably 4.5 - 5 μg / ml.

[0010] The method for preparing the CAR-T cells of the present invention further includes transferring the cells after co-culture to a culture medium without tumor antigen, anti-CD28 antibody and anti-CD6 antibody for conventional culture. The conditions for conventional culture are as follows: the culture temperature is 37°C, the CO2 concentration for culture is 5%, the culture medium is AIM-V + 2% FBS medium, the culture days are 5 to 10 days, and preferably 5 to 6 days; the culture medium contains cytokines, and the final concentration of the cytokines is 50 to 1000 U / ml, preferably 50 to 200 U / ml; the cytokines are selected from any one or more of IL-2, IL-15, IL-12 or IL-21, and preferably IL-2.

[0011] The anti-CD28 antibody of the present invention is a human anti-CD28 antibody, a murine anti-CD28 antibody or a rabbit anti-CD28 antibody, preferably a human anti-CD28 antibody; further, the anti-CD28 antibody is an anti-CD28 monoclonal antibody or an anti-CD28 polyclonal antibody, preferably an anti-CD28 monoclonal antibody.

[0012] The anti-CD6 antibody of the present invention is a human anti-CD6 antibody or a murine anti-CD6 antibody, preferably a human anti-CD6 antibody; further, the anti-CD6 antibody is an anti-CD6 monoclonal antibody or an anti-CD6 polyclonal antibody, preferably an anti-CD6 monoclonal antibody.

[0013] The method for transferring the nucleic acid containing the chimeric antigen receptor encoding sequence targeting the tumor antigen into PBMCs of the present invention is selected from any one of viral particle infection, electroporation, liposome transfection, calcium phosphate transfection and gene gun transfection, and preferably electroporation.

[0014] The tumor antigens targeted by the chimeric antigen receptors of the present invention are tumor-specific antigens and / or tumor-associated antigens. The tumor-specific antigens or tumor-associated antigens include but are not limited to any one or combination of the following: CD19, CD20, CEA, GD2, FR (Flavin reductase), PSMA (prostate-specific antigen), PMEL (premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, MART-1 (mucin A), ERBB2, MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family proteins) family, GAGE (growth hormone-releasing factor family proteins) family, AFP, MUC1, MUC16, CD22, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, EGP-2, EGP-40, PSA (kallikrein-related peptidase 3), MSLN (mesothelin), EGFR, ERBB3, ERBB4, AFU, HER family, EpCAM, IGFR1, CA242 or CA50, etc., preferably the HER family, and more preferably EGFR.

[0015] The nucleic acid containing the chimeric antigen receptor encoding sequence targeting tumor antigens in the present invention is DNA or mRNA. The DNA is preferably an expression vector, and the expression vector is selected from recombinant eukaryotic expression vectors or recombinant viral vectors. The recombinant eukaryotic vectors include, but are not limited to, pNB vectors (transposon plasmids), pRS vectors, pUC vectors, pSV2 vectors, pRSV vectors, pcDNA3.1 vectors, pVAX1 vectors, pEGFP vectors, or pCMVp-NEO-BAN vectors, etc. The recombinant viral vectors include, but are not limited to, recombinant adenovirus vectors or lentiviral vectors, etc. The mRNA contains a 5' end cap structure, 5' UTR, an open reading frame (ORF) encoding the chimeric antigen receptor, 3' UTR, and a polyA tail.

[0016] Compared with the prior art, the advantages of the present invention are as follows: The CAR-T preparation method of the present invention overcomes the production difficulties such as low transfection rate, less activation, poor proliferation, and difficult quality control of CAR-T cells caused by inconsistent quality of immune cells. Although the quality of immune cells is inconsistent, especially ZAP70 weakly expressing cells, by adopting the preparation method of the present invention, the proliferation and activation levels of CAR-T cells of abnormal quality immune cells can be enhanced to the same activity level as normal immune cells, and the present invention has no adverse effect on the proliferation and activation levels of CAR-T cells of normal immune cells. Description of the Drawings

[0017] Figure 1 Schematic diagram of the expression of human EGFR antigen on the surfaces of PanC-1 cells, BXPC-3 cells, Aspc1 cells of pancreatic cancer, HepG2, Huh-7 cells, Hep3B cells of liver cancer, and H460 cells, H292 cells, H23 cells of lung cancer in Example 1.

[0018] Figure 2 Statistical results of the expression levels of human EGFR antigen on the surfaces of PanC-1 cells, BXPC-3 cells, Aspc1 cells of pancreatic cancer, HepG2, Huh-7 cells, Hep3B cells of liver cancer, and H460 cells, H292 cells, H23 cells of lung cancer in Example 1.

[0019] Figure 3 Schematic diagram of the pNB338B-EGFR vector containing the chimeric antigen receptor sequence targeting the EGFR antigen in Example 2.

[0020] Figure 4 Schematic diagram of the ZAP70 expression in PBMC cells from different donors in Example 2.

[0021] Figure 5Schematic diagram of flow cytometry detection of CAR transfection in PBMC cells under different CAR-T preparation methods in Example 2. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0022] Figure 6 Statistical chart of the results of multiple flow cytometry detections of CAR transfection in PBMC cells under different CAR-T preparation methods in Example 2. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0023] Figure 7 Trend chart of in vitro culture and amplification of CAR-T cells prepared from PBMC cells under different CAR-T preparation methods in Example 3. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0024] Figure 8 Schematic diagram of flow cytometry detection of the surface activation index CD137 of CAR-T cells prepared from PBMC cells under different CAR-T preparation methods in Example 3. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFRCAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0025] Figure 9 Statistical chart of the results of flow cytometry detection of the surface activation index CD107α of CAR-T cells prepared from PBMC cells under different CAR-T preparation methods in Example 3. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0026] Figure 10Statistical chart of the flow cytometry detection results of the surface activation index CD107α of CAR-T cells prepared from PBMC cells under different CAR-T preparation methods in Example 3. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0027] Figure 11 Schematic diagram of the flow cytometry detection results of CD62L and CCR7 molecules in CAR-T cells with positive CD45RO on the cell surface prepared from PBMC cells under different CAR-T preparation methods in Example 3. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0028] Figure 12 Detection of cytokine release levels after co-incubation of CAR-T cells prepared from PBMC cells under different CAR-T preparation methods in Example 4 with tumor cells with different EGFR antigen expression levels. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0029] Figure 13 Schematic diagram of the killing results of CAR-T cells prepared from PBMC cells under different CAR-T preparation methods in Example 4 on target cells at different effector-to-target ratios. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody.

[0030] Figure 14 Statistical chart of the killing results of CAR-T cells prepared from PBMC cells under different CAR-T preparation methods in Example 4 on target cells at different effector-to-target ratios. Among them, the control group was incubated by contacting with anti-CD3 antibody and anti-CD28 antibody, the EGFR CAR-T group was incubated by contacting with EGFR antigen and anti-CD28 antibody, and the EGFR CAR-T / CD6 group was incubated by contacting with EGFR antigen, anti-CD28 antibody and anti-CD6 antibody. Detailed implementation methods

[0031] In the present invention, the term "Adoptive Cell Transfer Therapy (ACT)" refers to a therapy in which sensitized lymphocytes (with specific immunity) or products of sensitized lymphocytes (such as transfer factor and immune ribonucleic acid, etc.) are transfused into individuals with low cellular immune function (such as cancer patients) to enable them to acquire anti-tumor immunity.

[0032] The term "chimeric antigen receptor (CAR)" refers to a class of engineered receptors that transplant a defined specificity onto immune effector cells, usually T cells, and enhance the function of T cells.

[0033] The term "coding sequence" is the part of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (such as CAR). The boundaries of the coding sequence are usually determined by the ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame at the 5' end of the mRNA and the transcription termination sequence immediately downstream of the open reading frame at the 3' end of the mRNA. The coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0034] The term "nucleic acid construct" is an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The term "expression vector" is a nucleic acid construct or vector that is recombinantly or synthetically produced for expressing a nucleic acid of interest in target cells.

[0035] The term "antibody" is an immunoglobulin molecule that specifically binds to an antigen, and can be a complete immunoglobulin of natural or recombinant origin, or can be an immunoreactive part of an immunoglobulin. Antibodies can exist in various forms, including but not limited to monoclonal antibodies, polyclonal antibodies, Fv, Fab, scFv, humanized antibodies, heavy chain antibodies, or single domain antibodies.

[0036] The term "monoclonal antibody" is an antibody that exhibits a single binding specificity and binds only to a single antigen epitope.

[0037] The term "polyclonal antibody" is a mixture of antibodies containing multiple antibodies secreted by different B cell lineages in vivo.

[0038] The term "humanized antibody" is a non-human antibody whose sequence has been modified to have a higher similarity compared to its native human antibody sequence.

[0039] The term "non-immobilized" means that a protein antigen or antibody is not covalently or non-covalently linked to any matrix. The matrix can be a fixed matrix, such as a solid well plate, culture dish, glass slide, etc., or a suspended matrix, such as magnetic beads, nanoparticles, etc.

[0040] The term "immobilization" refers to the connection of an antigen or antibody to a matrix in a covalent or non-covalent manner. The matrix can be a fixed matrix, such as a solid well plate, culture dish, glass slide, etc., or a suspended matrix, such as magnetic beads, nanoparticles, etc.

[0041] The term "Single Chain Variable Fragment (scFv)": A single-chain antibody usually composed of a VH and a VL linked together by a peptide chain.

[0042] The term "major histocompatibility complex (MHC)": A collective term for a group of genes encoding major histocompatibility antigens in animals.

[0043] The term "Peripheral blood mononuclear cell (PBMC)": Cells in peripheral blood with a single nucleus, including lymphocytes and monocytes.

[0044] The conventional method for preparing CAR-T cells in the art is as follows: First, PBMC is isolated, then T lymphocytes are co-activated and amplified using CD3 and anti-CD28 antibodies as the first signal and the second signal respectively, and finally the transgenic CAR is introduced into T cells by viral particles or electroporation. The quality of T lymphocytes and PBMC cells in patients is uneven and uncontrollable due to factors such as changes in the internal environment or external interference in the body, resulting in problems such as low transfection efficiency, low activation, poor proliferation, and difficult quality control during the preparation process of CAR-T cells, leading to difficulties in clinical translation.

[0045] Prior art has found that weak expression of ZAP70 leads to the inability of the CD3 first signal and the CD28 second signal of activated T lymphocytes to synergistically play an activating role, thereby affecting the proliferation and activation in the process of CAR-T cell preparation. At the same time, prior art has shown that CD6 in the T cell activation pathway is a cell surface receptor expressed on immature thymocytes, peripheral blood T cells and B1a cells, and is co-localized with the TCR / CD3 complex at the immunological synapse (Biophysical Characterization of CD6-TCR / CD3 Interplay in T Cells. Frontiers in immunology, MBM, M., M.SFB, C.FB, T.R.J, K.JS, J.B, W.JJ, B.R, F.CG & C.A, 2018, 9, 2333.). CD6 can respond to the signaling cascade triggered by the activation of the TCR / CD3 complex and can also act as a co-stimulatory molecule to promote T cell activation and proliferation. Therefore, when the expression of ZAP70 in T cells is poor, on the basis of the existing CAR-T activation protocol, a new process (adding CD6 antibody) is adopted. According to the existing criteria for judging whether the quality of CAR-T cells meets the standard, such as CAR expression, in vitro proliferation, cell phenotype, and anti-tumor effect, etc., the optimization effect of the new process on CAR-T cells is evaluated from multiple angles, and the related mechanism of action is explored, providing an experimental basis for the clinical treatment research of CAR-T therapy.

[0046] Based on this, the method for preparing CAR-T cells of the present invention is as follows: contacting and culturing PBMC cells or T lymphocytes with tumor antigen, anti-CD28 antibody and anti-CD6 antibody, and the PBMC cells or T lymphocytes contain nucleic acids encoding chimeric antigen receptors targeting tumor antigens intracellularly. The CAR-T cells prepared by this method have uniform quality and similar activities, solving the problems of low transfection efficiency, less activation, poor proliferation, and difficult quality control of CAR-T cells caused by low expression of ZAP70, and meeting the requirements of cGMP production management and large-scale production.

[0047] The tumor antigens targeted by the chimeric antigen receptor of the present invention are tumor-specific antigens and / or tumor-associated antigens, and the tumor-specific antigen or tumor-associated antigen includes but is not limited to any one or combination of the following: CD19, CD20, CEA, GD2, FR (Flavin reductase), PSMA (prostate-specific antigen), PMEL (premelanosome protein), CA9 (carbonic anhydrase IX), CD171 / L1-CAM, MART-1 (mucin A), ERBB2, MAGE (melanoma-associated antigen E1) family proteins, BAGE (B melanoma antigen family proteins) family, GAGE (growth hormone-releasing factor family proteins) family, AFP, MUC1, MUC16, CD22, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, EGP-2, EGP-40, PSA (kallikrein-related peptidase 3), MSLN (mesothelin), EGFR, ERBB3, ERBB4, AFU, HER family, EpCAM, IGFR1, CA242 or CA50, etc., preferably the HER family, and more preferably EGFR.

[0048] The nucleic acid containing the coding sequence of the chimeric antigen receptor targeting tumor antigen of the present invention is DNA or mRNA, which realizes the expression coding of the chimeric antigen receptor in host cells, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion, etc. When the nucleic acid is DNA, it is a nucleic acid construct operably linked to one or more regulatory sequences, guiding the expression coding of the target sequence chimeric antigen receptor in host cells, preferably an expression vector, selected from recombinant eukaryotic expression vectors or recombinant viral vectors, and the recombinant eukaryotic vectors include but not limited to pNB vectors (transposon vectors), pRS vectors, pUC vectors, pSV2 vectors, pRSV vectors, pcDNA3.1 vectors, pVAX1 vectors, pEGFP vectors or pCMVp-NEO-BAN vectors, etc., and the recombinant viral vectors include but not limited to recombinant adenovirus vectors or lentiviral vectors, etc. The transposon vector can be a eukaryotic expression vector containing a transposon element selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty, and this type of transposon vector contains the 5' inverted terminal repeat sequence of the corresponding transposon and the 3' inverted terminal repeat sequence of the corresponding transposon. The transposase is selected from the transposases of the piggybac, sleeping beauty, frog prince, Tn5 or Ty transposon systems. When using transposases from different transposon systems, the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to this transposon system, which is a conventional technical choice easily determined by those skilled in the art. In some embodiments of the present invention, the expression vector is a pNB transposon vector, the transposase is a transposase from the piggybac system, and the 5' inverted terminal repeat sequence and 3' inverted terminal repeat sequence of the transposon are the 5' inverted terminal repeat sequence and 3' inverted terminal repeat sequence of the piggybac transposon. The mRNA contains a 5' cap structure, 5'UTR, an open reading frame (ORF) encoding the chimeric antigen receptor, 3'UTR and a polyA tail.

[0049] In some embodiments of the present invention, the extracellular antigen-binding region of the chimeric antigen receptor targeting tumor antigen is an scFv against the human epidermal growth factor receptor family, preferably an scFv against epidermal growth factor receptor 1. The DNA coding sequence of the scFv against epidermal growth factor receptor 1 is preferably positions 78 - 800 of SEQ ID NO:1.

[0050] The anti-CD28 antibody of the present invention is a human anti-CD28 antibody, a murine anti-CD28 antibody or a rabbit anti-CD28 antibody, preferably a human anti-CD28 antibody; further, the anti-CD28 antibody is an anti-CD28 monoclonal antibody or an anti-CD28 polyclonal antibody, preferably an anti-CD28 monoclonal antibody.

[0051] The anti-CD6 antibody of the present invention is a human anti-CD6 antibody or a murine anti-CD6 antibody, preferably a human anti-CD6 antibody. Further, the anti-CD6 antibody is a monoclonal anti-CD6 antibody or a polyclonal anti-CD6 antibody, preferably a monoclonal anti-CD6 antibody.

[0052] During the contact culture process of the present invention, the tumor antigen, anti-CD28 antibody, and anti-CD6 antibody are in a non-immobilized state or an immobilized state, preferably an immobilized state. The tumor antigen, anti-CD28 antibody, and anti-CD6 antibody are linked to the matrix in a covalent or non-covalent manner. The matrix can be a fixed matrix, such as a solid well plate, culture dish, or glass slide, etc., or a suspended matrix, such as magnetic beads, nanoparticles, etc.

[0053] When in the immobilized state, the coating concentration of the tumor antigen is 1 - 3 μg / ml, preferably 1.5 - 2 μg / ml; the coating concentration of the anti-CD28 antibody is 1 - 3 μg / ml, preferably 1.5 - 2 μg / ml; the coating concentration of the anti-CD6 antibody is 1 - 3 μg / ml, preferably 1.5 - 2 μg / ml.

[0054] The contact culture of the present invention is a conventional T cell culture method in the art, with the conditions: culture temperature 37°C, culture CO2 concentration 5%, culture medium AIM-V + 2% FBS medium, culture days 5 - 10 days, preferably 5 - 6 days; the culture medium contains cytokines, and the final concentration of the cytokines is 100 - 1000 U / ml, preferably 450 - 600 U / ml; the cytokines are selected from any one or more of IL-2, IL-15, IL-12, IL-21, preferably IL-2.

[0055] The CAR-T cell culture method of the present invention further includes transferring the cells after contact culture to a conventional T cell culture medium without tumor antigen, anti-CD28 antibody, and anti-CD6 antibody for conventional culture, with the conditions: culture temperature 37°C, culture CO2 concentration 5%, culture medium AIM-V + 2% FBS medium, culture days 5 - 10 days, the culture medium contains cytokines, and the final concentration of the cytokines is 50 - 1000 U / ml, preferably 50 - 200 U / ml; the cytokines are selected from any one or more of IL-2, IL-15, IL-12, IL-21, preferably IL-2.

[0056] The method for transferring the nucleic acid containing the chimeric antigen receptor coding sequence targeting the tumor antigen of the present invention into PBMC cells is selected from any one of viral particle infection, electroporation, liposome transfection, calcium phosphate transfection, and gene gun transfection, preferably electroporation. In certain embodiments of the present invention, the chimeric antigen receptor targeting the EGFR antigen is introduced into PBMC cells by electroporation with the recombinant pNB338B-EGFR vector containing its coding sequence.

[0057] The source of the PBMC cells of the present invention can be a conventional source in the art, for example, isolated from the blood of a donor subject by Ficoll density gradient centrifugation, or can be commercially available PBMCs.

[0058] Example 1: Detection of the expression abundance of tumor-associated antigen EGFR on the surface of tumor cells

[0059] 1 Culture of tumor cells

[0060] 1.1 Resuscitate the tumor cell lines required for the experiment: The human gastric cancer cell line HGC27, the human ovarian cancer cell line SKOV3, the human pancreatic cancer cell lines PanC 1, BXPC-3, and Aspc-1, the human liver cancer cell line Hep3B, the human lung cancer cell lines H460, H292, and H23, and the colon cancer cell line COLO 205 were all purchased from ATCC; the human liver cancer cell lines HepG2 and Huh7 were purchased from the Chinese Academy of Sciences;

[0061] 1.2 Culture different types of tumor cells in appropriate prepared media (generally using DMEM or RPMI-1640 medium and adding 5%-10% inactivated fetal bovine serum), and place them in a 37°C, 5% carbon dioxide cell culture incubator for culture;

[0062] 1.3 Culture for several generations, with good cell status and the tumor cells fused to about 80%, then digest and count.

[0063] 2 Detection of the expression of EGFR on the surface of different tumor cells by flow cytometry

[0064] 2.1 According to the counting result in 1.3, take an appropriate amount of tumor cell suspension and add it to a 1.5 ml EP tube, with 5×10 5 cells per tube;

[0065] 2.2 According to the recommended usage amount of the EGFR antibody, add the EGFR flow antibody to the cell suspension and resuspend it to 50 μl, mix well, and incubate in the dark at 4°C for 30 min;

[0066] 2.3 Add 1 ml of PBS to the cell suspension after incubating the antibody, centrifuge at 3000 rpm at room temperature for 3 min, discard the supernatant, and repeat the operation once;

[0067] 2.4 Aspirate 400 μl of PBS to resuspend the cells, transfer them to a flow tube, adjust the parameters of the flow cytometer, set the gate to select the target cell population, and collect the flow cytometry experimental data;

[0068] 2.5 Use Kaluza Analysis software to analyze the detection results, where Figure 1Flow cytometry detection results of EGFR tumor-associated antigens on the surfaces of different tumor cells;

[0069] 2.6 Use GraphPad Prism 8.0.1 software to statistically analyze the experimental data of multiple detections of each tumor cell, Figure 2 which is the statistical result of multiple flow cytometry detections of EGFR tumor-associated antigens on the surfaces of different tumor cells.

[0070] The above Figure 1 and Figure 2 results show that EGFR is expressed to varying degrees on the surfaces of three different tumor cell lines of pancreatic cancer, liver cancer, and lung cancer. Among them, the two cell lines of pancreatic cancer PanC 1 and lung cancer H292 highly express EGFR, the four cell lines of pancreatic cancer BXPC-3 / Aspc1 and liver cancer Huh-7 / Hep3B moderately express EGFR, and the three cell lines of liver cancer HepG2 and lung cancer H460 / H23 lowly express or do not express EGFR.

[0071] Example 2: Construction and activation of T cells stably expressing EGFR CAR

[0072] 1 Isolation and cryopreservation of peripheral blood mononuclear cells

[0073] 1.1 Ficoll aliquoting: According to the sample volume, prepare several 50 ml centrifuge tubes filled with ficoll solution, 15 ml per tube for standby;

[0074] 1.2 Use a 50 ml syringe to suck out the blood at the bleeding port of the blood bag, and add the sucked blood to a clean 50 ml centrifuge tube according to the sample volume for dilution;

[0075] 1.3 Dilute 30 ml of whole blood to 60 ml with DPBS, and then aliquot 20 ml per tube into 50 ml centrifuge tubes;

[0076] 1.4 Under light protection, gently add the diluted blood to the upper layer of ficoll that has been fully heated in a 20°C water bath, avoiding mixing the two solutions. Each centrifuge tube contains 35 ml of the mixed solution; centrifuge at 800 g for 20 min (the centrifuge speed increase is 1 and the speed decrease is 0). After centrifugation, layering will occur;

[0077] 1.5 The cell layer where PBMC is located is white. At this time, use a 1000 μl pipette to aspirate the cells in this layer into another clean 50 ml centrifuge tube;

[0078] 1.6 After adding 3 volumes of DPBS and mixing well, place it in a centrifuge and centrifuge at 1500 rpm for 10 min, then discard the supernatant, and add an appropriate amount of DPBS to resuspend;

[0079] 1.7 Take 10 μl of the cell suspension, mix it thoroughly with 10 μl of 0.4% trypan blue solution, and then add it to a cell counting chamber compatible with a cell counter for counting. After that, add 3 volumes of DPBS and centrifuge at 1500 rpm for 10 min, then discard the supernatant;

[0080] 1.8 Cell adhesion: After centrifuging and collecting the cells, resuspend them in AIM-V medium at a density of 5×10^6 cells / ml. Select an appropriate culture flask according to the resuspended volume for cell adhesion culture. Place the culture flask in an incubator at 37°C and 5% CO2 for 2 - 4 h or overnight;

[0081] 1.9 Collection of suspended cells: After culturing, collect the culture medium and suspended cells into a 50 ml centrifuge tube, and wash the culture flask once with DPBS. Collect the washing solution into the 50 ml centrifuge tube, centrifuge at 1500 rpm for 10 min, then discard the supernatant, and add an appropriate amount of DPBS to resuspend thoroughly.

[0082] 1.10 Take 10 μl of the cell suspension, mix it thoroughly with 10 μl of 0.4% trypan blue solution, and then add it to a cell counting chamber compatible with a cell counter for counting. After counting, centrifuge the cells at 1500 rpm for 10 min, then discard the supernatant;

[0083] 1.11 After centrifuging and collecting the cells, resuspend them in cell cryopreservation solution at a volume of 1×10 7 cells / ml. Add 1 ml of the cell and cryopreservation solution mixture to a cryotube, place it in a cell cryopreservation box, and then put it in a -80°C refrigerator for 24 h. The next day, transfer the cells to liquid nitrogen for long-term storage.

[0084] 2 Detection of ZAP70 expression in peripheral blood mononuclear cells

[0085] To detect the ZAP70 expression in PBMC, Western blot was used for determination.

[0086] 2.1 Protein extraction

[0087] 2.1.1 Collect the cells into a 1.5 ml EP tube, centrifuge at 1000 rpm for 3 min, discard the supernatant, add an appropriate amount of normal saline or DPBS to wash the cells, centrifuge at 1000 rpm for 3 min, and discard all the supernatant, leaving the cell pellet.

[0088] 2.1.2 Add lysis buffer to resuspend the cells (80 ul / 1×10 6 ), and lyse on ice for 20 min.

[0089] 2.1.3 Centrifuge at 4°C and 12000 rpm for 10 min, aspirate the supernatant (take 1 - 3 ul for BCA quantification

[0090] 2.2 BCA Quantification

[0091] 2.2.1 Take 25 μl of protein standards and protein samples to be measured at each dilution concentration and add them to the microplate (detection range 20 - 2000 μg / ml).

[0092] 2.2.2 Add 200 μl of working solution to each well, shake on the oscillator for 30 s to fully mix the solution.

[0093] 2.2.3 Seal the microplate and incubate at 37 °C for 30 min.

[0094] 2.2.4 Cool the microplate to room temperature and measure the absorbance of the sample at 562 nm using a microplate reader.

[0095] 2.2.5 Subtract the absorbance of the blank standard at 562 nm from the absorbance of each standard protein and the protein sample to be measured at 562 nm.

[0096] 2.2.6 Plot a standard curve by graphing the average absorbance of the BSA standard at 562 nm after blank control correction against its concentration. Determine the concentration of the sample to be measured based on its absorbance on the standard curve.

[0097] 2.3 Sample Boiling

[0098] 2.3.1 Add an appropriate amount of 1× loading buffer to the collected supernatant and incubate in a metal bath at 98 °C for 10 min.

[0099] 2.3.2 Place on ice for 2 min after treatment and store short - term at -20 °C and long - term at -80 °C

[0100] 2.4 Preparation of SDS - PAGE Gel (Prepared according to the rapid gel preparation kit of Yamei Company)

[0101] 2.5 Membrane Transfer

[0102] 2.5.1 After electrophoresis, gently remove the glass plate, cut off the excess gel, place the cut gel block in the electrotransfer buffer to keep it moist; cut an appropriate size of PVDF membrane and activate it in methanol for 2 min; cut 2 pieces of thick filter paper slightly larger (0.3 - 0.5 cm longer and wider) than the PVDF membrane.

[0103] 2.5.2 Semi - dry transfer: Equilibrate the thick filter paper, PVDF membrane, and gel in the transfer buffer for 10 min, assemble the transfer equipment in the following order (thick filter paper - PVDF membrane - gel - thick filter paper), moisten with some transfer solution in the electrophoresis tank, set the transfer conditions and start transfer. (16 v, 25 min).

[0104] 2.6 Blocking: Remove the PVDF membrane from the electroporator and place it in pre-prepared 5% skim milk or 5% BSA. Block at room temperature on a low-speed shaker for 2 h.

[0105] 2.7 Primary antibody incubation: Add the primary antibody diluted according to the corresponding dilution ratio (prepared with 5% skim milk or TBST), and incubate overnight at 4°C.

[0106] 2.8 Membrane washing: Recover the primary antibody. Add an appropriate amount of 1×TBST and shake on a shaker for 10 min, repeat three times.

[0107] 2.9 Secondary antibody incubation: Add the secondary antibody conjugated with HRP of the corresponding species and incubate at room temperature for 1 h.

[0108] 2.10 Membrane washing: Recover the secondary antibody. Add an appropriate amount of 1×TBST and shake on a shaker for 10 min, repeat three times.

[0109] 2.11 Development: Determine the amount of luminescent solution according to the size of the PVDF membrane. Take equal volumes of solution A and solution B, mix well and store for later use. Use forceps to take out the membrane, lay it on a layer of plastic wrap, add the mixed solution in the center of the membrane, and place it in an AI600 chemiluminescence imaging system for photographing.

[0110] 2.12 Data analysis.

[0111] The above detection results are as Figure 4 shown. Among the detection results of PBMCs from 5 randomly selected different donors, 2 donors' PBMCs showed strong ZAP70 expression, and 3 donors showed weak ZAP70 expression.

[0112] 3 Preparation of EGFR CAR-T cells

[0113] Use the electroporation technique to transfect the plasmid EGFR CAR plasmid constructed and stored in our laboratory (as Figure 3 , for the pNB338B vector plasmid structure, see CN201711476630.6, for the construction, see CN201711459160.2 or CN201711462801.X, the EGFRCAR sequence is as shown in SEQ ID NO:1), where the EGFR scFv coding sequence refers to positions 78 - 800 of SEQ ID NO:1, and transfect the pNB vector (transposon vector) into PBMCs to prepare EGFR CAR-T cells. Electroporation is completed using a LONZA 4D electroporator.

[0114] 3.1 Cell resuscitation: Take out PBMC cells from liquid nitrogen; quickly place them in a 37°C water bath and melt within 1 minute; use a 1 ml pipette to suck out the liquid in the tube, add it to a 15 ml centrifuge tube containing 5 ml of pre-prepared fresh AIM-V + 2% FBS medium, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, then add 1 ml of DPBS to resuspend and count.

[0115] 3.2 Plasmid incubation and electroporation

[0116] 3.2.1 Prepare electroporation solution: Take the corresponding LONZA electroporation solution and mix it in a ratio of nucleofection solution: additive = 82:18;

[0117] 3.2.2 Take out the EGFR CAR plasmid stored in the -20°C refrigerator and melt it. After shaking and mixing evenly, perform a transient centrifugation, calculate the amount required for electroporation (6 μg of EGFR CAR plasmid is used for each electroporation), mix it evenly with 100 μl of electroporation solution, and let it stand for 5 minutes;

[0118] 3.2.3 Take a 12-well plate, add 1 ml of fresh AIM-V + 2% FBS medium to each well, shake the culture plate to cover the bottom of the plate with the medium, and place the culture plate in a CO2 incubator for 30 minutes;

[0119] 3.2.4 Transfer the cells into a sterilized 1.5 ml EP tube, add 5×10 6 cells to each tube, centrifuge at 3000 rpm for 3 minutes, completely remove the supernatant for electroporation. Add 100 μl of the plasmid-electroporation solution mixture to the cells, place it in an electroporator, select the program F1-115 (human T cells, unstin, HE) for electroporation; use the micropipette in the kit to transfer the electroporated cell suspension to the 12-well plate with added medium, mix evenly, and place it in a 37°C, 5% CO2 incubator for 4 - 6 hours.

[0120] 4 Activate and culture EGFR CAR-T cells

[0121] 4.1 Antibody coating

[0122] Before electroporation, take out human CD3 antibody, human CD28 antibody, and human recombinant EGFR antigen stored in the -20°C refrigerator and melt them for standby. Add the antibodies to DPBS, with concentrations of 5 μg / ml respectively. After mixing evenly, add 1 ml to each well of a six-well plate. The control T cell group is coated with human CD3 antibody + human CD28 antibody; the EGFR CAR-T cell group is coated in two different ways: one is coated with human EGFR antigen + human CD28 antibody, and the other is coated with human EGFR antigen + human CD28 antibody + CD6 antibody. Place the coated plate in a 37°C incubator for more than 4 hours.

[0123] 4.2 EGFR CAR-T Cell Activation

[0124] 4 - 6 h after electroporation, take out the antibody-coated six-well plate from the incubator, discard all the liquid in the culture plate, add 1 ml of DPBS for washing, remove the DPBS, transfer the electroporated cells in the twelve-well plate to the antibody-coated six-well plate, add 2 ml of AIM-V + 2% FBS medium to each well, add an appropriate amount of IL-2, and place it in an incubator at 37 °C and 5% CO2 for about 5 days, where the final concentration of IL-2 is 500 U / ml.

[0125] 4.3 Conventional Culture of EGFR CAR-T Cells

[0126] After the activation of EGFR CAR-T cells, transfer the cells to a culture plate without antigen coating, and culture the T cells in each group using AIM-V + 2% FBS + 100 U / ml IL-2 medium. The cell culture concentration is maintained between 5×10 5 ~1×10 6 cells per milliliter, and place it in an incubator at 37 °C and 5% CO2 for culture.

[0127] 4.4 Detection of the Positive Rate of EGFR CAR-T Cells

[0128] 13 days after the electroporation of EGFR CAR-T cells and subculture, use flow cytometry to detect the transfection efficiency of CAR in T cells. The specific operation steps are as follows:

[0129] 4.4.1 Add the T cell suspension of each group to a 1.5 ml EP tube, with 1×10 6 cells in each tube;

[0130] 4.4.2 Add 2 μl of biotinylated human EGFR flow antibody, mix well, and incubate at 4 °C in the dark for 30 min;

[0131] 4.4.3 Add 1 ml of PBS, centrifuge at 3000 rpm at room temperature for 3 min, discard the supernatant, and repeat the operation once;

[0132] 4.4.4 Add 1 μl of PE-streptavidin secondary antibody flow antibody, mix well, and incubate at 4 °C in the dark for 30 min;

[0133] 4.4.5 Add 1 ml of PBS, centrifuge at 3000 rpm at room temperature for 3 min, discard the supernatant, and repeat the operation once;

[0134] 4.4.6 Aspirate 400 μl of PBS to resuspend the cells, transfer them to a flow tube, adjust the parameters of the flow cytometer, set the gate to select the target cell population, and collect the experimental data of flow cytometry detection;

[0135] 4.4.7 Analyze the test results using Kaluza Analysis software, where Figure 5 are the flow cytometry test results of the positive rates of CAR expression by T cells in each group;

[0136] 4.4.8 Use GraphPad Prism 8.0.1 software to statistically analyze the experimental data of multiple detections of each tumor cell, Figure 6 Statistical results of the multiple flow cytometry test results of the positive rates of CAR expression by T cells in each group.

[0137] The above experimental results confirm that the CD6 antibody has no obvious effect on the transfection efficiency of CAR in PBMC with high ZAP70 expression. The transfection efficiency of T cells activated by EGFR antigen + CD28 antibody (EGFR CAR-T) or EGFR antigen + CD28 antibody + CD6 antibody (EGFR CAR-T / CD6) can reach 70%-80% ( Figure 5 and Figure 6 ). However, the CD6 antibody has a relatively obvious enhancing effect on the transfection efficiency of CAR in PBMC with weak ZAP70 expression. The transfection efficiency of the EGFR CAR-T / CD6 group can reach 60%-80%, which is about 20% higher than that of the original EGFR CAR-T group ( Figure 5 and Figure 6 ).

[0138] Example 3: Effects of Different Activation Methods on the Proliferation, Activation and Differentiation of EGFR CAR-T Cells

[0139] After finding that the CD6 antibody as an activator can significantly increase the positive rate of PBMC with weak ZAP70 expression, further study the effects of the CD6 antibody on the proliferation, activation and differentiation functions of CAR-T cells prepared from PBMC with weak ZAP70 expression.

[0140] 1 Detect the proliferation of EGFR CAR-T cells

[0141] To detect the proliferation of T cells after transfection, continuously monitor the growth status of T cells after electroporation. Using the number of T cells on the 5th day as the starting number, count the T cells in each group cultured to the 5th / 7th / 9th / 11th / 12th / 13th day, and calculate their proliferation multiples. The results are as Figure 7 shown. Analysis found that all groups of T cells grew well during the in vitro culture and amplification process after stimulation and activation, and could be amplified 32-40 times after 13 days of in vitro culture. Moreover, the proliferation effect of the EGFR CAR-T / CD6 group was slightly better than that of the EGFR CAR-T group or the control T group ( Figure 7 ).

[0142] 2 Detect the indicators related to activation on the surface of EGFR CAR-T cells

[0143] After activating T cells by different activation methods and culturing them until the 11th day after electroporation, using the cells in the control T group as a control, collect the cells in the EGFR CAR-T group and the EGFR CAR-T / CD6 group and use flow cytometry to detect the expression of the surface activation indicators CD137 and CD107α. The specific experimental steps are as follows:

[0144] 2.1 Take 1×10 6 T cell suspensions of each group and add them to 1.5 ml EP tubes;

[0145] 2.2 According to the recommended usage amount of the CD137 or CD107α antibody, add the CD137 or CD107α flow antibody to the cell suspension and resuspend it to 100 uL, mix well, and incubate at 4°C in the dark for 30 min;

[0146] 2.3 Add 1 ml of PBS to the cell suspension after incubating the antibody, centrifuge at 3000 rpm at room temperature for 3 min, discard the supernatant, and repeat the operation once;

[0147] 2.4 Aspirate 400 μl of PBS to resuspend the cells, transfer them to a flow tube, adjust the parameters of the flow cytometer, set the gate to select the target cell population, and collect the experimental data of flow cytometry detection;

[0148] 2.5 Use Kaluza Analysis software to analyze the detection results, where Figure 8 are the flow cytometry detection results of activation-related indicators such as CD137 or CD107α on the surface of different T cells;

[0149] 2.6 Use GraphPad Prism 8.0.1 software to statistically analyze the experimental data of multiple detections of each tumor cell, Figure 9 and Figure 10 are the statistical results of multiple flow cytometry detections of activation-related indicators such as CD137 and CD107α on the surface of different T cells.

[0150] The above experimental results show that compared with the cells in the EGFR CAR-T group, the activation state of the cells in the EGFR CAR-T / CD6 group is better, indicating that the CD6 antibody can enhance the activation efficiency of T lymphocytes.

[0151] 3 Detect the indicators related to T cell differentiation on the surface of EGFR CAR-T cells

[0152] Use flow cytometry to detect the differentiation of central memory T cells in the EGFR CAR-T group and the EGFR CAR-T / CD6 group of cells after being stimulated by specific antigens in vitro. The specific experimental steps are as follows:

[0153] 3.1 Specific antigen stimulates T cells

[0154] Use plates coated with specific EGFR antigen, and add the same number of resting control T cell groups, EGFR CAR-T cell groups, and EGFR CAR-T / CD6 cell groups to the coated plates respectively. After stimulation for 24 hours, collect the cells of each group.

[0155] 3.2 Use flow cytometry to detect the expression levels of CD62L and CCR7 molecules in CD45RO-positive cells on the cell surface

[0156] 3.2.1 Collect the T cells of each group after stimulation. Take 1×10 6 T cell suspensions of each group and add them to 1.5 ml EP tubes;

[0157] 3.2.2 Add 5 μl of CD45RO, CD62L, and CCR7 flow antibodies to the cell suspensions respectively and make up the volume to 100 μl. Mix well and incubate in the dark at 4°C for 30 min;

[0158] 3.2.3 Add 1 ml of PBS to the cell suspensions after incubating with antibodies, centrifuge at 3000 rpm at room temperature for 3 min, discard the supernatant, and repeat the operation once;

[0159] 3.2.4 Aspirate 400 μl of PBS to resuspend the cells, transfer them to flow tubes, adjust the parameters of the flow cytometer, set the gate to select the target cell population, and collect the experimental data of flow cytometry detection;

[0160] 3.2.5 Use Kaluza Analysis software to analyze the detection results, where Figure 11 are the flow cytometry detection results of CD62L and CCR7 in CD45RO-positive cells of different T cells.

[0161] The above experimental results show that, taking the control T cell group as the control, the central memory T cells (CCR7+, CD62L+) in the EGFR CAR-T and EGFR CAR-T / CD6 cell groups can rapidly differentiate into effector memory T cells (CCR7-, CD62L-) after being stimulated by specific antigens ( Figure 11 ), among which the expression level of central memory T cells in the EGFR CAR-T / CD6 group is higher ( Figure 11 ), and the cell differentiation level is lower, indicating that the CD6 antibody plays an important role in maintaining the low differentiation level of T lymphocytes. From the comprehensive analysis of the above results, it can be known that the CD6 antibody can improve the proliferation and activation ability of T lymphocytes to a certain level and maintain its low differentiation level.

[0162] Example 4: Effects of different activation methods on the anti-tumor activity of EGFR CAR-T cells

[0163] Detection of cytokine release levels after co-incubation of T cells with tumor cells with different EGFR antigen expression levels

[0164] To explore the anti-tumor effect of EGFR CAR-T / CD6 cells in vitro, we first selected the PanC 1 tumor cell line with high EGFR antigen expression and the Hep G2 tumor cell line with low EGFR antigen expression according to the results of tumor surface antigen detection in Figure 2 . These cell lines were co-cultured with control T, EGFR CAR-T, and EGFR CAR-T / CD6 cells, respectively, and the secretion of IFN-γ in the supernatant was detected. The specific operation steps are as follows:

[0165] 1. Co-incubation of T cells with tumor cells

[0166] 1.1 Tumor cell culture: Culture the tumor cell lines PanC 1 (high expression of EGFR antigen) and HepG2 (low expression of EGFR antigen). When the growth state is good and the tumor cells are fused to about 80%, digest and count the cells;

[0167] 1.2 Seeding of tumor cells: Seed 1×10 5 tumor cells of PanC 1 or HepG2 evenly into a 12-well plate and allow them to adhere for 4 hours;

[0168] 1.3 Addition of effector cells: Inoculate 1×10 5 control T, EGFR CAR-T, and EGFR CAR-T / CD6 cells into the wells seeded with tumor cells of PanC 1 or HepG2, respectively. After co-culturing for 24 hours, collect the supernatant.

[0169] 1.2 Detection of cytokine levels in the supernatant of co-incubated cells using a multi-cytokine kit

[0170] Use the cytokine kit Human Th1 / Th2 Cytokine Kit II to detect the expression level of T cell cytokines. The specific experimental operations are as follows:

[0171] 1.2.1 Take 10 μl of the supernatant into a 1.5 ml centrifuge tube and dilute the sample with 40 μl of detection diluent (1×);

[0172] 1.2.2 Calculate and take out the corresponding magnetic beads according to the amount and mix them, then take 50 μl and add it to the sample;

[0173] 1.2.3 Take 50 μl of antibody (HumanTh1 / Th2-II PE Detection Reagent) and add it to the sample, and incubate at room temperature in the dark for 3 hours;

[0174] 1.2.4 Take 1 ml of washing buffer to wash the sample antibody mixture, centrifuge at 400 g for 5 min, discard the supernatant completely, take 300 μl of washing buffer for resuspension, collect using a flow cytometer and analyze the results using FACP Array v3 analysis software.

[0175] The above experimental results showed that EGFR CAR-T and EGFR CAR-T / CD6 group cells could recognize PanC 1 tumor cell lines with high expression of EGFR antigen and secrete a large amount of IFN-γ, but when co-cultured with Hep G2 tumor cell lines with low expression of EGFR antigen, the level of IFN-γ secreted was extremely low ( Figure 12 ). Adding anti-CD6 antibody as an activator after T cell electroporation did not affect the potential of EGFRCAR-T cells to recognize specific tumor antigens. And compared with EGFR CAR-T cells, EGFR CAR-T / CD6 cells secreted a higher level of IFN-γ ( Figure 12 ), and the results indicated that anti-CD6 antibody as an activator could enhance the anti-tumor effect of CAR-T cells.

[0176] 2 Detection of the anti-tumor activity of T cells against tumor cells with high expression of EGFR tumor-associated antigens

[0177] We further used the xCELLigence RTCA DP Instrument (Ansen Biotech (Hangzhou) Co., Ltd., China) to conduct experiments to evaluate the killing effect of CAR-T cells on the tumor cell line PanC1 with high expression of EGFR antigen. The specific experimental operations are as follows:

[0178] 2.1 Target cell culture: Routinely culture the tumor cells PanC 1. When the cells grow well, digest the cells and count them;

[0179] 2.2 Instrument zero adjustment: Add 50 uL of tumor cell culture medium to each well of the RTCA electrode culture plate for zero adjustment before use;

[0180] 2.3 Laying target cells: Lay the PanC 1 cell suspension into each well of the RTCA electrode culture plate at a rate of 1×10 4 cells / 50 uL, and run the instrument;

[0181] 2.4 Adding effector cells: After culturing the tumor cells for 24 hours, pause the instrument, and add control T, EGFR CAR-T, and EGFR CAR-T / CD6 cells with effector-to-target ratios of 1:1, 2:1, and 4:1 to each well, 50 uL per well, set three replicates, and start the instrument;

[0182] 2.5 Data acquisition: After co-culturing the cells for 24 hours, observe the cell killing situation, stop the program, analyze, and save the data;

[0183] 2.6 Analyze the killing results using the xCELLigence RTCA DP Instrument.

[0184] Compared with the blank control group T, at a low effector-to-target ratio (1:1), the killing effect of the two groups of EGFR CAR-T cells was stronger, and the killing efficiency of CAR-T increased significantly with the increase of the effector-to-target ratio. And it showed a consistent trend with the detection results of IFN-γ secretion. EGFR CAR-T / CD6 cells showed stronger anti-tumor effects than EGFR CAR-T cells within a certain range of effector-to-target ratios ( Figure 13 and Figure 14 ). In summary, the CD6 antibody can enhance the anti-tumor effect of CAR-T cells.

[0185] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention. Sequence Listing <110> Shanghai Cellular Therapy Group Co., Ltd. <120> Preparation Method of a CAR-T Cell <130> 202886 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1509 <212> DNA <213> Artificial Sequence <400> 1 aattcgccac catggcctta ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc 60 acgccgccag gccgagcgac atcttgctga ctcagtctcc agtcatcctg tctgtgagtc 120 caggagaaag agtcagtttc tcctgcaggg ccagtcagag tattggcaca aacatacact 180 ggtatcagca aagaacaaat ggttctccaa ggcttctcat aaagtatgct tctgagtcta 240 tctctgggat cccttccagg tttagtggca gtggatcagg gacagatttt actcttagca 300 tcaacagtgt ggagtctgaa gatattgcag attattactg tcaacaaaat aataactggc 360 caaccacgtt cggtgctggg accaagctgg agctgaaagg tggaggcggt tcaggcggag 420 gtggcagcgg cggtggcggg tcgcaggtgc agctgaagca gtcaggacct ggcctagtgc 480 agccctcaca gagcctgtcc atcacctgca cagtctctgg tttctcatta actaactatg 540 gtgtacactg ggttcgccag tctccaggaa agggtctgga gtggctggga gtgatatgga 600 gtggtggaaa cacagactat aatacacctt tcacatccag actgagcatc aacaaggaca 660 attccaagag ccaagttttc tttaaaatga acagtctgca atctaatgac acagccatat 720 attactgtgc cagagccctc acctactatg attacgagtt tgcttactgg ggccaaggga 780 ctctggtcac tgtctcttcg ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc 840 cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc 900 cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg gacttcgcct 960 gtgatatcta catctgggcg cccctggccg ggacttgtgg ggtccttctc ctgtcactgg 1020 ttatcaccct ttactgcaac cacaggagta agaggagcag gctcctgcac agtgactaca 1080 tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc tatgccccac 1140 cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca gacgcccccg 1200 cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga agagaggagt 1260 acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag ccgagaagga 1320 agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg gaggcctaca 1380 gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc ctttaccagg 1440 gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc ctgccccctc 1500 gctgataag 1509

Claims

1. A method for preparing CAR-T cells, characterized in that the steps Comprising: Contacting and culturing PBMC cells or T cells with tumor antigen, anti-CD28 antibody, and anti-CD6 antibody, wherein the PBMC cells or T cells contain nucleic acid encoding a chimeric antigen receptor targeting the antigen. Wherein, The tumor antigen, anti-CD28 antibody, and anti-CD6 antibody are in an immobilized state; the coating concentration of the tumor antigen is 1 - 10 μg / ml, the coating concentration of the anti-CD28 antibody is 1 - 10 μg / ml, and the coating concentration of the anti-CD6 antibody is 1 - 10 μg / ml.

2. The preparation method according to claim 1, characterized in that, The coating concentration of the tumor antigen is 4.5 - 5 μg / ml, the coating concentration of the anti-CD28 antibody is 4.5 - 5 μg / ml, and the coating concentration of the anti-CD6 antibody is 4.5 - 5 μg / ml.

3. The method for preparing CAR-T cells according to claim 1, wherein The conditions for the contact culture are: culture temperature 37°C, culture CO2 concentration 5%, culture medium AIM-V + 2% FBS medium, the culture medium contains cytokines, and the culture days are 5 - 10 days.

4. The method for preparing CAR-T cells according to claim 1, wherein, The final concentration of the cytokine is 100 - 1000 U / ml; the cytokine is selected from any one or more of IL-2, IL-15, IL-12, or IL-21.

5. The method for preparing CAR-T cells according to claim 4, wherein, The final concentration of the cytokine is 450 - 600 U / ml.

6. The preparation method of the CAR-T cell according to claim 4, wherein, The cytokine is IL-2.

7. The method for preparing CAR-T cells according to claim 1, wherein, Also included is, after the contact culture, transferring the cells obtained from the contact culture to a culture medium without tumor antigen, anti-CD28 antibody, and anti-CD6 antibody for routine culture.

8. The method for preparing CAR-T cells according to claim 1, wherein, The anti-CD28 antibody is a human anti-CD28 antibody, a murine anti-CD28 antibody, or a rabbit anti-CD28 antibody.

9. The preparation method of the CAR-T cell according to claim 8, characterized in that, The anti-CD28 antibody is a human anti-CD28 antibody.

10. The preparation method of the CAR-T cell according to claim 8, characterized in that, The anti-CD28 antibody is an anti-CD28 monoclonal antibody or an anti-CD28 polyclonal antibody.

11. The preparation method of the CAR-T cell according to claim 8, characterized in that, The anti-CD28 antibody is an anti-CD28 monoclonal antibody.

12. The method for preparing CAR-T cells according to claim 1, wherein, The anti-CD6 antibody is a human anti-CD6 antibody or a murine anti-CD6 antibody.

13. The preparation method of the CAR-T cell according to claim 12, characterized in that, The anti-CD6 antibody is a human anti-CD6 antibody.

14. The preparation method of the CAR-T cell according to claim 12, characterized in that, The anti-CD6 antibody is an anti-CD6 monoclonal antibody or an anti-CD6 polyclonal antibody.

15. The preparation method of the CAR-T cell according to claim 12, wherein, The anti-CD6 antibody is an anti-CD6 monoclonal antibody.

16. The method for preparing CAR-T cells according to claim 1, wherein, The method for transferring the nucleic acid encoding the chimeric antigen receptor targeting the antigen into PBMC is selected from any one of viral particle infection, electroporation, liposome transfection, calcium phosphate transfection, and gene gun transfection.

17. The method for preparing CAR-T cells according to claim 16, wherein The method for transferring the nucleic acid into PBMC is electroporation.

18. The method for preparing CAR-T cells according to claim 1, wherein The tumor antigens targeted by the chimeric antigen receptor are tumor-specific antigens and / or tumor-associated antigens, which are any one or combination of the following: CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171 / L1-CAM, MART-1, ERBB2, MAGE family proteins, BAGE family, GAGE family, AFP, MUC1, MUC16, CD22, CD30, CD33, CD44v7 / 8, CD70, VEGFR1, VEGFR2, EGP-2, EGP-40, PSA, MSLN, EGFR, ERBB3, ERBB4, AFU, HER family, EpCAM, IGFR1, CA242 or CA50.

19. The method for preparing CAR-T cells according to claim 18, wherein, The tumor antigen is the HER family.

20. The method for preparing CAR-T cells according to claim 18, wherein, The tumor antigen is EGFR.

21. The method for preparing CAR-T cells according to claim 1, characterized in that, The nucleic acid containing the coding sequence of the chimeric antigen receptor targeting the tumor antigen is DNA or mRNA.

22. The method for preparing CAR-T cells according to claim 21, wherein, The DNA is an expression vector.

23. The method for preparing CAR-T cells according to claim 22, wherein The expression vector is selected from recombinant eukaryotic expression vectors or recombinant viral vectors.

24. The method for preparing CAR-T cells according to claim 23, wherein The recombinant eukaryotic expression vector is a pNB vector, pRS vector, pUC vector, pSV2 vector, pRSV vector, pcDNA3.1 vector, pVAX1 vector, pEGFP vector or pCMVp-NEO-BAN vector, and the recombinant viral vector is a recombinant adenovirus vector or a lentiviral vector.

25. The method for preparing CAR-T cells according to claim 21, wherein, The mRNA contains a 5' cap structure, 5' UTR, open reading frame encoding the chimeric antigen receptor targeting the tumor antigen, 3' UTR and polyA tail.

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