Enhanced allogenic universal CAR-gamma delta T cell targeting EGFR, expressing CCR6 and secreting PD-1 single-chain antibody as well as preparation method and application of allogenic universal CAR-gamma delta T cell

Abstract

The invention belongs to the field of medicines, and relates to a gamma delta T cell containing a chimeric antigen receptor EGFR-CCR6-E27CAR. The chimeric antigen receptor comprises a single-chain antibody ScFv of a targeted EGFR (epidermal growth factor receptor), a CD8 hinge region, a CD8 transmembrane region, 4-1BB, a CD3 zeta chain, CCR6 and a secreting type ScFv of a targeted PD-1. The invention also comprises a protein and a nucleic acid sequence of the chimeric antigen receptor, and a preparation method and application of the vector, the protein, the nucleic acid and the cell. In an in-vitro cell model and an in-vivo mouse, the CAR-gamma delta T cell disclosed by the invention shows relatively strong tumor cell killing ability, can remarkably control in-vivo growth of tumors and remarkably prolong the lifetime of the mouse, and lays a foundation for the development of drugs for long-term control of in-vivo tumors of patients.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and relates to an allogeneic universal CAR-γδT cell with enhanced anti-tumor activity targeting EGFR on the surface of tumor cells, a chimeric antigen receptor expression vector contained therein, a preparation method thereof, and application thereof in immunotherapy of tumor cells expressing EGFR. BACKGROUND

[0002] Cancer has become an important disease affecting human health and average life expectancy, among which lung cancer is one of the most common cancers in the world with the second highest incidence rate after breast cancer, and is also a major tumor disease related to cancer-related deaths worldwide. Globally, there are an estimated 2 million new cases of lung cancer each year, and 1.8 million deaths. Among lung cancer patients, more than 85% are non-small cell lung cancer (NSCLC). The treatment methods for NSCLC usually include surgery, radiotherapy, chemotherapy alone or in combination, and immunotherapy such as molecular targeted therapy. However, the five-year survival rate of patients currently ranges from 30% to 80%, and therefore, finding more effective treatment methods for lung cancer, especially non-small cell lung cancer, remains the direction of efforts by numerous researchers and the biopharmaceutical industry.

[0003] The reinfusion of autologous T lymphocytes modified by chimeric antigen receptors has achieved remarkable results in hematological malignancies, and has been successfully commercialized. However, compared with hematological malignancies, the therapeutic effect of CAR-T therapy in solid tumors including NSCLC has not progressed significantly, mainly due to the lack of tumor-specific antigens, antigen escape, antigen heterogeneity, immunosuppressive tumor microenvironment, and low levels of CAR-T cell transport and infiltration into tumor tissue, among other well-known factors.

[0004] In addition, due to the need for autologous T lymphocytes, the cost of CAR-T therapy is expensive and the preparation period is long, which cannot meet the urgent medication needs of patients in the acute phase. Therefore, how to overcome these obstacles and optimize and promote CAR-T therapy for solid tumors including NSCLC is an urgent problem to be solved.

[0005] Currently, autologous cell therapy using CAR-αβT, immune checkpoint inhibitors, and chemokine receptors has been preliminarily explored. However, there is no report or patent on allogeneic universal targeting of EGFR-positive tumors based on γδT cells. SUMMARY

[0006] The technical problem to be solved by the present application is to prepare a safe and effective CAR-γδT cell with anti-tumor activity targeting EGFR on the surface of tumor cells, which can avoid graft-versus-host reaction.

[0007] Another technical problem to be solved by the present application provides a preparation method of the above-mentioned CAR-γδT cell.

[0008] The chemokine CCR6 has been proved to promote T lymphocyte infiltration into tumor tissue in previous studies, especially in CCL20 high expression non-small cell lung cancer. At the same time, the PD-1 monoclonal antibody can significantly improve the immune exhaustion of T lymphocytes and increase the anti-tumor activity of T lymphocytes. However, how to integrate these functions without mutual exclusion is an important direction that cell therapy is trying to achieve at present.

[0009] The present application is based on the allogeneic universal seed cell γδT cell, constructs the CAR structure targeting EGFR by lentivirus infection, and can express the chemokine CCR6 which promotes γδT infiltration into tumor tissue at the same time, constructs the EGFR-CCR6-E27 CAR-γδT cell which overcomes the immune cell exhaustion of secreted PD-1 single chain antibody.

[0010] The present application is completed on the basis of the above-mentioned research.

[0011] Specifically, the present application provides a genetically modified enhanced CAR-γδT cell, which expresses a chimeric antigen receptor targeting EGFR on the surface, expresses a chemokine CCR6, and secretes a PD-1 single chain antibody.

[0012] In one aspect, the present application provides a genetically modified γδT lymphocyte, which contains a chimeric antigen receptor expression vector, the chimeric antigen receptor expression vector comprising: a single chain antibody ScFv targeting EGFR, a CD8 hinge region, a CD8 transmembrane region, 4-1BB, CD3ζ chain, a chemokine CCR6.

[0013] Preferably, the chimeric antigen receptor expression vector further contains a secreted PD-1 single chain antibody.

[0014] In a preferred embodiment of the present application, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 1.

[0015] The amino acid sequence of the CD8 hinge region is shown in SEQ ID NO. 2.

[0016] The amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO. 3.

[0017] The amino acid sequence of the 4-1BB is shown in SEQ ID NO. 4.

[0018] The amino acid sequence of the CD3 zeta chain is shown as SEQ ID NO. 5.

[0019] The amino acid sequence of the chemokine CCR6 is shown as SEQ ID NO. 6; or

[0020] The amino acid sequence of the secreted PD-1 single-chain antibody (secreted ScFv targeting PD-1) is shown as SEQ ID NO. 7.

[0021] SEQ ID NO. 1:

[0022] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIGWVRQAPGQGLEWMGGIIPIFGIANYAQKFQGRVTITADESTS

[0023] SAYMELSSLRSEDTAVYYCAREEGPYCSSTSCYAAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQDPAASVA

[0024] LGQTVKITCQGDSLRSYFASWYQQKPGQAPTLVMYARNDRPAGVPDRFSGSKSGTSASLAISGLQPEDEADYYCAAWDDSLNGYLFGAGTKLTVL;

[0025] SEQ ID NO. 2: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

[0026] SEQ ID NO. 3: IYIWAPLAGTCGVLLLSLVITLYC;

[0027] SEQ ID NO. 4: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

[0028] SEQ ID NO. 5:

[0029] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR;

[0030] SEQ ID NO. 6:

[0031] MSGESMNFSDVFDSSEDYFVSVNTSYYSVDSEMLLCSLQEVRQFSRLFVPIAYSLICVFGLLGNILVVITFAFYKKA

[0032] RSMTDVYLLNMAIADILFVLTLPFWAVSHATGAWVFSNATCKLLKGIYAINFNCGMLLLTCISMDRYIAIVQATKSF

[0033] RLRSRTLPRSKIICLVVWGLSVIISSSTFVFNQKYNTQGSDVCEPKYQTVSEPIRWKLLMLGLELLFGFFIPLMFMI

[0034] FCYTFIVKTLVQAQNSKRHKAIRVIIAVVLVFLACQIPHNMVLLVTAANLGKMNRSCQSEKLIGYTKTVTEVLAFLHCCLNPVLYAFIGQKFRNYFLKILKDLWCVRRKYKSSGFSCAGRYSENISRQTSETADNDNASSFTM.

[0035] SEQ ID NO.7:

[0036] MEVQLVESGGGLIQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNYISMFDSWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQRPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDYVFGIGTKVTVLG

[0037] Accordingly, the present application also provides a nucleic acid encoding the chimeric antigen receptor expression vector. In addition, the present application also provides a vector, a plasmid, a cell, an exosome, a tissue, etc. containing the nucleic acid.

[0038] In another aspect, the present application also provides a method for preparing an enhanced allogeneic universal CAR-γδT cell targeting EGFR and expressing CCR6 and secreting a single-chain antibody of PD-1. The method comprises the following steps:

[0039] (1) co-transfect cells with the chimeric antigen receptor expression vector and the lentivirus packaging plasmid to obtain EGFR-CCR6-E27 CAR lentivirus expressing the chimeric antigen receptor expression vector;

[0040] (2) transduce the gamma delta T lymphocytes with the lentivirus obtained in step (1).

[0041] Preferably, step (1) comprises: co-transfecting cells with a three-plasmid packaging system psPAX2, PMD2.G and the chimeric antigen receptor expression vector, collecting the virus-containing supernatant after overnight culture, and centrifuging; and resuspending the virus precipitate with cell culture medium.

[0042] In the present application, the chimeric antigen receptor can be artificially synthesized using a kit according to its amino acid sequence, or the corresponding chimeric antigen receptor expression vector can be used to insert the corresponding coding nucleic acid of the chimeric antigen receptor into an expression vector or a related expression vector system for expression to obtain the chimeric antigen receptor.

[0043] Preferably, the chimeric antigen receptor expression vector EGFR-CCR6-E27 CAR comprises: a single-chain antibody ScFv targeting EGFR, a CD8 hinge region, a CD8 transmembrane region, 4-1BB, a CD3 zeta chain, a chemokine CCR6 and a secreted PD-1 single-chain antibody.

[0044] The sorting method of gamma delta T lymphocytes (or simply gamma delta T cells) comprises: resuspending peripheral blood mononuclear cells in T cell expansion medium, adding zoledronic acid, CD3 / CD28 antibody, rhIL-2, rhIL-15 and serum, culturing for 2-3 days, removing the CD3 / CD28 antibody, removing the zoledronic acid on the 5th-6th day, detecting the gamma delta T lymphocyte markers and obtaining the gamma delta T cells.

[0045] The lentivirus transduction of gamma delta T lymphocytes comprises: co-transfecting cells with a three-plasmid packaging system psPAX2, PMD2.G and the chimeric antigen receptor expression vector. The virus-containing supernatant is collected after overnight culture and centrifuged. The virus precipitate is resuspended with cell culture medium and used to infect gamma delta T cells to obtain EGFR-CAR-E27-CCR6-gamma delta T cells.

[0046] Preferably, step (2) comprises:

[0047] (1) separating PBMCs from peripheral blood by density gradient centrifugation;

[0048] (2) stimulating the obtained PBMCs with zoledronic acid, IL-2 and IL-15, and replacing the complete culture medium every 1-3 days;

[0049] (3) After 5-7 days of culture, the proportion of γδT positive cells is detected;

[0050] (4) The γδT cells are enriched from the PBMCs using a γδT cell separation kit;

[0051] (5) The purity of the γδT cells is detected;

[0052] (6) The EGFR-CCR6-E27 CAR lentivirus prepared in claim 12 is added to the culture environment of the γδT cells (MOI=20);

[0053] (7) After overnight lentivirus infection, the complete culture medium containing IL-2 and IL-15 is replaced.

[0054] In a preferred embodiment of the present application, the specific method is as follows: the EGFR-CCR6-E27 CAR expression vector and the commercial lentivirus packaging plasmid PSPX2 and PMD.2G are transfected into 293T cells, and the cell supernatant is harvested at 48 h and 72 h, respectively, and the lentivirus is obtained from the supernatant by ultracentrifugation; the PBMCs are separated from the peripheral blood by density gradient centrifugation; the obtained PBMCs are stimulated and cultured using zoledronate, IL-2 and IL-15, and the complete culture medium is replaced every 2 days; after 7 days of culture, the proportion of γδT positive cells is detected by cell flow cytometry; the γδT cells are enriched from the PBMCs cultured for 7 days using a commercial γδT cell separation kit; the purity of the γδT cells is detected by cell flow cytometry; the EGFR-CCR6-E27 CAR lentivirus prepared in claim 12 is added to the culture environment of the γδT cells (MOI=20); after 24 hours of lentivirus infection, the complete culture medium containing IL-2 and IL-15 is replaced; after 72 hours of lentivirus infection, the expression of the chimeric antigen receptor and CCR6 is detected by cell flow cytometry. It is detected that the γδT cells amplified in the present application have strong proliferation ability. Figures 1-8

[0055] Meanwhile, the enhanced CAR-γδT cells prepared by the present application are used to evaluate the effect of treating non-small cell lung cancer expressing EGFR, CCL20 and PD-L1 in vivo and in vitro. The specific technical solutions are as follows:

[0056] The present application also includes the evaluation of the in vivo and in vitro effect of the enhanced CAR-γδT cells targeting EGFR and expressing CCR6 and secreting PD-1 single-chain antibody, including the following steps: the prepared enhanced CAR-γδT cells are co-incubated with A549 cells expressing CCL20 and PD-L1-luc at different effector target ratios for 6 h, and then the expression level of luciferase is detected by a microplate reader to determine the killing effect of the CAR-γδT cells at different effector target ratios.

[0057] ​In the application, in the EGFR-CAR expression vector, EGFR-scFv, CD8aH, CD8aTM, 4-1BB, CD3zeta are sequentially connected; in the EGFR-CAR-E27 expression vector, EGFR-scFv, CD8aH, CD8aTM, 4-1BB, CD3zeta, CCR6, E27 are sequentially connected; the structure of the EGFR-CCR6-E27 CAR expression vector is specifically shown in the following figure Figure 9 .

[0058] The prepared enhanced CAR-gammadelta T cells and A549 cells expressing CCL20 and PD-L1-luc are incubated at a ratio of 1:1 for 24h, 48h or 72h, and then the expression level of luciferase is detected by a microplate reader to determine the killing effect of CAR-gammadelta T cells at different incubation times. The prepared enhanced CAR-gammadelta T cells are co-incubated with patient-derived tumor cells expressing CCL20 and PD-L1 at different effector-target ratios for 24h, and then the activity of LDH is detected by a microplate reader using a commercial LDH kit to determine the killing effect of CAR-gammadelta T cells on patient-derived tumor cells at different effector-target ratios. A549 cells expressing CCL20 and PD-L1-luc are transplanted into severely immunodeficient NOD mice, and 3 days after the transplantation of the cells, enhanced CAR-gammadelta T cells are injected, and the injection is performed every 3 days for 3 times. The change of tumor in mice over time is monitored by in vivo imaging of mice. The inhibitory effect of enhanced CAR-gammadelta T cells on tumor cells in vivo is determined. Patient-derived tumors expressing CCL20 and PD-L1-luc are transplanted into severely immunodeficient NOD mice, and after the mice are successfully modeled, enhanced CAR-gammadelta T cells are injected, and the injection is performed every 3 days for 6 times. The change of tumor over time is measured by an external vernier caliper. The detection results prove that the enhanced CAR-gammadelta T cells of the application can inhibit tumor cells in vivo, and the chemotactic factor element CCR6 on the surface of EGFR-CAR-E27-CCR6-gammadelta T cells can be efficiently expressed.

[0059] In a third aspect, the application further provides the use of the genetically modified gammadelta T lymphocyte, i.e., the use of the genetically modified gammadelta T lymphocyte in the preparation of a medicament for alleviating or treating tumors.

[0060] Preferably, the medicament is a live cell medicament for alleviating or treating non-small cell lung cancer cells.

[0061] Preferably, the medicament is a medicament for inhibiting the proliferation, growth, migration, killing of tumor cells or invasion of other tissue cells of tumor cells.

[0062] Preferably, the use comprises the following steps:

[0063] obtaining the γδ T lymphocytes;

[0064] obtaining tumor cells;

[0065] co-culturing the γδ T lymphocytes with the tumor cells. The co-culturing time is usually no less than 4 hours, can be no less than 6 hours, 9 hours, 12 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days or more, for example, 8-72 hours or 12-24 hours, etc.

[0066] In the present application, E:T refers to the ratio of effector cells to target cells. In one embodiment of the present application, it refers to the ratio of effector cells (e.g., γδ T lymphocytes containing EGFR-CAR, EGFR-CAR-E27, EGFR-CAR-E27-CCR6) to target cells (e.g., A549-PDL1-Luc-CCL20 cells). When co-cultured, the ratio of γδ T lymphocytes to tumor cells is (0.5-10): 1, more preferably, γδ T lymphocytes are more than tumor cells, for example, the ratio of the two is (1-5): 1, for example, 2:1, 3:1, 4:1, 6:1, 8:1, etc.

[0067] Preferably, the tumor is derived from EGFR-positive, PD-L1-positive and CCL20-positive tumor cells. For example, non-small cell lung cancer cells, lung adenocarcinoma cells that express EGFR, PD-L1 and CCL20 on the cell surface. In one preferred embodiment of the present application, the tumor cells used are derived from A549 and overexpress PD-L1 and CCL20. In another preferred embodiment of the present application, the tumor cells used are derived from purified lung cancer tumor cells A049 from a non-small cell lung cancer patient, which cells express up to EGFR, PD-L1 and CCL20.

[0068] The genetically modified γδ T lymphocytes of the present application have a significant inhibitory or killing effect on tumor cells, but not all genetically modified γδ T lymphocytes have the same significant effect in vivo. Surprisingly, EGFR-CAR-E27-CCR6-γδ T cells have a significant tumor growth control effect on autologous or allogeneic mouse models CDX (human tumor cell line xenograft, Cell-Derived Xenograft) and PDX (Patient-Derived Xenografts), significantly prolonging the survival of mice. Further studies have shown that the vectors and CAR-γδ T cells of the present application have good specificity and safety.

[0069] In the present application, the CDX model preparation method is as follows: A549-PDL1-Luc-CCL20 target cells are subcutaneously injected into the axillary part of immunodeficient mice, and from the fourth day, MOCK gamma delta T, EGFR-E27-CCR6-CAR-gamma delta T, EGFR-E27-CAR-gamma delta T, EGFR-CAR-gamma delta T four groups of effector cells are injected, once every three days, and tumor growth is detected.

[0070] In the present application, the PDX model preparation method is as follows: the patient-derived tumor tissue block is cut into 2*2*2mm 3 After the average size of the tumor reaches 100mm3, the Vehicle group is given PBS, the Mock-T group is given 10*106 gamma delta T, the CAR-T H group is given 10*10 6 EGFR-CAR-E27-CCR6-gamma delta T cells, and the CAR-T L group is given 3*106 EGFR-CAR-E27-CCR6-gamma delta T, each group is injected with effector cells once a week, a total of 3 times, and the experiment ends 14 days after the injection ends, and the mice are euthanized. Measure the tumor volume and tumor inhibition rate.

[0071] Graft-versus-host disease (GVHD) is due to T lymphocytes in the allogeneic donor graft after transplantation, which greatly enhances the immune response of the recipient to the recipient antigens, and launches a cytotoxic attack on the recipient target cells, among which the skin, liver and intestine are the main target targets.

[0072] The present application successfully constructs allogeneic universal CAR-gamma delta T cells targeting EGFR and expressing CCR6 and secreting PD-1 monoclonal antibody in vitro. In vitro cell models and in vivo CDX and PDX models, it is found that EGFR-CCR6-E27CAR-gamma delta T cells show stronger cell killing ability than gamma delta T, EGFR-CAR-gamma delta T, EGFR-CCR6-CAR-gamma delta T cells, can significantly control the in vivo growth of tumors, and significantly prolong the survival of mice, and can be used as an anti-tumor drug to lay the foundation for long-term control of the development of tumors in patients. BRIEF DESCRIPTION OF DRAWINGS

[0073] In order to more clearly illustrate the technical solutions of the present application, the following will briefly introduce the drawings needed in the embodiments. Obviously, each drawing in the following description is directed to some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0074] Figure 1 Peripheral blood mononuclear cells PBMCs are activated for 3-4 days, and cell clumping can be observed.

[0075] Figure 2 is the proportion of γδT from the beginning of activation to peripheral blood mononuclear cells PBMC.

[0076] Figure 3 is the count of peripheral blood mononuclear cells PBMC and γδT cells from the beginning of activation.

[0077] Figure 4 is the proportion of γδT before and after sorting.

[0078] Figure 5 is the flow identification of γδT cells after sorting using Human PE-conjugated anti-CD45RA antibody, Human FITC-conjugated anti-CD62L antibody staining.

[0079] Figure 6 is the proportion of different memory subtypes of γδT cells after sorting.

[0080] Figure 7 is the proportion of positive cells of γδT cells after sorting.

[0081] Figure 8 is the proportion of positive cells of γδT cells after sorting.

[0082] Figure 9 is the CCR6 expression result of γδT cells after infection with virus. The upper half is the expression efficiency of EGFR-CAR, CCR6 of γδT, EGFR-CAR-γδT, EGFR-CAR-E27-γδT, EGFR-CAR-E27-CCR6-γδT four groups of cells detected by cell flow after 72 hours of lentivirus infection. The lower part is the structural composition diagram of EGFR-CAR-E27-CCR6.

[0083] Figure 10 is the killing effect statistics of effector cells and A549-PD-L1-CCL20-luciferase target cells co-incubation. Among them, the effector cells are EGFR-CAR-αβT and EGFR-CAR-γδT cells, the effector target ratio is 1:1, 1:5, 1:10, and the co-incubation time is 8h.

[0084] Figure 11The killing effect of four groups of effector cells γδT, EGFR-CAR-γδT, EGFR-CAR-E27-γδT, EGFR-CAR-E27-CCR6-γδT on A549-PD-L1-CCL20-luciferase target cells after 8h co-incubation at the effector to target ratio of 0, 0.5:1, 1:1, 1:5.

[0085] Figure 12 1x10 5 The killing effect of four groups of effector cells γδT, EGFR-CAR-γδT, EGFR-CAR-E27-γδT, EGFR-CAR-E27-CCR6-γδT on 1x10 5 The killing effect of different groups of CAR-γδT on A549-PD-L1-CCL20-luciferase cells after 24h, 48h or 72h co-incubation.

[0086] Figure 13 1x10 5 PDX-A049-CCL20-PD-L1 cells derived from non-small cell lung cancer patients were inoculated into 24-well plates, and 0, 1x10 5 , 5x10 5 or 1x10 6 The killing effect of different groups of CAR-γδT on PDX-049-CCL20-PD-L1 cells after 24h co-incubation.

[0087] Figure 14 The flow chart of CDX model animal experiment.

[0088] Figure 15 The live imaging of CDX model animal experiment.

[0089] Figure 16 The luciferase fluorescence value statistics chart of live imaging of CDX model animal experiment.

[0090] Figure 17 The survival curve of CDX model animal experiment.

[0091] Figure 18 The GvHD scoring table of CDX model animal experiment.

[0092] Figure 19 The hematoxylin-eosin (HE) staining paraffin section of heart, liver, spleen, lung and kidney of CDX model animal experiment.

[0093] Figure 20 The flow chart of PDX model animal experiment.

[0094] Figure 21 is a tumor volume statistical graph of PDX model animal experiment.

[0095] Figure 22 is a tumor inhibition rate statistical graph of PDX model animal experiment.

[0096] Figure 23 is a tumor weight statistical graph of PDX model animal experiment.

[0097] Figure 24 is a tumor arrangement photograph of PDX model animal experiment.

[0098] Figure 25 is a GvHD scoring table of PDX model animal experiment.

[0099] Figure 26 is a hematoxylin-eosin (HE) staining paraffin section of heart, liver, spleen, lung and kidney of PDX model animal experiment. DETAILED DESCRIPTION

[0100] The present application belongs to the technical field of tumor cell treatment, and particularly relates to a preparation method of enhanced EGFR-targeting CCR6-expressing PD-1 single-chain antibody-secreting allogeneic universal CAR-gammadelta T cells and application thereof in non-small cell lung cancer. Tests show that the enhanced EGFR-targeting CCR6-expressing PD-1 single-chain antibody-secreting CAR-gammadelta T cells of the present application for resisting non-small cell lung cancer can significantly inhibit the in-vivo and in-vitro growth of non-small cell lung cancer, can be used for non-small cell lung cancer cell immunotherapy, and has a good application prospect.

[0101] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0102] Example 1: In-vitro efficient expansion of gammadelta T cells

[0103] After the fresh blood sample is placed at 4 degrees for 30 min, it is centrifuged at 600 g for 10 min to separate into clear layers. The upper yellow serum is centrifuged at 12000 rpm for 3 min, filtered through a 0.22 um filter membrane, and then 1% penicillin-streptomycin double antibody is added after being repeated three times, and the mixture is stored at 4 degrees. The lower red plasma is mixed with RPMI 1640 medium at a ratio of 1:1, and then the mixture is mixed uniformly by blowing. The diluted blood is added to the mixed solution of the gammadelta T cell culture medium and the feeder cell culture medium at a ratio of 2:1. PaquePLUS TMLympholyte separation, 800g, 20min, 9 for acceleration, 1 for deceleration. After centrifugation, the tube can be observed to have 4 layers, the top layer is yellow and transparent, the second layer is milky white and misty (which is the target cell layer), the third layer is colorless and transparent, and the bottom layer is red. Carefully discard the top layer, and transfer the milky white misty layer to a new 15mL centrifuge tube, about 3mL per tube. Add 9mL RPMI 1640 medium to each 15mL centrifuge tube, 500g, 10min, 8 for acceleration, 3 for deceleration. Then resuspend with RPMI 1640 medium, 300g, 10min centrifugal cleaning twice, count, and the obtained is PBMC (peripheral blood mononuclear cell).

[0104] PBMC was resuspended in Stemcell ImmunoCult 6 -XF T Cell ExpansionMedium medium at a density of 1*10 TM / mL, and supplemented with 1ug / mL zoledronate (MCE), 5ul / mL ImmunoCult TM Human CD3 / CD28 TCell Activator CD3 / CD28 antibody (Stemcell TM ), 10ng / mL of rhIL-2 (coastal protein TM ), 10ng / mL of rhIL-15 (coastal protein TM ), 5% autologous serum. On the 3rd day, remove the CD3 / CD28 antibody, on the 5th-6th day, remove the zoledronate, and replace the complete medium every 2 days at 140g for 10min. From the 3rd-4th day, cell clumping can be observed Figure 1 ), and from the 5th-6th day, cell proliferation can be observed. From the 0th day of expansion, cells were stained every 3 days using flow antibody PE Mouse Anti-Human TCRγδ antibody, and flow cytometry was used to determine the proportion of γδ T cells in all cells. It can be concluded that the proportion of γδ T cells increased from about 2% on the 0th day to 18.4% on the 9th day and 72.4% on the 27th day Figure 2 ). The absolute number of γδ T cells increased 300-fold on the 9th day and 10,000-fold on the 27th day Figure 3 .

[0105] Use Meitianyin TMThe magnetic bead TCRy / d+T cell isolation kit human was sorted on day 7, and the cells were stained with a flow antibody PE Mouse Anti-Human TCRy / d antibody before and after sorting. The proportion of y / d T cells was detected by flow cytometry, and the proportion of y / d T cells after sorting was more than 90% ( Figure 4 ). In order to determine the proportion of different memory subtypes, the sorted y / d T cells were stained with a human PE-conjugated anti-CD45RA antibody and a human FITC-conjugated anti-CD62L antibody for flow identification ( Figure 5 , 6), and it was found that 64.5% were naive T cells Tn (CD45RA + CD62L + ), 26.7% were effector T cells Teff (CD45RA + CD62L -) , 6.4% were effector memory T cells Tem (CD45RA - CD62L + ), and 1.4% were central memory T cells Tcm (CD45RA - CD62L -) . More than 60% were naive T cells, which proved that the y / d T cells amplified by the application had strong proliferation capacity. Anti-CD279, anti-LAG3 and anti-Tim3 antibodies were used for staining and flow identification to determine the proportion of positive cells of different T cell exhaustion surface markers ( Figure 7 , Figure 8 ). The positive rates of CD279, LAG3 and Tim3 were 3.6%, 6.8% and 12.4% respectively, which proved that the surface expression of the sorted y / d T cells was low.

[0106] Example 2 Preparation of EGFR-CAR-y / d T cells, EGFR-CAR-E27-y / d T cells and EGFR-CAR-E27-CCR6-y / d T cells

[0107] To obtain lentivirus particles expressing EGFR-CAR, EGFR-CAR-E27, EGFR-CAR-E27-CCR6, HEK293T / 17 cells were co-transfected with three plasmid packaging system psPAX2, PMD2.G and three CAR vector plasmids EGFR-CAR, EGFR-CAR-E27, EGFR-CAR-E27-CCR6 at a mass ratio of pMD2.G:psPAX2:CAR plasmid = 3.4:6.8:10 ug per 10 cm dish in the presence of transfection reagent polyethyleneimine (PEI). The culture medium of transfected cells was DMEM + 5% FBS, and after 8-12 h of transfection, the supernatant was discarded, and the new culture medium was replaced and 0.625 uM of small molecule drug M344 was added to improve lentivirus yield. After 48 h, 72 h of culture, the virus-containing supernatant was collected, and the Beckman Coulter TM After 25000 rpm centrifugation for 2 h on the XPN-100 ultracentrifuge SW28 rotor, the supernatant was aspirated and discarded. The lentivirus pellet was resuspended using blank RPMI 1640 medium overnight in a 4-degree refrigerator and used the next day, or resuspended using lentivirus frozen liquid overnight and stored in a -80 refrigerator.

[0108] 293FT-17 cells were infected at 8 ug / mL polystyrene by gradient dilution of the virus solution, and lentivirus titer was calculated by flow cytometry. When the 293FT-17 infection efficiency is less than 20%, it is considered that each positive cell is obtained by infection of one virus particle. At this time, the formula for calculating the functional activity titer of the virus is as follows:

[0109] Virus activity titer TU / mL -1 = (percentage of positive cells x number of inoculated cells x 1000) / volume of virus concentrate.

[0110] γδΤ cells were infected with lentivirus particles containing EGFR-CAR, EGFR-CAR-E27, EGFR-CAR-E27-CCR6 at MOI = 20 (multiplicity of infection), and after 24 hours, the Stemcell ImmunoCult TM -XFT Cell Expansion Medium complete medium. After 72 hours of lentivirus infection, the expression of EGFR-CAR, CCR6 was detected by cell flow cytometry, and the EGFR-CAR positive rates of the four groups of γδΤ, EGFR-CAR-γδΤ, EGFR-CAR-E27-γδΤ, EGFR-CAR-E27-CCR6-γδΤ cells were 3.32%, 65.3%, 63.1%, and 52.1%, respectively, and the positive rates of the chemokine CCR6 element were 3.02%, 3.35%, 4.85%, and 48.7%, respectively.Figure 9 ) were proved. The successful preparation of EGFR-CAR-γδΤ, EGFR-CAR-E27-γδΤ, EGFR-CAR-E27-CCR6-γδΤ cells were proved with high lentivirus transfection efficiency, and the high expression of chemokine element CCR6 on the surface of EGFR-CAR-E27-CCR6-γδΤ cells was also proved.

[0111] Example 3 EGFR-CAR-γδΤ cells have better killing effect than EGFR-CAR-αβΤ

[0112] Both γδΤ and αβΤ cells are used to kill tumor cells. In order to compare the killing effect of EGFR-CAR-γδΤ and common EGFR-CAR-αβΤ on target cells, the inventors packaged lentivirus in the manner of Example 2, and infected expanded γδΤ or αβΤ cells respectively. 1x10 5 A549-PD-L1-CCL20-luciferase target cells (the main element sequence of PD-L1-CCL20-luciferase is EF-1a-CD274 (PD-L1)-T2A-CCL20-P2A-luciferase) were inoculated into 24-well plates at 1x10 Figure 10 ) were found (EGFR-CAR-αβΤ (E:T = 1, 5 or 10: 6.7%, 21.4%, 44.8%), EGFR-γδΤ (E:T = 1, 5 or 10: 8.8%, 35.2%, 75.1%)). The results showed that the killing ability of EGFR-CAR-γδΤ cells was significantly higher than that of EGFR-CAR-αβΤ cells

[0113] Example 4 In vitro CDX tumor cell killing detection under different effector-target ratios

[0114] 1x10 5A549-PD-L1-CCL20-luciferase target cells were inoculated into 24-well plates, and 24 h later, four groups of effector cells, γδΤ, EGFR-CAR-γδΤ, EGFR-CAR-E27-γδΤ, and EGFR-CAR-E27-CCR6-γδΤ, were co-incubated with A549-PD-L1-CCL20-luciferase target cells at an effector-to-target ratio of 0, 0.5:1, 1:1, and 1:5 for 8 h, after which the expression level of luciferase was detected by a microplate reader, and the killing effect of different groups of CAR-γδΤ at different effector-to-target ratios was calculated. It was found that the cytotoxicity of effector cells to target cells increased with the increase of effector-to-target ratio, and under the same effector-to-target ratio, the killing ability of EGFR-CAR-γδΤ (E:T = 0.5, 1, or 5: 3.5%, 10.4%, and 47.5%), EGFR-CAR-E27-γδΤ (E:T = 0.5, 1, 5: 4.0%, 11.7%, and 55.4%), and EGFR-CAR-E27-CCR6-γδΤ (E:T = 0.5, 1, 5: 3.8%, 12.5%, and 65.4%) cells was significantly higher than that of the control group γδΤ cells (E:T = 0.5, 1, 5: 1.9%, 3.7%, and 7.6%) Figure 11 ).

[0115] Example 5 In Vitro CDX Tumor Cell Killing Detection at Different Incubation Times

[0116] 1x10 5 γδΤ, EGFR-CAR-γδΤ, EGFR-CAR-E27-γδΤ, and EGFR-CAR-E27-CCR6-γδΤ were co-incubated with 1x10 5 A549-PD-L1-CCL20-luciferase cells for 24 h, 48 h, or 72 h, after which the expression level of luciferase was detected by a microplate reader, and the killing effect of different groups of CAR-γδΤ at different incubation times was calculated. It was found that the cytotoxicity of effector cells to target cells increased with the increase of incubation time, and under the same incubation time, the killing ability of EGFR-CAR-γδΤ (24 h, 48 h, and 72 h were 19.6%, 38.4%, and 50.0%, respectively), EGFR-CAR-E27-γδΤ (24 h, 48 h, and 72 h were 26.8%, 47.8%, and 69.2%, respectively), and EGFR-CAR-E27-CCR6-γδΤ (24 h, 48 h, and 72 h were 26.3%, 47.8%, and 75.3%, respectively) cells was significantly higher than that of the control group γδΤ cells (24 h, 48 h, and 72 h were 8.2%, 20.3%, and 31.0%, respectively) Figure 12 ).

[0117] Example 6 Killing effect of different groups of CAR-γδT cells with different effector to target ratios on PDX-A049-CCL20-PD-L1 cells derived from tumor patient

[0118] 1 x 10 5 PDX-A049-CCL20-PD-L1 cells derived from non-small cell lung cancer patient were inoculated into 24-well plates, and 24 h later, 0, 1 x 10 5 , 5 x 10 5 or 1 x 10 6 number of different groups of CAR-γδT cells were co-incubated with PDX-A049-CCL20-PD-L1 cells for 24 h. Then the killing effect of different groups of CAR-γδT cells on PDX-A049-CCL20-PD-L1 cells was calculated by detecting the level of lactate dehydrogenase (LDH) in different groups. It was found that the toxicity of effector cells to target cells increased with the increase of effector to target ratio, and under the same effector to target ratio, the killing ability of EGFR-CAR-γδT (E:T = 1, 5, 10, respectively 8.6%, 30.1%, 52.8%), EGFR-CAR-E27-γδT (E:T = 1, 5, 10, respectively 48.0%, 10.4%, 70.2%), EGFR-CAR-E27-CCR6-γδT (E:T = 1, 5, 10, respectively 16.0%, 51.1%, 72.9%) cells was significantly higher than that of control γδT cells (E:T = 1, 5, 10, respectively 4.6%, 12.4%, 16.7%) Figure 13 ).

[0119] Example 7 CDX model animal experiment

[0120] In order to determine the inhibitory effect of enhanced CAR-γδT cells on tumor cells in vivo in a xenotransplantation model (CDX) established based on cell lines, the inventors performed an animal experiment according to the flowchart shown in Figure 14 5-week-old immunodeficient male mice B-NDG were purchased from Bao Saigu TM The A549-PDL1-Luc-CCL20 target cells were collected by centrifugation, resuspended in PBS, and washed with PBS for 3 times. On day 0, 1 x 10 6The cells (100 μL) were subcutaneously injected into the armpit of the mice. From the third day, live imaging was performed every 6 days. According to the fluorescence intensity on the third day, the mice were randomly divided into four groups, MOCK γδT, EGFR-E27-CCR6-CAR-γδT, EGFR-E27-CAR-γδT, and EGFR-CAR-γδT, according to the fluorescence intensity gradient. From the fourth day, the effector cells of the four groups, MOCK γδT, EGFR-E27-CCR6-CAR-γδT, EGFR-E27-CAR-γδT, and EGFR-CAR-γδT, were injected, 5 x 10 6 The effector cells were resuspended in ImmunoCult TM -XF T Cell Expansion Medium, and the MOCK group was injected with 100 uL of blank medium. Through in vivo injection of 150 mg / kg of luciferase substrate for live imaging, it was found that the enhanced EGFR-E27-CCR6-CAR-γδT significantly inhibited the growth of the CDX model compared with the EGFR-E27-CAR-γδT, EGFR-CAR-γδT, and MOCK γδT groups (P < 0.05) (FIG. 6A), and the tumor inhibition rates were 17.2%, 44.5%, and 71.2% at 45 days after tumor inoculation (FIG. 6B). Figure 15 , Figure 16 Through the preparation of a survival curve, it was found that the enhanced EGFR-E27-CCR6-CAR-γδT significantly prolonged the survival of tumor-bearing mice compared with the other experimental groups, and the survival rate was 60% at 99 days after tumor inoculation (FIG. 6C). Figure 17

[0121] To evaluate whether the injection of effector cells caused graft-versus-host disease (GvHD), the potential of the enhanced CAR-γδT as an allogeneic and universal CAR-T therapy was evaluated, and the GvHD score was evaluated based on five indicators, including weight loss, posture, activity, skin condition, and skin exposure. The score of each indicator was 0-2, and the total score was the sum of the five indicators. The higher the score, the more severe the GvHD at the scoring time point. It was found that the mice injected with the effector cells of the four groups, MOCK γδT, EGFR-E27-CCR6-CAR-γδT, EGFR-E27-CAR-γδT, and EGFR-CAR-γδT, did not have GvHD, which proved the good potential of the enhanced CAR-γδT as an allogeneic and universal CAR-T therapy (FIG. 6D). Figure 18

[0122] ​​The paraffin sections stained by hematoxylin-eosin (HE) were observed to find that there was no obvious pathological damage in the heart, liver, lung and kidney of the mice in the MOCK γδT, EGFR-E27-CCR6-CAR-γδT, EGFR-E27-CAR-γδT and EGFR-CAR-γδT groups, which proved the good safety of the enhanced EGFR-E27-CCR6-CAR-γδT cell therapy Figure 19 ).

[0123] Example 8 PDX model animal experiment

[0124] In order to determine the inhibitory effect of the enhanced CAR-γδT cells on tumor cells in vivo in a xenotransplantation model (PDX) established based on patient-derived tumor cells, the inventors performed an animal experiment according to the flowchart shown in Figure 20 The patient-derived tumor tissue block obtained with informed consent was cut into 2*2*2mm 3 subcutaneously inoculated into NDG mice, and after 5 days, the average size of the tumors reached 100mm 3 After that, the mice were randomly divided into Vehicle, Mock-T, CAR-T H and CAR-T L groups, the Vehicle group was given PBS, the Mock-T group was given 10*10 6 γδT, the CAR-T H group was given 10*10 6 EGFR-CAR-E27-CCR6-γδT cells, and the CAR-T L group was given 3*10 6 EGFR-CAR-E27-CCR6-γδT cells, each group was injected with effector cells once a week, a total of 3 times, and the experiment ended 14 days after the injection was completed, and the mice were euthanized. After the start of injection of the effector, the tumor volume was measured every 2-3 days with a vernier caliper Figure 21 ). According to the absolute tumor volume of the Mock-T, CAR-T H and CAR-T L groups relative to the Vehicle group and the tumor weight after euthanasia, the tumor inhibition rates of each group were 12.%, 11.7% and 33.6% respectively Figure 22 ). At the same time, the body weight changes of the mice were recorded. After euthanasia, the tumors were removed, weighed and counted Figure 23 ) and photographed according to the grouping Figure 24 ).

[0125] In order to evaluate whether injection of effector cells causes graft-versus-host disease (GvHD), the enhanced CAR-gammadelta T cells are evaluated as allogeneic, universal CAR-T therapy potential, and GvHD scoring is performed by five indicators of weight loss amplitude, posture, activity, skin condition and skin exposure. The single indicator scoring is 0-2 points, and the five indicators are added to the total score. The higher the score, the more serious the GvHD at the scoring time point. The results show that the Vehicle, Mock-T, CAR-T H and CAR-T L four groups of mice in the application do not have GvHD phenomenon, which proves that the enhanced CAR-gammadelta T cells as allogeneic have good potential in universal CAR-T therapy Figure 25 )。

[0126] It is found by observing paraffin sections stained by hematoxylin-eosin (HE) that the Vehicle, Mock-T, CAR-T H and CAR-T L four groups of mice have no obvious pathological damage in heart, liver, lung and kidney, which proves that the enhanced EGFR-E27-CCR6-CAR-gammadelta T cell therapy has good safety Figure 26 )。

[0127] The above-described embodiments are merely specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can conceive of changes or replacements within the technical scope disclosed in the present application without creative labor, and such changes or replacements should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims in the present application.

Claims

1. A genetically modified γδT lymphocyte, characterized in that, The γδT lymphocytes contain or express chimeric antigen receptors, which include: a single-chain antibody ScFv targeting EGFR, the CD8 hinge region, the CD8 transmembrane region, 4-1BB, and the CD3ζ chain.

2. The γδT lymphocytes according to claim 1, characterized in that, The chimeric antigen receptor also contains a secretory PD-1 single-chain antibody.

3. The method for preparing γδT lymphocytes according to claim 1, characterized in that, The preparation method includes: (1) The chimeric antigen receptor expression vector and the lentiviral packaging plasmid were co-transfected into cells to obtain a lentivirus expressing the chimeric antigen receptor. (2) Use the lentivirus obtained in step (1) to transduce γδT cells.

4. The preparation method according to claim 3, characterized in that, Step (1) includes: co-transfecting cells with the three plasmid packaging system psPAX2, PMD2.G and the chimeric antigen receptor expression vector, culturing overnight, collecting the virus-containing supernatant, and centrifuging; resuspending the virus pellet in cell culture medium. Step (2) includes: (1) PBMCs were separated from peripheral blood by density gradient centrifugation; (2) The obtained PBMCs were cultured with zoledronic acid, IL-2 and IL-15 stimulation, and the complete culture medium was changed every 1-3 days. (3) Incubate for 5-7 days; (4) Using a γδT lymphocyte isolation kit, γδT cells were enriched from PBMCs; (5) Detection of γδT lymphocyte purity; (6) Add the EGFR-CCR6-E27 CAR lentivirus (MOI=20) as described in claim 3 to the culture environment of γδT lymphocytes; (7) Replace the lentivirus infection with complete culture medium containing IL-2 and IL-15 after overnight infection.

5. A chimeric antigen receptor, characterized in that, This chimeric antigen receptor includes: a single-chain antibody ScFv targeting EGFR, the CD8 hinge region, the CD8 transmembrane region, 4-1BB, the CD3ζ chain, the chemokine CCR6, and a secretory ScFv targeting PD-1; The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.1; The amino acid sequence of the CD8 hinge region is shown in SEQ ID NO.2; The amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO.3; The amino acid sequence of the 4-1BB is shown in SEQ ID NO.4; The amino acid sequence of the CD3ζ chain is shown in SEQ ID NO.5; The amino acid sequence of the chemokine CCR6 is shown in SEQ ID NO. 6; or The amino acid sequence of the secretory PD-1-targeting ScFv is shown in SEQ ID NO.

7.

6. The encoding nucleic acid of the chimeric antigen receptor as described in claim 5.

7. The application of the gene-modified γδT lymphocytes according to claim 1, characterized in that, The application of the aforementioned gene-modified γδT lymphocytes in the preparation of drugs to alleviate or treat tumors.

8. The application according to claim 7, characterized in that, The drug is a live cell drug that alleviates or treats non-small cell lung cancer cells; the drug is a drug that inhibits the proliferation, growth, migration, killing of tumor cells, or invasion of other tissue cells.

9. The application according to claim 7, characterized in that, Includes the following steps: Obtain the γδT lymphocytes as described in claim 1; Obtain tumor cells; The γδT lymphocytes described in claim 1 are co-incubated with tumor cells for a period of not less than 6 hours, wherein the ratio of the number of γδT lymphocytes to tumor cells is (1-10):

1.

10. The application according to claim 7, characterized in that, The tumor is derived from tumor cells expressing PD-1 or EGFR; or the drug is an anti-tumor drug targeting the autologous or allogeneic organism.

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