CN116284409A

CN116284409A - GPC3CAR-T cells and their use in the preparation of a medicament for the treatment of cancer

Info

Publication number: CN116284409A
Application number: CN202211461184.2A
Authority: CN
Inventors: 刘晓军; 赵阳兵
Original assignee: Shanghai Youtijisheng Biomedical Co ltd
Current assignee: Shanghai Youtijisheng Biomedical Co ltd
Priority date: 2022-07-08
Filing date: 2022-11-21
Publication date: 2023-06-23

Abstract

The invention discloses GPC3CAR-T cells and application thereof in preparation of medicines for treating cancers. Also disclosed is a GPC 3-targeting CAR molecule, a CAR molecule and switch receptor combination, and immune response cells containing the same. The CAR molecule comprises an extracellular domain that specifically binds GPC3, a CD8 hinge region, a CD8 transmembrane region, a costimulatory signal region, and a CD3 zeta signaling domain, the extracellular domain that specifically binds GPC3 comprising the amino acid sequence shown as SEQ ID No. 2 or SEQ ID No. 4. The CAR molecule can kill tumors in vitro with high efficiency, secrete a large amount of cytokines and promote the rapid proliferation of CAR-T cells, and has higher safety. The CAR molecule binds to the conversion receptor, so that the proliferation capacity of the CAR-T cells can be further enhanced, the killing function of the CAR-T cells is obviously improved, and the tumor is rapidly inhibited.

Description

GPC3CAR-T cells and their use in the preparation of a medicament for the treatment of cancer

Technical Field

The invention belongs to the field of cell therapy, and particularly relates to GPC3CAR-T cells and application thereof in preparation of medicines for treating cancers.

Background

T cell immunotherapy is a therapeutic approach to adoptive transfer of naturally occurring or genetically engineered autologous or allogeneic T cells to cancer patients to initiate tumor regression. Unlike monoclonal antibody drugs that bind to specific targets, adoptive T cell immunotherapy uses patient tumor infiltrating lymphocytes or peripheral blood lymphocytes as drugs, which are screened, expanded and genetically modified in vitro and then infused back to the patient. The reinfusion T cells can recognize and lyse cancer cells in vivo and continue to expand, thereby achieving the purposes of stimulating the patient's autoimmune system and treating cancer.

Israel immunologists Eshhar and his team synthesized the single chain variable region of the monoclonal antibody with the cd3ζ chain into a chimeric receptor (chimeric antigen receptor, CAR) that was transferred into T cells that recognized cell membrane surface tumor antigens in a non-MHC-restricted manner and killed cancer cells. The structure of CARs has been continually improved over thirty years, from a first generation of CARs that do not initially contain a co-stimulatory molecule, to a second generation of CARs that incorporate a co-stimulatory molecule, to a third generation of CARs that co-express a cytokine or a converter molecule, and to a fourth generation of CARs that co-express a cytokine or converter molecule. The complete cure rate of the CD19 CAR-T in treating refractory recurrent acute lymphoblastic leukemia reaches 90%. Given the remarkable success of CAR-T in clinical trials of hematological tumors, more research has turned to solid tumor treatment.

Hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of the most common malignant tumors, and the annual cases of new and dead liver cancer in our country are more than 50% of the world. Glypican-3 (GPC 3) is a proto-oncoglycoprotein GPC3 that is highly expressed in embryonic liver and plays an important role in embryogenesis and growth. In view of the high expression of GPC3 in liver cancer cells and the correlation with the occurrence and development of liver cancer and poor prognosis, GPC3 is getting more and more attention as a potential therapeutic target for liver cancer.

GPC3 is highly expressed in liver cancer cells, but is not expressed in normal liver, but the low expression of GPC3 is detected in important tissues and organs such as lung, kidney and heart. The off-Target (TAA) toxic side effects faced by CAR-T cell therapy of solid tumors are typically due to low expression of tumor-associated antigens (tumor associate antigen, TAA) in normal tissues. Antibodies with moderate affinity should be carefully selected for tumor-associated antigens, improving the ability of CAR-T cells to differentially recognize tumor cells and normal tissues, and for TAA-targeted CAR-T, safety screening prior to clinical trials, to prevent off-target toxicity.

CAR-T cell immunotherapy has achieved remarkable results in hematological tumors, but has encountered many challenges in the treatment of solid tumors, one of the important reasons being the tumor's inhibitory microenvironment. Tumor cells utilize the regulatory mechanism of the immune system, and inhibit the function of immune cells by up-regulating and expressing immunosuppressive molecules such as PD-1 (Programmed cell death protein 1), CTLA-4 (cytotoxin T-lymphocyte-associated protein 4), tim3 (T-cell immunoglobulin mucin-3), tigit (T cell immunoglobulin and ITIM domain) and the like or secreting immune regulatory molecules such as IDO (indoleamine 2, 3-dioxygenase), adenosine, TGF-beta (Transforming growth factor-beta) and the like, so that immune monitoring is avoided. In addition, tumor-associated hypoxic microenvironments and suppressors, comprising: treg (T regulatory cells), TAMS (Tumor associated macrophage), MDSC (Myeloid-derived suppressor cells), CAF (Cancer Associated Fibroblasts), etc., all induce the disability and failure of CAR-T cells.

Disclosure of Invention

In order to solve the problem of lack of high-efficiency targeted GPC3 CAR-T cells in the prior art, the invention provides GPC3 CAR-T cells and application thereof in preparation of medicines for treating cancers. This study designed and constructed 5 GPC3 CARs based on the DNA sequences of 5 GPC3 antibodies: verx.bbz, verh.bbz, ver.bbz, gc33.bbz and m11f1.Bbz. Antibodies used to construct these 5 CARs differ in affinity for GPC3, but all target 10 amino acids total from 544 th to 553 th of the C-terminal end of GPC3 (PKDNEISTFH). The 5 intracellular segments of GPC3CAR are composed of 4-1BB and CD3 zeta chain intracellular segments, belonging to the second generation of CAR. GPC3 CAR-T cells by co-culture with GPC3 positive tumor cells, the function of GPC3 CAR-T cells to kill tumors and secrete cytokines was examined. Meanwhile, 5 GPC3 CARs are screened, and CARs with moderate affinities are selected, so that the CAR-T cells can identify tumor tissues and normal tissues differently, and the curative effect and safety of the CAR-T cells are improved.

GPC3 is GPI anchor protein, furin protease can make GPC3 core protein at Arg ³⁵⁸ And Ser ³⁵⁹ Cut into an N-terminal subunit of 40KD and a C-terminal subunit of 30 KD. Yoshitaka et al confirmed that the majority of free GPC3 (sgsc 3) detected in serum was from the N-terminal subunit by immunoprecipitation combined with amino acid sequencing. The GPC3CAR designed in the research targets an epitope of 10 amino acids in total of 544-553 of the C-terminal subunit, and the loss of an antigen target caused by the formation of sGPC3 is avoided to a certain extent.

Thus, in a first aspect the invention provides a CAR molecule targeting GPC3, wherein the CAR molecule comprises an extracellular domain, a transmembrane region and an intracellular region which specifically binds GPC3, the extracellular domain which specifically binds GPC3 comprising an amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 4.

In some specific embodiments, the transmembrane region is a CD8 transmembrane region, the intracellular region comprises a 4-1BB costimulatory signal region and a CD3 zeta signaling domain, the CD8 transmembrane region and the extracellular domain that specifically binds GPC3 are linked by a CD8 hinge region.

In some preferred embodiments, the amino acid sequences of the CD8 hinge region, CD8 transmembrane region, costimulatory signal region, and CD3 zeta signaling domain are shown as SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively.

In some more preferred embodiments, the amino acid sequence of the CAR molecule is as shown in SEQ ID NO. 15 or SEQ ID NO. 16.

In some further preferred embodiments, the nucleotide sequence encoding the CAR molecule is set forth in SEQ ID NO. 1 or SEQ ID NO. 3.

The present study utilizes the opposite mechanism of action of TGF-beta and IL-12 in immune regulation, and the structural features that TGF-beta receptor (TGFbR 1/TGFbR 2) and IL-12 receptor (IL 12Rb1/IL12Rb 2) are both heterotetramers, two transforming receptors TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2 (collectively referred to as TGFbR/IL12R transforming receptors) for transforming TGF-beta signals into IL-12 signals are constructed. Further, the function of T cells expressing the conversion receptor was examined, and the anti-tumor function of GPC3 CAR-T cells coexpressing the TGFbR/IL12R conversion receptor and in vitro and HepG2 liver cancer NSG mouse models was examined.

The present study also compared the newly constructed TGFbR/IL12Rb switch receptor to PD1-CD28 switch receptor, dnTGFbR2, tested CAR-T cells in vitro and CAR-T cells co-expressing the switch receptor for tumor killing and cytokine secretion functions, and analyzed differences in phenotype, cytokine and chemokine secretion, expression profile of genes. Meanwhile, in a HepG2 liver cancer NSG mouse model, the inhibition effect of GPC3 CAR-T cells and GPC3 CAR-T cells (PD 1-CD28+GPC3 CAR-T, TGFbR/IL12R+GPC3 CAR-T and dnTGFbR2+GPC3 CAR-T) of a co-expression conversion receptor on tumor growth is compared.

Thus, in a second aspect the invention provides a CAR molecule and a switch receptor combination, wherein the CAR molecule is a GPC 3-targeting CAR molecule according to the first aspect of the invention, the switch receptor comprising a first polypeptide associated with a negative signal and a second polypeptide associated with a positive signal; the first polypeptide is selected from CTLA4, PD-1, BTLA, TIM-3, and TGF beta R, and the second polypeptide is selected from CD28, ICOS, 4-1BB, and IL-12R.

In some specific embodiments, the amino acid sequence of the switch receptor is as shown in or has at least 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 18, SEQ ID NO. 20 or SEQ ID NO. 22;

In some preferred embodiments, the amino acid sequence of the switch receptor is as shown in SEQ ID NO. 18 or SEQ ID NO. 20;

in some more preferred embodiments, the nucleotide sequence encoding the switch receptor is set forth in SEQ ID NO. 17 or SEQ ID NO. 19.

A third aspect of the invention provides a nucleic acid construct or combination thereof, wherein it encodes a GPC 3-targeting CAR molecule according to the first aspect of the invention; or encodes a CAR molecule and a switch receptor combination according to the second aspect of the invention.

In some specific embodiments, the nucleic acid construct comprises a nucleotide sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 3.

In some preferred embodiments, the nucleotide sequence encoding the switch receptor is set forth in SEQ ID NO. 17, SEQ ID NO. 19 or SEQ ID NO. 21.

In some preferred embodiments, the nucleotide sequence encoding the GPC 3-targeting CAR molecule and the nucleotide sequence encoding the switch receptor are located in the same nucleic acid construct or separate nucleic acid constructs.

In a fourth aspect the present invention provides a recombinant expression vector comprising a nucleic acid construct according to the third aspect of the present invention or a combination thereof;

In some preferred embodiments, the starting vector of the recombinant expression vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, and viral vectors of adeno-associated viral vectors; preferably a retroviral vector and/or a lentiviral vector.

In a fifth aspect the invention provides an immune response cell, wherein the immune response cell comprises a GPC 3-targeting CAR molecule according to the first aspect of the invention.

In some embodiments, the immunoresponsive cell further comprises a switch receptor having an amino acid sequence as set forth in SEQ ID NO. 18, SEQ ID NO. 20, or SEQ ID NO. 22.

In some specific embodiments, the immune response cell is a T cell, a natural killer cell, a hematopoietic stem cell, or a hematopoietic progenitor cell.

In some preferred embodiments, the immune response cell is a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, or a regulatory T cell.

In some more preferred embodiments, the immune response cells are from a human.

A sixth aspect of the invention provides a kit comprising one or more of a GPC 3-targeting CAR molecule according to the first aspect of the invention, a CAR molecule and switch receptor combination according to the second aspect of the invention, a nucleic acid construct according to the third aspect of the invention or a combination thereof, a recombinant expression vector according to the fourth aspect of the invention and an immune responsive cell according to the fifth aspect of the invention.

The seventh aspect of the invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises an immunoresponsive cell according to the fifth aspect of the invention; and a pharmaceutically acceptable carrier or excipient.

An eighth aspect of the invention provides a kit of parts comprising a kit a and a kit B, wherein the kit a comprises an immunoresponsive cell comprising a CAR molecule targeting GPC3 according to the first aspect of the invention and the kit B comprises an immunoresponsive cell comprising a switch receptor as defined by the CAR molecule and switch receptor combination according to the second aspect of the invention.

A ninth aspect of the invention provides the use of a CAR molecule targeting GPC3 according to the first aspect of the invention, a CAR molecule and switch receptor combination according to the second aspect of the invention, a nucleic acid construct according to the third aspect of the invention or a combination thereof, a recombinant expression vector according to the fourth aspect of the invention and an immune response cell according to the fifth aspect of the invention in the manufacture of a medicament for a tumour.

In some preferred embodiments, the tumor is selected from liver cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, leukemia and lymphoma, preferably liver cancer.

In a tenth aspect the invention provides a method of treating a tumor in an individual in need thereof, wherein a therapeutically effective amount of an immune responsive cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention, is administered to the individual.

In some preferred embodiments, further comprising administering to the subject a lymphocyte clearance chemotherapy, e.g., administering to the subject a therapeutically effective amount of cyclophosphamide and/or fludarabine;

The invention also provides the use of an immunoresponsive cell according to the fifth aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention for the manufacture of a medicament for the treatment of a tumour.

Preferably, the medicament further comprises a lymphocyte scavenger chemotherapeutic agent, such as cyclophosphamide and/or fludarabine.

More preferably, the tumor is selected from liver cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, leukemia and lymphoma, preferably liver cancer.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

1. the CAR molecule can kill tumors in vitro efficiently, secrete a large amount of cytokines and promote the rapid proliferation of CAR-T cells, effectively inhibit the growth of the tumors, and has higher safety.

2. The CAR molecule is combined with a conversion receptor, so that the proliferation capacity of CAR-T cells can be further enhanced, the killing function of the CAR-T cells is obviously improved, and tumors are rapidly inhibited. For example, TGFbR/IL12R conversion receptor can convert the inhibition signal of TGF-beta into the stimulation signal of IL-12, and can obviously increase the secretion of cytokines such as IFN-gamma; the PD1-CD28 conversion receptor can convert the inhibition signal of PD1/PD-L1 into the activation signal of CD28, so as to protect CAR-T cells from being induced to be disabled or failure; thereby enhancing the anti-tumor function.

Drawings

FIG. 1 constructs GPC3 CAR expression vectors to detect expression of CAR on the surface of T cells. (a) GPC3 CAR RNA expression vector. (B) GPC3 CAR lentiviral expression vector. (C) GPC3 CAR in vitro transcribed mRNA electrotransformation T cells, flow cytometry detection electrotransformation of the next day T cell surface CAR expression, not electrotransformed T cells as negative control. (D) Lentiviral transduced cells encoding GPC3 CAR, flow cytometry detected T cell surface CAR expression tenth day after transduction, non-transduced T cells as negative controls.

FIG. 2 shows the in vitro tumor killing function of GPC3 CARs. (A) T cells electroporated with different GPC3 CARs were incubated with GPC 3-positive tumors HepG2-CBG and GPC 3-negative tumors a549-ESO for 4 hours at a ratio of E: t=1:2, and flow cytometry examined the expression of CD 3-positive cells CD107a with NO electroporated T cells (NO EP) as negative control (left panel). Tumor cells HepG2 and A549-ESO GPC3 antigen staining (right panel). (B) ELISA detects cytokines in the supernatant of the co-culture of T cells and tumor cells. After overnight incubation (3 replicates per group) of T cells electrotransformed with different GPC3 CARs with GPC 3-positive tumors HepG2-CBG and GPC 3-negative tumors a549-ESO at a ratio E: t=1:1, ELISA examined the levels of cytokines IL-2 (upper panel) and IFN- γ (lower panel) in the supernatants. (C) The killing test of targeting HepG2 tumor cells based on luciferase detects the function of GPC3 CARs to kill tumors. T cells (NOEP as negative control) electroporated with 5 GPC3 CARs respectively were incubated overnight at different E: T ratios with HepG2 tumor cells, and the percentage of CAR-T cells killing tumor cells at the different E: T ratios was calculated based on fluorescence detected by the plate reader. (D) Electrotransport VerH.BBZ and VerX.BBZ CAR-T cells were cultured with hepatoma cells as shown (A549-ESO is GPC3 negative control) at a ratio of E: T=1:2 for 4 hours, respectively, and the flow cytometer examined the expression of T cell CD137, with non-electrotransport T cells (NO EP) as negative control for T cells (left panel). Liver cancer cells and A549-ESO GPC3 antigen staining (right panel).

FIG. 3 shows the measurement of GPC3 expression in 14 normal tissues derived from lung, heart, nerve, kidney and skin. Agarose gel electrophoresis showed GPC3 RT-PCR results. HepG2 is a GPC3 positive control, and A549 is a GPC3 negative control. The GAPDH gene is an internal control gene. (B) GPC3 quantitative PCR results. The rectangular plot shows the fold change in GPC3 expression of primary cells from 14 normal tissues analyzed for double delta Ct. A549 cells were negative controls and GAPDH was an internal control gene.

FIG. 4 shows the measurement of the expression of GPC3 in A549 cells electroporated GPC3 mRNA. (A) Flow cytometry detects the expression of a549 cell surface GPC3 antigen electrotransformed with different doses of GPC3 mRNA. The agarose gel electrophoresis of (B) showed GPC3 RT-PCR results. HepG2 and Caco-2 were GPC3 positive control, and A549 was GPC3 negative control. The GAPDH gene is an internal control gene. (C) GPC3 quantitative PCR results. The bar graph shows the change in the fold expression of GPC3 in cells after 16 hours of incubation, when double delta Ct assay A549 cells were electrotransformed with different doses of GPC3 mRNA. A549 cells were negative controls and GAPDH was an internal control gene.

FIG. 5 detects the density of GPC3 recognized by different GPC3 CAR-T cells. (A) A549 cells were electrotransformed with different doses of GPC3mRNA as shown, co-cultured for 4 hours with 5 GPC3 CAR-T cells shown in the figure, and the flow cytometer detected the expression of CD3 positive cells CD107a, with NO electrotransformed T cells (NO EP) as negative controls for T cells. (B) The bar graph shows the percentage of cd107a+ and cd3+ cells. (C) T cells were electroporated with 10 μg GPC3 CAR mRNA and (D) a549 cells electroporated with different doses of GPC3mRNA were cultured overnight and the supernatant was assayed for cytokines by ELISA. (E) Electrotransport of different doses of a549 cells stimulated GPC3 CAR-T cell proliferation. CFSE labeled resting CD 4T cells were electroporated with 10 μg GPC3 CAR mRNA and a549 cells were incubated for 5 days at a ratio of E: t=1:1 at different doses of GPC3mRNA than shown in the figures. Expression of CFSE on the surface of T cells was diluted in a multiple ratio with proliferation of T cells. Flow cytometry detects the expression of T cell CFSE, the percentage of proliferating T cells shown in the figure.

FIG. 6 VerH.BBZ CAR-T cells inhibit the growth of NSG mouse HepG2 tumor cells. (A) Day 0 mice were subcutaneously injected 1 x 10 in the back ⁶ HepG2-CBG tumor cells were examined weekly for fluorescence in vivo imaging starting on day 6 of tumor injection. (B) As shown in the figure, fromTumor size was measured twice weekly starting on day 3 after tumor inoculation, and HepG2 tumor size was calculated. (C) Flow cytometry examined the transduction efficiency of lentiviral transduced verh.bbz CAR-T cells. (D) Anti-tumor activity of lentivirally transduced verh.bbz CAR-T cells was detected in NSG mouse model of HepG2 hepatoma cells. On day 10 post tumor inoculation, mice were injected 1×10 via tail vein ⁷ Mouse, 5X 10 ⁶ Mouse, 1X 10 ⁶ CAR positive T cells of mice, non-transduced T cells as negative controls. Fluorescence in vivo imaging assays were performed weekly starting on day 6 of tumor inoculation.

FIG. 7 detects expression of tumor cell immunosuppressive molecules and inhibition of CAR-T cell proliferation by TGF- β1. (A) IFN-gamma induces up-regulated expression of PD-L1 by HepG2 tumor cells and A549 tumor cells. A549 and HepG2 tumor cells were cultured overnight in media containing different concentrations of IFN- γ, and flow cytometry examined the expression of PD-L1. (B) expression of TGF-beta by HepG2 tumor cells. The Mouse Anti-TGF-beta antibody detects the expression of TGF-beta in HepG2 tumor cells (peak 1), and the isotype antibody is a negative control (peak 2). (C) CAR-T cell CFSE proliferation assay. CFSE labeled resting CD 4T cells were electroporated with 10 μg verx.bbz or 10 μg verh.bbz CAR mRNA after overnight culture. The electrotransformed T cells and the irradiated HepG2 tumor cells are cultured in R10 culture media containing different concentrations of TGF-beta 1 according to the ratio of 2:1, and the expression of the CFSE of the CAR-T cells on the 5 th day is detected by a flow cytometer.

FIG. 8 TGF-beta receptor and TGFbR/IL12R switch receptor schematic. (A) schematic representation of TGF-beta receptor. TGFbRI and TGFbRII bind TGF- β to generate TGF- β signals, activating the Smad (SMAD Family Member) signaling pathway. (B) schematic of TGFbR1-IL12Rb1 and TGFbR2-IL12Rb 2. The extracellular and transmembrane domains of TGFbRI and TGFbRII fused to the intracellular domains of IL-12 receptors b1 and b2, respectively, constitute TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2, cloned into pGEM.64A RNA in vitro transcription vectors and MSGV retroviral vectors. TGFbR1/IL12R1 and TGFbR2/IL12R2 bind TGF- β to generate IL-12 signaling, activating the STAT4 (Signal transducer and activator of transcription 4) signaling pathway.

FIG. 9 TGFbR/IL12R switch receptor can block TGF-beta signaling while simultaneously converting to IL12 signaling. (A) Flow cytometry detected pSmad2/3 expression in T cells following TGF- β1 induction. T cells were electrotransferred to 5. Mu.g TGFbR1/IL12Rb 1+5. Mu.g TGFbR2/IL12Rb2 mRNA or 10. Mu.g dnTGFbR2 mRNA and after electrotransfer the T cells were incubated overnight at 37 ℃. TGF- β1 was added to the medium the following day to a final concentration of 10ng/ml and after half an hour pSmad2/3 was stained intra-cellularly. NO EP was treated without TGF- β1 as a negative control and NO EP was treated with TGF- β1 as a positive control. (B) TGFbR/IL12R switch receptor induces NK cells to secrete IFN-gamma. NK cells were electrotransformed with 5. Mu.g TGFbR1/IL12Rb 1+5. Mu.g TGFbR2/IL12R2 mRNA or 10. Mu.g dnTGFbR2 mRNA, and the electrotransformed NK cells were cultured in R10 or a medium containing 10ng/ml TGF-. Beta.1R10 for 2 hours, co-cultured with K562 cells at a ratio of 1:1, and after 16 hours, supernatants were collected and assayed for secretion of IFN-. Gamma.by ELISA. (C) TGF- β inhibits CAR-T cell proliferation. Resting CD 4T cells were labeled with CFSE and cultured overnight, the following day were electroporated with 5. Mu.g of VerH.BBZ mRNA, 5. Mu.g of VerH.BBZ+5. Mu.g of TGFbR1/IL12Rb 1+5. Mu.g of TGFbR2/IL12Rb2 mRNA, 5. Mu.g of TGFbR1/IL12Rb 1+5. Mu.g of TGFbR2/IL12Rb2 mRNA, respectively, and the electroporated T cells were cultured with irradiated HepG2 tumor cells in R10 medium containing varying concentrations of TGF-. Beta.1 in the ratio of E: T=2:1, and flow cytometry examined expression of the 5 th day of CFSE.

Figure 10 flow assay of CAR expression and conversion receptor expression and ELISA assay of cytokine secretion. (A) Expression of lentivirus and retrovirus co-transduced T cell CARs. The CD3/CD28 magnetic beads stimulated normal donor T cells, the next day transduced lentiviruses encoding GPC3 CAR, the second and third days transduced retroviruses encoding the transduction receptor. The tenth day flow meter detects CAR expression and the T cells without transduction are negative controls. (B) Expression of T cell transduction receptors co-transduced by lentiviruses and retroviruses. Anti-TGFbR2-APC antibodies detect TGFbR/IL12R and dnTGFbR2 expression, and Anti-PD1-APC antibodies detect PD1-CD28 expression. ELISA detects cytokines in the supernatant of the co-culture of T cells and tumor cells. Verx.bbz, verh.bbz CAR-T cells and CAR-T cells co-expressing the conversion receptor were cultured overnight (3 replicates per group) with 4 tumor cells HepG2, hepG2-PD-L1, MDA231, a549 in the ratio E: t=1:1 as shown in the figures. The following day ELISA was used to detect the IL-2 (C) and IFN-gamma content (D) in the culture supernatants, and the untransduced T cells were used as negative controls.

Figure 11 detection of in vitro killing function of GPC3 CAR-T cells and GPC3 CAR-T cells co-expressing a switch receptor. RTCA can dynamically detect the killing function of VerH.BBZ CAR-T cells and VerH.BBZ CAR-T cells which co-express the conversion receptor on HepG2 tumor cells under different E:T ratios. xcelligent detects the cell number of HepG2 tumor cells on E-plate 96 in real time (10000 starting HepG2 tumor cells) and draws a killing curve. (A-E) killing curves for VerH.BBZ, PD1-CD28+VerH.BBZ, dnTGFbR2+VerH.BBZ and TGFbR/IL12R CAR-T cells, respectively, at different E:T ratios. (B) (C) and (D) are killing curves of CAR-T cells expressing PD1-CD28, dnTGFbR, TGFbR/IL12R conversion receptors at different E:T ratios. (F) Tumor killing curves of CAR-T cells co-transduced with the receptor at an E: T ratio of 0.3:1.

FIG. 12 transduce receptors markedly enhanced the phenotype of central memory cells and secretion of TH1/TH2 cytokines. (A) flow-through detection of T cell surface molecular markers. Irradiated HepG2 tumor cells stimulated verh.bbz CAR-T cells co-expressing the conversion receptor in an e:t=2:1 ratio. Collection of T cells on day 7 of culture the cells were stimulated a second time with the same conditions and after 16 hours the cells were collected and flow cytometry examined the expression of CD62L, CD RA, CD27, CD69 molecules (B) rectangular plots showing the proportion of different types of cells in each group and the results of statistical analysis. (p <0.05, < p <0.01, < p < 0.001.) (C) Luminex analyzed cell culture supernatant for the content of 30 cytokines.

FIG. 13 RNA-seq reveals that genes differentially expressed by CAR-T cells expressing a switch receptor are associated with T cell function. (A) The PCA plot shows the results of statistical analysis of gene reads after sequencing of 13 RNA samples. (B) Volcanic images show genes differentially expressed in the VerH.BBZ, PD1-CD28+VerH.BBZ, TGFbR/IL12R+VerH.BBZ, dnTGFbR2+VerH.BBZ CAR-T groups compared to the NO TD control group. All genes (DEGs) that are significantly differentially expressed are represented by their log2 value for Fold Change (FC) as the x-axis and the lg value for negative significance of the gene expression difference (p-value) as the y-axis. The dashed line represents the threshold value for selecting the DEGs gene: FD absolute value is more than 0.6, P is less than 0.05. The up-regulated gene (FD > 0.6) is shown in red and the down-regulated gene (FD < -0.6) is shown in the region indicated by a. (C) Volcanic images show genes differentially expressed in the PD1-CD28+VerH.BBZ, TGFbR/IL12R+VerH.BBZ, dnTGFbR2+VerH.BBZ CAR-T groups compared to VerH.BBZ. (D) Volcanic images show genes differentially expressed in the TGFbR/IL12R+VerH.BBZ group compared to VerH.BBZ. Table 2-1 shows differentially expressed up-regulated genes (region shown b) of pre-alignment 50 and Table 2-2 shows differentially expressed down-regulated genes (region shown a) of pre-alignment 50. (E) The venn plot shows Meta analysis results of the 4 CAR-T groups differentially expressed genes (left panel), and Meta analysis results of the differentially expressed gene-enriched signaling pathways (right panel) compared to the NO TD control group. The iPathway guide software uses KEGG database for meta analysis. Tables 2-3 list the signaling pathways enriched for the differential expressed genes of the TGFbR/IL12R+VerH.BBZ group compared to the NO TD control group. (F) The Venn diagram shows the meta analysis results of differential expression genes in the PD1-CD28+VerH.BBZ, TGFbR/IL12R+VerH.BBZ, dnTGFbR2+VerH.BBZ group (left panel), and the meta analysis results of differential expression gene-enriched signaling pathways (right panel) compared to VerH.BBZ.

Fig. 14 CAR-T cells co-expressing the transduction receptor showed enhanced anti-tumor activity in NSG mice HepG2 liver cancer tumor model. (A) fluorescence imaging to detect the tumor size in mice. Animal experiments were divided into 5 groups, namely, verH.BBZ CAR-T cells transduced by lentivirus, and VerH.BBZ CAR-T cells expressing the conversion receptors PD1-CD28 and TGFbR-IL12R, dnTGFbR transduced by lentivirus and retrovirus, and the untransduced T cells were negative control groups. Mice were subcutaneously injected 1 x 10 on the right dorsal side ⁶ HepG2-CBG tumor cells, mouse tail intravenous injection 1X 10 on day 10 ⁶ CAR positive T cells. Tumor size was detected by fluorescence imaging at day 9, day 17, day 25 and day 32 after tumor inoculation. (B) The fluorescence imaging results were plotted as a dot-matrix plot according to imaging time, with each dot representing the fluorescence imaging results of one mouse. (C) results of statistical analysis of the results of fluorescence imaging of mice. The table shows the t detection results (×p) between two groups of mice at 2 fluorescence imaging time points<0.05，**p<0.01)。

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If any potential ambiguity exists, the definitions provided herein take precedence over any dictionary or extraneous definitions. The use of "or" means "and/or" unless stated otherwise.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor that is engineered to be expressed on immune cells and to specifically bind to an antigen. CARs may be used as adoptive cell transfer therapies. T cells are removed from the patient and modified to express a receptor specific for the antigen or a particular form of antigen. The CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region.

The term "encoding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) as a template for use in biological processes for synthesizing other polymers and macromolecules having defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand (which has the nucleotide sequence identical to the mRNA sequence and is generally provided in the sequence listing) and the non-coding strand (which serves as a template for transcription of a gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA.

As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

An "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or included in liposomes) and viruses (e.g., sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate the recombinant polynucleotide.

The term "antibody", as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins (e.g., binding fragments of antibodies). Antibodies are typically tetramers of immunoglobulin molecules. Antibodies of the invention can exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, fv, fab and F (ab) 2, as well as single chain antibodies (scFv) and humanized antibodies (Harlow et al, 1999,In:Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY;Harlow et al, 1989,In:Antibodies:ALaboratory Manual,Cold Spring Harbor,New York;Houston et al, 1988,Proc.Natl.Acad.Sci.USA 85:5879-5883; bird et al, 1988,Science 242:423-426).

The term "antibody fragment" refers to a portion of an intact antibody and to the epitope variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, and multispecific antibodies.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (such as Fv, fab, fab ', F (ab') 2 or other antigen-binding subsequence of an antibody) that contains minimal sequence from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (e.g., mouse, rat or rabbit) (donor antibody) having the desired specificity, affinity and capacity. In some cases, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications may further improve and optimize antibody performance. Typically, a humanized antibody will comprise substantially all of the following: at least one and typically two variable domains, wherein all or substantially all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all FR regions are those of a human immunoglobulin sequence. Humanized antibodies may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See Jones et al, nature,321:522-525,1986 for further details; reichmann et al, nature,332:323-329,1988; presta, curr.Op.struct.biol.,2:593-596,1992. "fully human" refers to an immunoglobulin, such as an antibody or binding fragment thereof, in which the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody.

As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Five members included in this class of proteins are IgA, igG, igM, igD and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the primary immunoglobulin produced in the primary immune response of most subjects. It is the most potent immunoglobulin in agglutination reactions, complement fixation and other antibody responses, and is important in combating bacteria and viruses. IgD is an immunoglobulin that does not have known antibody functions but can be used as an antigen receptor. IgE is an immunoglobulin that mediates tachyphylaxis by causing the release of mediators from mast cells and basophils upon exposure to allergens.

As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in the sample. For example, an antibody that specifically binds an antigen from one species may also bind antigens from one or more species. However, this cross-species reactivity does not itself alter the specific class of antibodies. In another example, antibodies that specifically bind to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity does not itself alter the specific class of antibodies. In some examples, the term "specifically binds" or "specifically binds" is used with reference to an interaction of an antibody, protein, or peptide with a second chemical species, to refer to the interaction being dependent on the presence of a particular structure (e.g., an epitope) on the chemical species; for example, antibodies recognize and bind specific protein structures rather than generally recognizing and binding proteins. If the antibody is specific for epitope "a", the presence of a molecule comprising epitope a (or free, unlabeled a) in a reaction comprising labeled "a" and antibody will reduce the amount of labeled a bound to the antibody.

As used herein, the term "identity" refers to subunit sequence identity between two polymeric molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. When two amino acid sequences have identical residues at the same position, e.g., if each position in both polypeptide molecules is occupied by arginine, they are identical at that position. Two amino acid sequences are typically expressed in percent identity or degree of identity with identical residues in the same aligned positions. Identity between two amino acid sequences is a direct function of the number of matching or identical positions, and if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, then the identity of the two sequences is 50%; if 90% of the positions (e.g., 9 out of 10) are matched or identical, then the identity of the two amino acid sequences is 90%.

As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigenic molecule as a foreign and induce antibody formation and/or activate lymphocytes to remove the antigen.

As used herein, the term "immunosuppression" refers to a reduction in the overall immune response.

The term "isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely separated from coexisting materials in its natural state, is "isolated. The isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment (such as, for example, a host cell).

As used herein, "lentivirus" refers to a genus of the retrovirus family. Among retroviruses, lentiviruses are the only ones that can infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, so they are one of the most effective methods of gene delivery vehicles. HIV, S1V and FIV are all examples of lentiviruses. Lentiviral derived vectors provide a means to accomplish significant levels of gene transfer in vivo.

As used herein, the term "modified" refers to a change in the state or structure of a molecule or cell of the invention. The molecule may be modified in a variety of ways, including chemically, structurally and functionally. The cell may be modified by introducing a nucleic acid. Reference herein to an immune cell comprising or including a CAR molecule means that the immune cell is modified by the introduction of a nucleic acid encoding the CAR molecule.

In the context of the present invention, the following abbreviations are used for the nucleobases which usually occur. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise specified, a "nucleotide sequence" of amino acid sequences encoding a protein includes all nucleotide sequences that are degenerate versions of each other and that encode identical amino acid sequences. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may include the intron(s) in some versions.

The term "operably linked" or "operatively linked (operatively linked)" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence.

As used herein, the terms "polynucleotide", "nucleic acid construct" are defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One of ordinary skill in the art is generally aware that a nucleic acid is a polynucleotide that can be hydrolyzed to monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art and by synthetic means, including, but not limited to, recombinant means, i.e., cloning from recombinant libraries or cellular genomic nucleic acid sequences using common cloning techniques, PCR, and the like.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can constitute the protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, e.g., which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, and also refers to longer chains, which are commonly referred to in the art as proteins, there being many types of proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

The term "stimulation" refers to a primary response induced by binding a stimulatory molecule (e.g., a TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling via the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as the down regulation of TGF- β and/or reorganization of cytoskeletal structures, and the like.

"stimulatory molecule", as the term is used herein, refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell.

"costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

As used herein, a "co-stimulatory signal" refers to a signal that binds to a primary signal (such as a TCR/CD3 linkage), resulting in up-or down-regulation of T cell proliferation and/or a key molecule.

The term "subject" is intended to include living organisms (e.g., mammals) within which an immune response may be elicited. As used herein, a "subject" or "patient" may be a human or non-human mammal. Non-human mammals include, for example, domestic animals and pets such as sheep, cattle, pigs, dogs, cats and murine mammals. Preferably, the subject is a human.

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to a compound, formulation, material, or composition as described herein that is effective to achieve a particular biological result, or to provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, the amount of the composition, when administered to a mammal, may cause a detectable level of immunosuppression or immune tolerance as compared to the immune response detected in the absence of the composition of the invention. Immune responses can be readily assessed in a number of ways recognized in the art. Those skilled in the art will appreciate that the amount of the compositions administered herein will vary and can be readily determined based on a number of factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

Example 1

Experimental materials and methods

1. Experimental materials

1.1 animals

NOD-SCIDR-/- (NSG) mice of 6-10 weeks of age were purchased from and bred in the Proteus transformation center animal house of university of pennsylvania under SPF conditions. The feeding and experimental treatment followed the institutional animal ethics committee of pennsylvania university and the animal laboratory manual.

1.2 cells

Cell lines	Cell origin	Culture medium
			HepG2	Human liver cancer cell	RPMI+10％FBS
Caco-2	Human colorectal cancer cell	MEM+20％FBS
			C3A	Human liver cancer cell	RPMI+10％FBS
PLC/PRF/5	Human liver cancer cell	RPMI+10％FBS
			SNU475	Human liver cancer cell	RPMI+10％FBS
A549-ESO	Human lung cancer cell	RPMI+10％FBS
			SK-OV3	Human ovarian cancer cells	RPMI+10％FBS

The above cell lines are all conventional in the art and are commercially available.

1.3 major reagents

1.3.1 antibodies

1.3.2 other important reagents

1.4 Main instruments

1.5 primer sequences

1.5.1 real-time quantitative PCR detection primers

1.5.2 GPC3 CAR sequences

CD8 hinge region amino acid sequence: (SEQ ID NO: 11)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 a transmembrane domain amino acid sequence: (SEQ ID NO: 12)

IYIWAPLAGTCGVLLLSLVITLYC

4-1BB co-stimulatory domain amino acid sequence: (SEQ ID NO: 13)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD3 zeta amino acid sequence: (SEQ ID NO: 14)

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

1.5.2.1 VerX.BBZ nucleotide sequence (SEQ ID NO: 1)

ATGGCCCTGCCCGTGACCGCCCTCCTGTTGCCGCTTGCCCTGCTCCTCCACGCCGCGAGACCGCAGGTGCAGCTGGTGCAGAGCGGGGCCGAGGTCAAGAAGCCCGGGGCTTCCGTGAAGGTGAGCTGTAAGGCCTCCGGGTATACGTTCACCGATTACGAGATGCACTGGGTGCGCCAAGCCCCCGGGCAGGGCCTGGAGTGGATGGGCGCTCTGGATCCAAAGACGGGGGATACTGCCTATAGTCAGAAGTTCAAAGGCAGGGTCACACTGACCGCCGACAAATCCACATCTACCGCCTACATGGAGTTATCCTCGCTGACCAGCGAGGACACGGCAGTGTATTACTGCACGCGCTTCTATTCCTACACGTACTGGGGGCAAGGGACCCTGGTGACGGTGTCCTCCGGCGGCGGCGGTAGTGGCGGGGGTGGCTCCGGCGGGGGGGGCAGTGATGTCGTGATGACTCAGTCCCCCCTGTCGCTGCCTGTGACGCCCGGCGAGCCCGCCAGCATCTCCTGCCGATCCTCCCAGTCACTGGTGCATTCCAACGGGAATACTTACCTGCACTGGTATCTCCAGAAGCCCGGCCAGAGCCCTCAGCTCCTGATCTACAAGGTCTCTAATAGGTTCTCCGGCGTCCCCGATCGGTTTAGCGGCTCCGGCAGCGGCACAGACTTTACACTCAAGATTTCCCGCGTGGAAGCTGAGGACGTCGGAGTCTACTATTGCTCCCAGAATACCCACGTCCCGCCAACCTTCGGCCAGGGAACAAAACTGGAGATCAAAACCACGACGCCAGCGCCGCGACCCCCCACCCCGGCGCCGACAATCGCATCGCAGCCCCTGAGCCTGAGGCCCGAGGCATGTAGGCCCGCCGCAGGCGGAGCCGTCCACACCAGGGGGCTCGACTTTGCATGCGATATCTACATTTGGGCCCCTCTGGCCGGCACCTGCGGAGTGCTCCTCCTCAGTCTGGTGATCACACTCTACTGTAAGAGGGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTCCAGACCACCCAGGAAGAAGATGGGTGCAGCTGCAGATTCCCCGAGGAGGAGGAAGGGGGGTGCGAATTGCGCGTTAAGTTCTCGCGTTCCGCCGACGCCCCAGCTTACAAGCAGGGCCAGAACCAGCTTTATAACGAGCTCAATTTGGGCCGGAGGGAGGAGTACGACGTACTCGACAAGCGCCGCGGCCGCGACCCCGAAATGGGTGGAAAGCCCCGGCGCAAAAACCCCCAGGAGGGGCTGTACAACGAGCTGCAGAAGGATAAGATGGCTGAGGCCTACTCTGAGATCGGTATGAAGGGCGAGCGGCGCCGCGGCAAGGGACACGATGGCCTGTACCAGGGGCTGTCCACGGCCACAAAGGATACGTACGACGCTCTGCACATGCAGGCCCTGCCCCCCCGCTAA

VerX scFv amino acid sequence (SEQ ID NO: 2):

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGS GGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK

VerX.BBZ amino acid sequence (SEQ ID NO: 15):

MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

1.5.2.2 VerH.BBZ nucleotide sequence (SEQ ID NO: 3)

ATGGCCCTGCCCGTCACTGCCCTCCTTCTGCCACTCGCCCTCCTCCTGCACGCAGCACGACCCCAGGTGCAGCTGGTCGAAAGCGGCGCCGAGGTGAAGAAGCCTGGTGCCTCCGTCAAGGTGTCCTGCAAGGCGTCAGGCTACACCTTCACTGACTACGAGATGCACTGGGTACGCCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCGCCTTAGACCCCAAGACCGGCGACACCGCTTATTCCCAGAAGTTTAAAGGGCGTGTCACCCTGACAGCCGACAAATCCACATCAACCGCGTATATGGAGCTGTCCTCTCTGACCAGCGAGGACACCGCCGTCTATTACTGCACTCGCTTCTATTCCTACACGTACTGGGGCCAGGGTACGCTAGTGACCGTGTCAAGTGGCGGGGGGGGCAGTGGGGGCGGCGGAAGTGGCGGGGGGGGCTCCGACGTCGTCATGACCCAGAGTCCCCTGTCCCTGCCCGTGACCCCGGGCGAACCCGCCTCTATTAGCTGCCGGTCCTCGCAGTCTCTCGTGCACTCGAACGGGAACACCTACCTGCACTGGTACCTGCAAAAGCCCGGGCAGAGTCCGCAGCTGCTGATCTATAAGGTTAGCAACAGGTTTTCCGGGGTCCCCGATCGCTTCAGCGGGTCGGGGTCGGGTACGGATTTCACCCTCAAGATCTCGCGAGTGGAGGCAGAAGACGTTGGCGTGTACTACTGCAGTCAGAATACCCATGTGCCCCCCACTTTCGGTCAGGGTACCAAACTGGAGATCAAGACCACGACGCCAGCGCCGCGACCCCCCACCCCGGCGCCGACAATCGCATCGCAGCCCCTGAGCCTGAGGCCCGAGGCATGTAGGCCCGCCGCAGGCGGAGCCGTCCACACCAGGGGGCTCGACTTTGCATGCGATATCTACATTTGGGCCCCTCTGGCCGGCACCTGCGGAGTGCTCCTCCTCAGTCTGGTGATCACACTCTACTGTAAGAGGGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTCCAGACCACCCAGGAAGAAGATGGGTGCAGCTGCAGATTCCCCGAGGAGGAGGAAGGGGGGTGCGAATTGCGCGTTAAGTTCTCGCGTTCCGCCGACGCCCCAGCTTACAAGCAGGGCCAGAACCAGCTTTATAACGAGCTCAATTTGGGCCGGAGGGAGGAGTACGACGTACTCGACAAGCGCCGCGGCCGCGACCCCGAAATGGGTGGAAAGCCCCGGCGCAAAAACCCCCAGGAGGGGCTGTACAACGAGCTGCAGAAGGATAAGATGGCTGAGGCCTACTCTGAGATCGGTATGAAGGGCGAGCGGCGCCGCGGCAAGGGACACGATGGCCTGTACCAGGGGCTGTCCACGGCCACAAAGGATACGTACGACGCTCTGCACATGCAGGCCCTGCCCCCCCGCTAA

VerH scFv amino acid sequence (SEQ ID NO: 4):

MALPVTALLLPLALLLHAARPQVQLVESGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGS GGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK

VerH.BBZ amino acid sequence (SEQ ID NO: 16):

MALPVTALLLPLALLLHAARPQVQLVESGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

1.5.2.3 BBZ sequence (SEQ ID NO: 5)

ATGGCCCTGCCAGTGACCGCCTTGCTGCTGCCCCTGGCCCTCCTGCTGCATGCTGCGAGGCCTCAGGTGCAGCTGGTCGAATCAGGGGCTGAGGTGAAGAAGCCTGGCGCCTCGGTGAAGGTCAGCTGTAAGGCGTCCGGCTACACCTTCACCGATTACGAGATGCACTGGGTCCGCCAGGCACCGGGCCAGGGCCTGGAGTGGATGGGGGCCCTGGATCCTAAGACCGGCGACACCGCTTACTCGCAGAAGTTCAAGGGTCGGGTCACCATCACCGCCGATGAATCTACGAGCACCGCTTACATGGAGCTGTCGTCCTTGCGGAGCGAAGACACTGCCGTATACTACTGCGCCAGATTCTACTCTTACACCTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCTGGCGGGGGCGGTTCCGGAGGGGGGGGCTCGGGCGGCGGCGGCTCTGACGTAGTGATGACCCAATCCCCACTGTCCCTGCCGGTGACACCTGGCGAGCCCGCCTCCATCTCGTGCCGCAGTTCGCAGTCCCTGGTGCACTCGAACGGGAACACATACCTGCACTGGTACCTTCAGAAACCCGGCCAGTCCCCCCAGCTGCTCATCTACAAGGTCTCCAATAGGTTTTCCGGAGTGCCCGACCGCTTCTCCGGCTCCGGCTCAGGAACTGATTTTACCCTGAAAATTTCCCGGGTGGAGGCCGAAGACGTCGGAGTGTACTACTGTTCCCAGAACACCCACGTCCCCCCCACCTTCGGCCAGGGGACCAAATTGGAAATTAAGACCACGACGCCAGCGCCGCGACCCCCCACCCCGGCGCCGACAATCGCATCGCAGCCCCTGAGCCTGAGGCCCGAGGCATGTAGGCCCGCCGCAGGCGGAGCCGTCCACACCAGGGGGCTCGACTTTGCATGCGATATCTACATTTGGGCCCCTCTGGCCGGCACCTGCGGAGTGCTCCTCCTCAGTCTGGTGATCACACTCTACTGTAAGAGGGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTCCAGACCACCCAGGAAGAAGATGGGTGCAGCTGCAGATTCCCCGAGGAGGAGGAAGGGGGGTGCGAATTGCGCGTTAAGTTCTCGCGTTCCGCCGACGCCCCAGCTTACAAGCAGGGCCAGAACCAGCTTTATAACGAGCTCAATTTGGGCCGGAGGGAGGAGTACGACGTACTCGACAAGCGCCGCGGCCGCGACCCCGAAATGGGTGGAAAGCCCCGGCGCAAAAACCCCCAGGAGGGGCTGTACAACGAGCTGCAGAAGGATAAGATGGCTGAGGCCTACTCTGAGATCGGTATGAAGGGCGAGCGGCGCCGCGGCAAGGGACACGATGGCCTGTACCAGGGGCTGTCCACGGCCACAAAGGATACGTACGACGCTCTGCACATGCAGGCCCTGCCCCCCCGCTAA

Ver scFv amino acid sequence (SEQ ID NO: 6):

MALPVTALLLPLALLLHAARPQVQLVESGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARFYSYTYWGQGTLVTVSSGGGGSGGGGS GGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK

1.5.2.4 GC33.BBZ nucleotide sequence (SEQ ID NO: 7)

ATGGCCTTACCTGTGACGGCCCTGCTTCTTCCGCTGGCCCTCCTGCTGCACGCCGCACGGCCGCAGGTGCAGCTGCAGCAGTCGGGGGCCGAGCTGGTGCGGCCCGGGGCCTCCGTCAAGCTGTCCTGCAAGGCCAGTGGGTACACCTTCACTGATTATGAGATGCACTGGGTGAAGCAGACCCCTGTGCATGGCCTGAAATGGATCGGCGCTCTCGACCCCAAGACAGGTGACACCGCCTACAGTCAGAAGTTTAAGGGCAAAGCAACCCTCACCGCCGACAAGTCGTCCTCGACCGCCTACATGGAGCTGAGGTCCCTGACCTCCGAGGATTCGGCCGTCTACTACTGCACCCGCTTCTACTCCTACACATACTGGGGCCAGGGTACCTTAGTGACCGTCTCGGCAGGCGGGGGGGGCTCCGGGGGTGGCGGCAGTGGTGGGGGGGGCTCCGACGTGGTGATGACACAGACCCCTCTGTCCCTCCCAGTTTCGCTCGGTGACCAGGCATCGATCTCGTGCCGCTCCTCACAGAGTCTGGTGCACTCCAACGGCAACACATATCTGCACTGGTACCTCCAGAAGCCCGGGCAGTCACCTAAGCTCCTCATCTACAAGGTGAGTAATCGATTCAGTGGCGTGCCAGACAGGTTCTCCGGCTCCGGGAGTGGAACGGATTTCACCCTGAAGATCTCCCGCGTGGAGGCGGAGGACCTGGGAGTCTACTTCTGCTCGCAGAACACCCACGTCCCGCCCACGTTTGGCAGTGGCACTAAGCTCGAGATCAAAACCACGACGCCAGCGCCGCGACCCCCCACCCCGGCGCCGACAATCGCATCGCAGCCCCTGAGCCTGAGGCCCGAGGCATGTAGGCCCGCCGCAGGCGGAGCCGTCCACACCAGGGGGCTCGACTTTGCATGCGATATCTACATTTGGGCCCCTCTGGCCGGCACCTGCGGAGTGCTCCTCCTCAGTCTGGTGATCACACTCTACTGTAAGAGGGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTCCAGACCACCCAGGAAGAAGATGGGTGCAGCTGCAGATTCCCCGAGGAGGAGGAAGGGGGGTGCGAATTGCGCGTTAAGTTCTCGCGTTCCGCCGACGCCCCAGCTTACAAGCAGGGCCAGAACCAGCTTTATAACGAGCTCAATTTGGGCCGGAGGGAGGAGTACGACGTACTCGACAAGCGCCGCGGCCGCGACCCCGAAATGGGTGGAAAGCCCCGGCGCAAAAACCCCCAGGAGGGGCTGTACAACGAGCTGCAGAAGGATAAGATGGCTGAGGCCTACTCTGAGATCGGTATGAAGGGCGAGCGGCGCCGCGGCAAGGGACACGATGGCCTGTACCAGGGGCTGTCCACGGCCACAAAGGATACGTACGACGCTCTGCACATGCAGGCCCTGCCCCCCCGCTAA

GC33 scFv amino acid sequence (SEQ ID NO: 8)

MALPVTALLLPLALLLHAARPQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAGGGGSGGGGS GGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIK

1.5.2.5 M11F1.BBZ sequence (SEQ ID NO: 9)

ATGGCTCTCCCTGTGACCGCCCTCTTGCTGCCGCTGGCCCTGCTGCTCCACGCAGCCCGCCCGCAGGTGACTCTTAAGGAGAGCGGCCCAGGAATCTTACAGCCTAGTCAGACCCTCAGTCTCACCTGCTCTTTCAGTGGCTTCTCCCTCTCCATATACGGCATGGGGGTGGGCTGGATCCGGCAGCCGAGCGGCAAGGGACTTGAGTGGCTGGCGAATATCTGGTGGAACGATGACAAATACTACAACTCAGCACTGAAGTCCCGTCTTACCATCTCCAAGGACACGAGTAATAACCAGGTCTTCCTGAAGATCTCGTCCGTGGACACCGCCGACACCGCCACCTACTACTGCGCACAGATCGGATACTTCTACTTCGACTACTGGGGCCAGGGGACAACCCTGACCGTGAGCAGTGGCGGCGGGGGGTCCGGAGGGGGTGGCTCGGGCGGTGGGGGAAGTGATGTGGTGATGACGCAGACGCCGCTGTCGCTGCCGGTGAGCCTCGGCGACCAGGCCTCGATTTCGTGCCGCTCCTCCCAGTCCCTCGTGCACTCCAACGGGAACACCTACCTGCATTGGTACCTGCAGAAGCCTGGCCAGTCGCCGAAGCTGCTCATCTACAAAGTTTCCAATCGCTTCTCCGGCGTGCCTGACCGGTTCAGCGGGTCGGGCTCCGGCACGGACTTCACTCTTAAGATCTCCCGTGTGGAAGCTGAGGACTTAGGCGTGTACTTCTGCTCCCAGAGCACTCATGTACCATGGACTTTTGGAGGGGGCACGAAACTCGAGATCAAGACCACGACGCCAGCGCCGCGACCCCCCACCCCGGCGCCGACAATCGCATCGCAGCCCCTGAGCCTGAGGCCCGAGGCATGTAGGCCCGCCGCAGGCGGAGCCGTCCACACCAGGGGGCTCGACTTTGCATGCGATATCTACATTTGGGCCCCTCTGGCCGGCACCTGCGGAGTGCTCCTCCTCAGTCTGGTGATCACACTCTACTGTAAGAGGGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTCCAGACCACCCAGGAAGAAGATGGGTGCAGCTGCAGATTCCCCGAGGAGGAGGAAGGGGGGTGCGAATTGCGCGTTAAGTTCTCGCGTTCCGCCGACGCCCCAGCTTACAAGCAGGGCCAGAACCAGCTTTATAACGAGCTCAATTTGGGCCGGAGGGAGGAGTACGACGTACTCGACAAGCGCCGCGGCCGCGACCCCGAAATGGGTGGAAAGCCCCGGCGCAAAAACCCCCAGGAGGGGCTGTACAACGAGCTGCAGAAGGATAAGATGGCTGAGGCCTACTCTGAGATCGGTATGAAGGGCGAGCGGCGCCGCGGCAAGGGACACGATGGCCTGTACCAGGGGCTGTCCACGGCCACAAAGGATACGTACGACGCTCTGCACATGCAGGCCCTGCCCCCCCGCTAA

M11F1 scFv amino acid sequence (SEQ ID NO: 10):

MALPVTALLLPLALLLHAARPQVTLKESGPGILQPSQTLSLTCSFSGFSLSIYGMGVGWIRQPSGKGLEWLANIWWNDDKYYNSALKSRLTISKDTSNNQVFLKISSVDTADTATYYCAQIGYFYFDYWGQGTTLTVSSGGGGSGG GGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK

1.5.3 GPC3 CAR and GPC3 cDNA cloning primers

1.5.4 GPC3 cDNA sequence (SEQ ID NO: 33)

AACCAAGCTTACCGCCATGGCTGGCACCGTCCGCACCGCGTGTCTGGTTGTAGCCATGCTCCTGTCCCTGGACTTCCCCGGTCAGGCCCAGCCACCCCCCCCTCCGCCCGACGCAACCTGCCACCAGGTACGTTCCTTCTTTCAGAGGCTGCAACCCGGCCTCAAGTGGGTCCCAGAGACCCCCGTGCCGGGGAGTGACCTCCAGGTGTGCCTGCCCAAGGGTCCCACGTGCTGCTCGCGGAAGATGGAAGAAAAGTACCAGCTCACCGCCAGACTCAACATGGAGCAGTTACTACAATCCGCCTCCATGGAGCTGAAGTTCCTAATCATCCAGAACGCCGCCGTCTTCCAGGAGGCATTCGAAATTGTGGTGCGGCACGCGAAGAATTACACGAACGCCATGTTCAAGAACAATTACCCCTCGCTCACTCCCCAGGCCTTCGAATTCGTCGGCGAGTTCTTCACGGACGTGAGTCTCTACATCCTGGGGTCCGACATCAACGTCGACGACATGGTTAACGAGCTGTTCGACTCCCTCTTCCCGGTGATCTACACTCAGCTGATGAACCCCGGACTGCCGGACTCTGCCCTGGATATCAACGAGTGCCTGCGCGGAGCCAGGAGAGACCTGAAGGTTTTCGGTAACTTTCCAAAGTTGATCATGACGCAGGTCTCCAAAAGCCTCCAGGTGACTCGGATCTTCCTCCAGGCCCTCAACCTGGGCATCGAGGTCATCAACACCACCGACCACCTGAAGTTTTCCAAGGACTGCGGCCGTATGCTGACCCGTATGTGGTACTGCTCCTACTGTCAGGGCCTGATGATGGTGAAGCCCTGTGGCGGCTACTGTAATGTGGTGATGCAGGGCTGCATGGCGGGCGTGGTCGAAATCGACAAGTATTGGCGCGAGTACATCTTATCCCTCGAAGAACTGGTAAATGGAATGTACCGCATTTATGACATGGAAAACGTACTCCTCGGCCTATTCTCCACCATCCATGACAGCATACAGTACGTGCAGAAGAACGCCGGGAAGTTAACCACCACCATCGGTAAGCTCTGCGCCCACTCCCAGCAGAGACAGTATCGGTCCGCCTACTATCCAGAGGACCTGTTTATCGACAAGAAGGTGCTGAAGGTCGCCCACGTGGAACACGAGGAGACTCTGTCCTCCAGGAGACGGGAGCTGATTCAAAAACTGAAATCGTTCATATCTTTCTACTCAGCTCTGCCCGGCTACATCTGTAGTCACTCCCCTGTGGCCGAGAATGATACGCTCTGCTGGAACGGTCAGGAACTGGTGGAGCGCTACTCCCAGAAAGCCGCTCGCAACGGAATGAAGAACCAGTTTAACCTGCACGAACTGAAGATGAAGGGGCCCGAGCCCGTGGTGTCCCAGATCATCGACAAGCTCAAGCACATTAACCAGCTGCTGCGCACCATGTCCATGCCAAAGGGCCGCGTGCTGGACAAGAACTTGGACGAGGAGGGCTTCGAGAGCGGCGACTGTGGGGACGATGAGGATGAGTGTATAGGCGGCTCCGGCGATGGCATGATCAAAGTGAAGAACCAGCTCAGATTCCTGGCCGAGCTGGCGTACGATCTCGACGTCGATGATGCCCCGGGGAACTCCCAGCAGGCAACGCCCAAGGACAATGAGATCTCCACCTTCCACAACTTGGGCAACGTACATTCGCCCCTGAAGCTCCTGACGTCCATGGCCATCTCAGTGGTGTGCTTCTTCTTCCTGGTGCACTAATAAGCGGCCGC

Example 2

2.1 Total RNA extraction, RNA reverse transcription, RT-PCR

The RNA kit extracts the total cellular RNA, which is then reverse transcribed into cDNA. cDNA1 template was mixed with detection primer (or control gene primer) and superscreenmix fluorescent quantitative PCR in proportion. PCR instrument amplification procedure: 94 ℃ 5';94℃30 ", 58℃30", 72℃30 ", 30 cycles; 72℃7',4℃60'. The PCR amplified fragment was detected by 2% agarose gel electrophoresis. The reagents and primers used are shown in the experimental materials (tables 1.3-1.5).

2.2 CAR plasmid construction

Construction of pD-A.GPC3 CAR (RNA in vitro transcription vector) and pTRP.GPC3 CAR (lentiviral vector) plasmid: first, according to the DNA sequences of the light chain and the heavy chain of GPC3 antibody disclosed in the patent publication, DNA fragments encoding scFv were designed and synthesized, and the scFv was connected with the 4-1BBZ transmembrane region and intracellular signaling region by PCR amplification. After double enzyme digestion of the pD-ARNA vector (or pTRP vector) and the PCR product, the pD-A vector (or pTRP vector) fragment and the target gene fragment are recovered by using a gel recovery kit, and after the pD-ARNA vector (or pTRP vector) fragment and the target gene fragment are connected, the pD-ARNA vector (or pTRP vector) fragment and the target gene fragment are transformed into competent cells, monoclonal is selected, plasmid is extracted, PCR, enzyme digestion and sequencing are performed to verify that the vector is successfully constructed.

2.3 In vitro transcription of mRNA

After linearization of the plasmid by single cleavage, the PCR recovery kit is used for recovery and purification, and the linearized plasmid is used as a template for in vitro transcription of mRNA. According to the instructions of the T7 mscript system (CellScript) kit, the DNA template was mixed with reagents such as buffer, T7 enzyme, etc., incubated at 37℃for 2 hours, and then digested with DNase for 15 minutes to degrade the DNA template, followed by tailing with polyA enzyme. The RNeasy kit purified mRNA. The purified mRNA was stored in sub-package in-80℃refrigerator.

2.4 CD3/CD28 magnetic bead expansion T cells

T cells used in this study were all healthy donor T cells, supplied by the university of pennsylvania human immunology center. The CD3/CD28 magnetic beads were washed three times with R10 medium and the preservative of the magnetic bead preservation solution was removed. The magnetic beads and T cells are mixed according to the ratio of 3:1, and the initial concentration of the T cell culture is 0.5-0.7X10 ⁶ /ml. On the third day, T cells were counted after being blown and mixed uniformly, and fresh R10 medium was added until the cell concentration was 0.5-0.7X10 ⁶ /ml. From day three, cells were counted every other day and fresh medium was added. The beads were removed on the fifth day. Continuing to culture T cells for the tenth day, the size of the cells was about 300. Mu.m ³ T cells are collected and transferred to a liquid nitrogen tank for preservation after being frozen by cold storage liquid.

2.5 T cell and tumor cell electrotransformation

Frozen (or fresh) T cells were thawed and cultured in R10 medium in an incubator at 37 ℃ for 4 hours (or overnight). Before electrotransformation, T cells were collected by centrifugation (1500 rpm,5 min), medium was removed, resuspended in OPTI-mem medium, the procedure was repeated twice (FBS in R10 medium was removed), and then T cells were resuspended in OPTI-mem medium to a cell concentration of 50X 10 ⁶ /ml. 100 μl of cells are mixed with a proper amount of RNA, and then quickly transferred to a 0.2cm electric shock cup, and placed into an electric shock tank with electric transfer conditions to complete electric transfer. The electrotransformation conditions of T cells were 500V/700. Mu.s.

The electrotransport process of tumor cells is the same as that of T cells, but the electrotransport conditions are slightly different. Electrotransformation conditions for HepG2 tumor cells were 300V/500. Mu.s.

2.6 fluorescent quantitative PCR

The template cDNA was mixed with detection primers and fluorescent quantitative PCR Premix (SYBR Green, TIANGEN) according to the instructions and operated in the dark. After mixing the samples, the target gene was detected using a real-time quantitative PCR instrument BioRad C1000TM Thermal Cycler CFX96TM setup program.

2.7 flow cytometry staining

Antibodies used for cell staining are listed in table 1.3.1. Cells were collected by centrifugation and suspended in flow buffer (PBS solution containing 2% FBS), appropriate amount of antibody (recommended dose by the company) was added, stained at 4℃for 30 minutes in the absence of light, washed once with buffer, and fluorescence was detected by flow cytometry (BD FacsCalibur) (Staining Cell Surface Targets, protocol A). The FlowJo software analyzes the streaming results (Treestar).

The intracellular staining was performed using the kit Foxp3/Transcription Factor Staining Buffer Set (eBioscience, cat#00-5523-00) following the staining method recommended by the kit.

2.8 ELISA experiments

After washing the tumor cells, the cell concentration was adjusted to 1X 10 by suspending in R10 medium ⁶ Individual cells/mL. Tumor cells were taken in 100 μl and added to a 96-well U-plate. After T-cell washing, the cell concentration was adjusted to 1X 10 by suspending in R10 medium ⁶ Individual cells/mL. 100 μ l T cells were added to designated wells of a 96-well U-plate and mixed with tumor cells for culture. Three replicates were cultured for each T cell and tumor cell. The 96-well plates were incubated at 37℃for 16 to 24 hours. After incubation, the supernatants were harvested and subjected to ELISA assays (eBioscience).

2.9 fluorescence-based killing experiments

Lentivirus encoding CBG-GFP transduces HepG2 tumor cells, and GFP positive cells are sorted to give a HepG2-CBG-GFP tumor cell line which can be used for luciferase-based killing assays. The method comprises the following steps: after washing HepG2-CBG-GFP tumor cells, the suspension was used in R10 medium to adjust the cell concentration to 1X 10 ⁵ Mu.l per well was added to 96-well white flat bottom fluorescent plates (Cat#3917, COSTAR) per ml. T cells were washed and suspended in R10 medium and the cell concentration was adjusted to 2X 10 ⁶ Individual cells/mL. In another 96-well U-shaped plate, T cells are diluted by a ratio of 2 times, and the concentration of the diluted T cells is sequentially 1 multiplied by 10 ⁶ /ml、0.5×10 ⁶ /ml、0.25×10 ⁶ /ml、0.125×10 ⁶ /ml、0.06×10 ⁶ /ml、0.03×10 ⁶ /ml、1.5×10 ⁴ /ml、0.75×10 ⁴ /ml，0.4×10 ⁴ /ml、0.2×10 ⁴ Per ml, two replicates per concentration. T cells diluted in a 100. Mu.l-fold ratio were transferred to corresponding positions of 96-well white flat-bottom fluorescent plates with 100. Mu.l tumor cells added thereto, and the E: T ratios of T cells and tumor cells after mixing were 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, 0.3:1, 0.15:1, 0.075:1, 0.04:1, 0.02:1, 100. Mu. l R10 medium were negative controls in this order. The plate was incubated overnight at 37℃in an incubator. Before plate reading, the culture medium is gently removed by a multi-channel pipette, PBS is used for cleaning once, 100 mu l of substrate is added, the plate reader reads the fluorescence of each hole, and the percentage calculation formula of killing is as follows: killing% = (100- (fluorescence value per well/negative control fluorescence average value) ×100))%.

2.10 CD107a experiment

Tumor cells were washed and suspended in R10 medium to adjust cell concentration to 2X10 ⁶ Individual cells/mL. Tumor cells were taken in 100ul and added to a 96-well U-shaped plate. T cells were washed and suspended in R10 medium to adjust the cell concentration to 1X 10 ⁶ Individual cells/mL. 100 μl T cells were added to designated wells of a 96-well U-plate and mixed with tumor cells (E: T=1:2). Mu.l of anti-CD 107a-PE antibody was added to each well. The 96-well U-plate was incubated at 37℃for 1 hour, then 10. Mu.l of Golgi Stop working solution (2. Mu.l of Golgi-Stop in 3mL of R10 medium) was added to each well, and the mixture was returned to the incubator for 2.5 hours. After each well 5. Mu.l of CD3-ACP and 5. Mu. lCD8-FITC antibodies were added and incubated for 0.5 hours, the samples were washed with FACS buffer and fluorescence was detected by flow cytometry.

2.11 CFSE-tagged T cell proliferation assay

The resting T cells of CD4 were collected by centrifugation and washed with PBS and suspended at a concentration of 1X 10 in PBS ⁷ /ml.1ml of the cell suspension was added to 120. Mu.l of CFSE solution (25 mmol/L CFSE), gently mixed and left at room temperature for 3.5 minutes. The labeling was stopped with 5% FBS (PBS solution of FBS), followed by two washes with stop buffer, and the labeled CD4 cells were cultured overnight in R10 medium containing 10IU/mL IL-2. CFSE-labeled T cells were electrotransformed into CAR RNAs the next day. T cells were cultured at 37℃for 2 to 4 hours after electrotransformation, collected by centrifugation, and after washing, the T cells were suspended in R10 medium (containing 10IU/mL of IL-2). After tumor cell irradiation, the cells were suspended in R10 medium at 1X 10 ⁶ /mL. CD 4T cells were mixed with tumor cells at a ratio of 1:1 (or 2:1) (5X 10) ⁵ 5X 10 of CD 4T cells ⁵ Individual tumor cells) were cultured in 48-well plates with a final volume of 1ml. T cells were counted every 2 days from day 3 and fresh medium was replenished. CFSE expression was flow-detected on day 5.

Example 3

1. Construction and functional detection of GPC3 CAR

3.1.1 Construction of GPC3 CAR and high expression on T cell surface

According to the antibody sequence of GPC3 that has been published in patent (US 2010/02483559 A1), 3 humanized murine antibody VerX, verH, ver,1 human murine chimeric antibody GC33 and 1 murine antibody M11F1 were selected, and 5 GPC3 CAR RNA vectors (a of fig. 1) and 5 lentiviral vectors (B of fig. 1) were each constructed, respectively. All GPC3 CARs were constructed on the second generation CAR structure of scfv.bbz, namely: the single chains of the light and heavy chains of the antibody are linked into single chain variable regions (single chain fragment of variable region, scFv) by hinge regions (linker) having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 34); the scFV is then linked to the intracellular domain by the transmembrane region (transmembrane region) of CD 8; the transmembrane region is linked to a costimulatory signal region within the cell (the intracellular region of the 4-1BB costimulatory molecule) and is terminally linked to the CD3ζ subunit.

GPC3 CAR RNA vectors were transcribed into mRNA in vitro, T cells were electrotransformed, and expression of the CAR was detected by flow cytometry after one day. The assay showed that GPC3 CAR expressed above 88.8% on the T cell surface, with NO electrotransport T cells (NO EP) as negative control (C of fig. 1).

Lentiviral vectors were used to transduce CD3/CD28 bead stimulated T cells after virus packaging. Flow cytometry examined lentiviral transduction for expression of T cell surface CARs on day ten, and staining results showed that GPC3 CAR transduction efficiency was between 69.7% -83.6% with NO transduced T cells (NO TD) as negative control (D of fig. 1).

5 GPC3 CARs were highly expressed on the surface of T cells by both electrotransduction and lentiviral transduction methods.

3.1.2 Function detection of GPC3 CARs

1) Verh.bbz and verx.bbz CAR-T cells specifically recognize GPC 3-positive tumors, promoting CD107a expression

Flow cytometry detection showed that HepG2 hepatoma cells were GPC 3-positive tumor cells, and a549 lung cancer cells were GPC 3-negative tumor cells (right panel a of fig. 2). 5 GPC3 CAR mRNA were electroporated into T cells, respectively, and cultured overnight in an incubator at 37℃and then co-cultured with HepG2 tumor cells (GPC 3+), A549 tumor cells (GPC 3-). CD107a staining results show that VerH.BBZ, verX.BBZ CAR-T cells can highly react with HepG2 tumor cells, and the ratio of CD107a positive CD4/CD 8T cells is 27.6%/47.1%, 29.1%/34.2%, respectively, and VerH.BBZ and VerX.BBZ CAR-T cells do not recognize A549 tumor cells; ver.BBZ and GC33.BBZ CAR-T cells can recognize HepG2 tumor cells, the ratio of CD107a positive CD4/CD 8T cells is 11.4%/23.8% and 32.8%/40.5%, respectively, and simultaneously, the ratio of CD107a positive CD4/CD 8T cells is 4.31%/18.3% and 8.07%/19.9% respectively; m11f1.bbz CAR can be expressed on T cell surfaces but cannot recognize HepG2 and a549 tumor cells (a left panel of fig. 2).

2) VerH.BBZ and VerX.BBZ CAR-T cells secrete large amounts of IL-2 and IFN-gamma after co-cultivation with GPC3 positive tumors

After 5 kinds of GPC3 CAR mRNA were electroporated into T cells, respectively, and cultured overnight in an incubator at 37℃and then co-cultured with HepG2 and A549 tumor cells, respectively, at a ratio of 1:1, the content of IL-2 and IFN-r in the supernatant was examined by ELISA (FIG. B). Co-culture of VerH.BBZ, verX.BBZ CAR-T cells with HepG2 tumor cells all produced large amounts of cytokines IL-2 and IFN-r, which secreted more cytokines than the two. Ver.BBZ, GC33.BBZ CAR-T cells secrete 3-4 times less cytokines than VerX.BBZ CAR-T cells. M11f1.bbz CAR-T cells were co-cultured with HepG2 cells, no cytokine secretion was detected in the supernatant. In contrast to the NO EP negative control group, all GPC3 CAR-T cells were co-cultured with a549 cells with NO or only background levels of secretion of IL-2 and IFN-r.

3) The killing test proves that the VerH.BBZ and VerX.BBZ CAR-T cells have stronger capability of killing tumor cells

The lentivirus encoding CBG (click beetle green) transduces HepG2 tumor cells, and the CBG positive HepG2 tumor cells (HepG 2-CBG) are obtained by flow separation and serve as tumor cells of killing experiments. After electrotransformation of T cells with 5 GPC3 CAR mrnas, respectively, the cells were co-cultured with HepG2-CBG for 16 hours at a ratio of effector cells to tumor cells (E: T ratio) from high to low (10:1, 5:1, 2.5:1, 1.25:1, 0.6:1, 0.3:1, 0.15:1, 0.075:1, 0.04:1, 0.02:1, double dilution), respectively. The killing test results show that: the killing capacities of the VerX.BBZ and the VerH.BBZ CAR-T cells are similar, the HepG2 tumor cells can be killed efficiently at a high E:T ratio, the killing function is reduced along with the reduction of the E:T ratio, and the killing rate of the tumor cells is close to 50% at the E:T ratio of 0.6:1; the killing function of ver.BBZ and GC33.BBZ CAR-T cells is relatively weak, the cracking ratio of tumor cells is lower than 80% when the E:T ratio is 10:1, the killing function is reduced along with the reduction of the E:T ratio, the cracking ratio of tumor cells is lower than 40% when the E:T ratio is 0.6:1, and the M11F1.BBZ CAR-T cells are similar to NO EP negative control groups and cannot kill tumor cells (C in figure 2).

4) VerH.BBZ and VerX.BBZ CAR-T effectively kill other 4 GPC3 positive hepatoma cells

To verify the specific tumor cell killing capacity of verx.bbz and verh.bbz CAR-T cells, four liver cancer tumor cell lines C3A, PLC/PRF/5, SNU475, SNU387 cells positive for GPC3 expression were selected for flow-through assays (D, right panel of fig. 2) co-cultured with verx.bbz and verh.bbz CAR-T, respectively (a 549 as negative control). The results show that the T cells electroporated with verx.bbz and verh.bbz CAR mRNA were co-cultured with 5 GPC 3-positive hepatoma cells, and that both verx.bbz and verh.bbz CAR-T cells CD137 were highly expressed in flow assays (D, left panel of fig. 2), demonstrating high antitumor activity.

2. Expression of GPC3 in Normal tissue cells and GPC3 CAR safety study

3.2.1. Normal tissue primary cell physiological level low expression GPC3 mRNA

RT-PCR detects the expression of GPC3 from 14 normal tissues such as lung, heart, nerve, kidney, skin and the like, hepG2 and A549 are respectively positive control and negative control, and GAPDH is an internal control gene.

Cells that were negative for the RT-PCR result were: hpmc (Human Pulmonary Microvascular Endothelial Cells, human lung microvascular endothelial cells), HSAEpC (Human Small Airway Epithelial Cells, human small airway endothelial cells), HPAEC (Human Pulmonary Artery Endothelial Cells ), HAoSMC (Human Aortic Smooth Muscle Cells, human aortic smooth muscle cells), HREpC (Human Renal Epithelial Cells, human kidney epithelial cells), HOB (human Osteoblasts ), hMSC-BM (Human Mesenchymal Stem Cells from Bone Marrow, human bone marrow mesenchymal stem cells), HACAT (Primary Epidermal Keratinocytes ), NHEM (Primary Normal Human Epidermal Keratinocytes, primary normal human epidermal keratinocytes); cells that were weakly positive as a result of RT-PCR were: HRCEpC (Human Renal Cortical Epithelial Cells, human renal cortex epithelial cells), hN2 (Primary human Neuron, human primary neurons), hpsmc (Human Pulmonary Artery Smooth Muscle Cells ); cells positive for RT-PCR were: hNP1 (human Neuronal progenitors, human neuronal progenitor), HCM (human cardiac myocytes, human cardiomyocytes); the result of RT-PCR of HepG2 liver cancer cell was strongly positive (A of FIG. 3).

The quantitative PCR results showed that: HCM (human cardiac muscle cell) GPC3 was expressed 22 times that of a549 cells, HRCEpC (human renal cortex epithelial cells), hN2 (human primary neurons), hNP1 (human neuronal progenitor cells) GPC3 was expressed 10-15 times that of a549 cells, hpsmc (human pulmonary artery smooth muscle cells) GPC3 was expressed 6 times that of a549 cells, and the remaining 9 normal tissue primary cells GPC3 was expressed less than 5 times that of a549 cells (B of fig. 3). The expression level of GPC3 of HepG2 hepatoma cells was 1410 times that of a549 cells (E of fig. 3). The results of RT-PCR and quantitative PCR show that human heart, kidney, lung and nerve tissue cells have low expression of GPC 3.

3.2.2 A549 cell electrotransformation different doses of GPC3 mRNA mimic cells expressing different levels of GPC3

RT-PCR and quantitative PCR show that GPC3 has low expression in some important tissues and organs, so that the safety of detecting GPC3CAR is necessary to prevent the toxic and side effects of CAR-T cells caused by off-target. Normal tissue primary cell material is difficult to obtain and complex to culture in vitro, so we constructed RNA in vitro transcription vectors of GPC3 to mimic normal cells expressing different levels of GPC3 by electrotransformation of GPC3 negative tumor cell a549 with different doses of GPC3 mRNA.

A549 tumor cells were electrotransferred to 10 μg, 3 μg, 1 μg, 0.3 μg, 0.1 μg, 0.03 μg and 0.01 μg of GPC3 mRNA, and after 16h incubation, the flow cytometer detected GPC3 expression. As shown in FIG. 4A, GPC3 expression correlated positively with the dose of mRNA for the electrotransformation. The electrotransport 10. Mu.g of GPC3 mRNA expressed highest, and the dose of mRNA was reduced, so was the expression of GPC3, and 0.1. Mu.g of mRNA was the lowest dose of mRNA detectable by flow cytometry.

A549 tumor cells were electrotransformed with different doses of GPC3 mRNA, RNA was extracted after 16h of culture, and the content of GPC3 RNA in electrotransformed a549 cells was detected by RT-PCR and quantitative PCR, respectively. As shown in the B gel electrophoresis results of FIG. 4, the dose of the electroporated GPC3 mRNA correlated positively with the intensity of the PCR amplified bands. As the dose of the electrotransport GPC3 mRNA was reduced, the intensity of the amplified band was reduced. The lowest dose at which GPC3 electrotransformation can be detected by RT-PCR is 0.01. Mu.g mRNA. HepG2 and Caco2 (human large intestine epithelial adenocarcinoma) are GPC3 positive tumors, and expression of GPC3 by HepG2 tumor cells is higher than that by Caco-2 tumor cells. GAPDH is an internal control gene (B of fig. 4).

As shown in FIG. 4C, the relative expression amount of GPC3 detected in the A549 cells of electrotransport GPC3 compared with that of non-electrotransport A549 was linearly dependent on the dose of electrotransport GPC3 mRNA. The relative expression amounts of GPC3 in A549 cells electrotransport 10. Mu.g, 3. Mu.g, 1. Mu.g, 0.3. Mu.g, 0.1. Mu.g, 0.03. Mu.g, and 0.01. Mu.g of GPC3 mRNA were 33018, 12139.7, 4505, 1195, and 82.5 times that of A549, in this order. The relative expression amount of GPC3 in A549 detected at different times after electrotransformation was decreased with the lapse of time, and the relative expression amount of GPC3 in A549 cells detected at 10. Mu.g of GPC3 mRNA for 4 hours, 24 hours, 48 hours was 39963.8 times, 11943 times and 1595 times in this order. The relative expression level of GPC3 in HepG2 tumor cells was 1410 times that of A549, and was between 1. Mu.g and 0.3. Mu.g of GPC3 mRNA for A549 electrotransport. GPC3 expressed the highest amount of human myocardial cell (HCM) GPC3 expressed was 22 times that of A549 cells, 0.03. Mu.g GPC3 mRNA was electrotransferred to less than A549 cells, and approximately 0.01. Mu.g GPC3 mRNA was electrotransferred (C of FIG. 4).

3.2.3 GPC3CAR security detection

1) CD107a results showed that both VerX.BBZ and VerH.BBZ CARs were safe GPC3 CARs

GPC3 CAR-T cells were co-cultured with A549 cells expressing different levels of GPC3 after electrotransformation, and sensitivity and safety of GPC3CAR were examined. Bz can effectively recognize HepG2 and electrotransport high dose GPC3mRNA a549 cells; co-culturing with A549 cells electrotransport 10 μg, 3 μg, 1 μg GPC3mRNA, with VerX.BBZ CAR-T cell CD107a values of 73.2%, 73.3%, 71.5%; continuing to decrease the GPC3mRNA dose, the CD107a value of verx.bbz CAR-T cells decreased rapidly, with the lowest dose of verx.bbz CAR-T cells recognizing GPC3 being 0.03 μg (upper panel a of fig. 5). Verh.bbz CAR-T cells can efficiently recognize HepG2 tumor cells and electrotransduce a549 cells of high dose GPC3mRNA, and are highly activated; co-culture with electrotransport 10. Mu.g, 3. Mu.g GPC3mRNA A549 cells, verX.BBZ CAR-T cell CD107a values of 66.6%,62.8%; continuing to decrease GPC3 dose, the CD107a value of VerH.BBZ CAR-T cells was rapidly decreased, and co-cultured with A549 cells electrotransport of 1 μg, 0.3 μg, 0.1 μg GPC3mRNA, with values of 45.5%, 26.6%, 12.5% for VerH.BBZ CAR-T cells CD107 a; the lowest dose of VerH.BBZ CAR-T cells recognizing GPC3 was 0.1 μg (upper panel of FIG. 5). Ver.bbz, gc33.bbz CAR-T cells can recognize HepG2 tumor cells and electrotransport high dose GPC3mRNA a549 cells; both ver.bbz CAR-T cells and gc33.bbz CAR-T cells non-specifically recognized non-electrotransformed a549 cells with CD107a values of 15.3% and 10.5%, respectively (upper panel of fig. 5). The CD107a experimental results are summarized in a rectangular chart (lower panel of fig. 5 a).

2) ELISA results demonstrated the safety of VerX.BBZ and VerH.BBZ CARs

GPC3 CAR-T cells were co-cultured overnight with A549 cells electrotransformed with different GPC3 mRNAs, and cytokines were detected in the supernatant by ELISA (FIG. 5B). VerX.BBZ CAR-T cells highly react with HepG2 and A549 cells electrotransport of 10 μg, 3 μg, 1 μg GPC3 mRNA, secreting large amounts of cytokines; a549 cells electrotransduce 3 μg, 1 μg GPC3 mRNA, compared to electrotransduce 10 μg, stimulated verx.bbz CAR-T to produce more IL-2 and IFN- γ, with verx.bbz CAR-T cells exhibiting some of the properties of high affinity CAR-T; co-culture of VerX.BBZ CAR-T cells with A549 cells electrotransport of less than 0.1. Mu.g GPC3 mRNA did not detect secretion of IL-2 and IFN-gamma. VerH.BBZ CAR-T cells are highly reactive with HepG2 tumor cells and A549 cells electrotransport of 10 μg, 3 μg GPC3 mRNA, secreting large amounts of cytokines; the cytokine detected in the supernatant was proportional to the dose of GPC3 for a549 cell electrotransport; co-culture of VerH.BBZ CAR-T cells with A549 cells electrotransport of less than 0.3 μg GPC3 mRNA did not detect secretion of IL-2 and IFN- γ. Ver. bbz, gc33.bbz CAR-T cells were co-cultured with HepG2 and electrotransduce various doses of GPC3 mRNA a549 cells, producing no or only very low doses of cytokines (B of fig. 5).

3) CFSE proliferation experiments show that VerH.BBZ is a safer GPC3 CAR

Whether CAR-T cells proliferate while killing tumor cells is an important indicator to detect CAR-T cell function. A549 cells electrotransformed with different doses of GPC3mRNA were co-cultured with CFSE-labeled CAR-T cells after irradiation, and the change in CAR-T cell surface CFSE was detected by flow cytometry on day 5. The CFSE intensity of the CAR-T cell surface markers decreases with proliferation of the cells, and the proportion of CFSE decrease is the proportion of proliferating cells. A549 cells electrotransport transformed by 10 mug and 1 mug of GPC3mRNA can stimulate the quick proliferation of VerX.BBZ and VerH.BBZ CAR-T cells, and the proliferation proportion of CATR-T cells is higher than 65%; decreasing the dose of electrotransport GPC3, the proportion of CAR-T cell proliferation decreases; a549 cells electrotransport 0.1 μg of GPC3mRNA activated verx.bbz, verh.bbz CAR-T cell proliferation ratios of 60.8%, 36.3%, respectively; further reducing the dose of GPC3 to 0.01 μg, the proportion of VerX.BBZ CAR-T cell proliferation was 27.8%, while VerH.BBZ CAR-T cells were not activated and proliferated. Co-culture of Ver.BBZ, GC33.BBZ CAR-T cells with A549 cells electrotransport of 10 μg GPC3mRNA had proliferation of 47.5% and 41.4%; the dose of GPC3 was reduced to 1. Mu.g, and Ver.BBZ, GC33.BBZ CAR-T cells were not activated and proliferated.

In summary, the lowest doses of VerX.BBZ CAR-T cell recognition A549 electrotransport GPC3 detected by CD107a, ELISA and CFSE proliferation experiments were 0.03 μg, 0.3 μg and 0.01 μg, respectively, and the lowest doses of VerH.BBZ CAR-T cell recognition were 0.1 μg, 1 μg and 0.1 μg, respectively. The control of the expression level of human cardiac myocytes GPC3 in normal tissue (0.03. Mu.g > HCM > 0.01. Mu.g) demonstrated that VerH.BBZ is a safe GPC3 CAR.

3. VerH.BBZ CAR-T cells can effectively inhibit the growth of NSG mouse HepG2 tumor cells

3.3.1 establishing an NSG mouse HepG2 liver cancer subcutaneous tumor-bearing animal model

Establishing a tumor animal model is an important means for developing tumor treatment related researches. At present, a transplantation type liver cancer model is commonly used in a laboratory, and can be divided into an in-situ type and an ectopic type according to the position of a transplantation tumor. Ectopic type is mostly formed by injecting a certain amount of cancer cells into the back of an experimental animal in a subcutaneous mode, the success rate of inoculation is high, the change of tumor volume is easy to measure, and local intervention is easy to administer. The experiment adopts an NSG mouse (NOD scid gamma mouse) HepG2 liver cancer cell subcutaneous tumor-bearing animal model. NOD/SCID mice are defective in T cell, B cell and NK cell functionsIs considered to be the most ideal recipient animal for transplantation, because it has low immunity and is easily subjected to xenograft. Culturing luciferase gene marked HepG2 liver cancer cell (HepG 2-CBG) under standard condition, digesting adherent cell before injection, centrifuging, collecting every 1×10 ⁶ The cells were suspended in 0.1mL of PBS, and NSG mice (males, 6-8 weeks old, and weighing 20 g-25 g) were subcutaneously injected on the right back. Subcutaneous tumor appearance was seen around one week after inoculation, and fluorescence biopsy (a of fig. 6) and two tumor size measurements (B of fig. 6) were performed weekly from the first week.

The results show 1X 10 subcutaneous injections at the back ⁶ All 5 mice with HepG2 tumor cells were successfully inoculated, and the tumor size was 350mm about 10 days after inoculation ³ Two weeks after inoculation, the tumor growth entered the fast logarithmic growth phase, and three weeks after inoculation the tumor size was 700mm ³ . According to the tumor growth curve, the CAR-T cell infusion time was selected on day 10 after tumor inoculation.

3.3.2 The dose of verh.bbz CAR-T cell infusion was significantly correlated with tumor elimination in mice

Verh.bbz lentivirus transduced CD3/CD28 magnetic beads stimulated T cells of the first day (CD 4: cd8=1:1), and transduced T cells were cultured under standard conditions. The tenth day flow cytometer detected that CAR-T cell transduction efficiency was 72% and that the untransduced cells were negative controls (C of fig. 6). Cells were collected by centrifugation on day ten, frozen in frozen liquid and stored in liquid nitrogen. The experiments were performed in three experimental groups and one control group, two mice per group. Three experimental groups were each injected with 1×10 mice ⁷ 、5×10 ⁶ 、1×10 ⁶ VerH.BBZ CAR positive T cells, control mice were injected with 1X 10 ⁷ Non-transduced T cells.

Day 1 mice were back vaccinated with 1X 10 ⁶ HepG2-CBG tumor cells, T cells were intravenously injected from the tail of tumor-bearing mice according to groups on day 10, and tumor sizes were detected by fluorescence imaging on

days

6, 13, 20, and 27 of inoculation (D of FIG. 5). The fluorescence imaging detection result shows that: on day 13, the tumors of the three CAR-T treated mice did not significantly change from that before T cell infusion, whereas the tumors of the control mice significantly increased; first, the20 days, 1×10 ⁷ The tumor of two mice in CAR-T group is obviously reduced by 5 multiplied by 10 ⁶ The CAR-T group had one mouse tumor disappeared and one mouse tumor had a slight increase of 1×10 compared to day 13 ⁶ The tumor of the two mice in the CAR-T group is slightly reduced compared with the tumor of the two mice in the 13 th day, and the tumor of the two mice in the control group is obviously increased; day 27, 1×10 ⁷ Tumor complete disappearance, 5×10, in two mice of CAR-T treated group ⁶ The mice with the tumor disappeared in the CAR-T treatment group had no tumor, and the tumor of the other mice was significantly reduced by 1×10 compared with day 13 ⁶ The tumors of both mice in the CAR-T treatment group significantly increased compared to day 20, indicating that CAR-T cells were unable to continue to control the tumor and that the tumors of the control group mice increased rapidly. The experimental results show that: the VerH.BBZ CAR-T cells can effectively kill tumor cells in mice, the elimination of tumors in mice is obviously related to the dose of CAR-T cell infusion, the high-dose CAR-T cells can completely inhibit tumors, the low-dose CAR-T cells can only partially control tumor growth, so that the CAR-T can treat solid tumors, and the amount of the reinfused CAR-T cells has an important influence on the treatment effect. From the perspective analysis of animal model design, although low dose CAR-T cells can only control tumor growth to a certain extent, space for further improving therapeutic effect is reserved for studies such as optimizing CAR design, CAR and switch molecule combination, CAR-T and antibody combination, etc.

Example 4

1. Experimental materials

4.1 cell lines: hepG2, caco-2, A549-ESO and MDA231, etc. see example 1.2

4.2 main reagents: antibodies are described in example 1, 1.3.1

4.3 Main instrumentation is described in example 1 at 1.4

4.4 converting receptor sequences

4.4.1PD1-CD28

Nucleotide sequence (SEQ ID NO: 17)

AtgcagatcccacaggcgccctggccagtcgtctgggcggtgctacaactgggctggcggccaggatggttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcacccaggccagccggccagttccaaaccctggtgttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccTAA

Amino acid sequence (SEQ ID NO: 18)

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

4.4.2 TGFbR1/IL12Rb1

Nucleotide sequence (SEQ ID NO: 19)

AtggaggcggcggtcgctgctccgcgtccccggctgctcctcctcgtgctggcggcggcggcggcggcggcggcggcgctgctcccgggggcgacggcgttacagtgtttctgccacctctgtacaaaagacaattttacttgtgtgacagatgggctctgctttgtctctgtcacagagaccacagacaaagttatacacaacagcatgtgtatagctgaaattgacttaattcctcgagataggccgtttgtatgtgcaccctcttcaaaaactgggtctgtgactacaacatattgctgcaatcaggaccattgcaataaaatagaacttccaactactgtaaagtcatcacctggccttggtcctgtggaactggcagctgtcattgctggaccagtgtgcttcgtctgcatctcactcatgttgatggtctatatcagggccgcacggcacctgtgcccgccgctgcccacaccctgtgccagctccgccattgagttccctggagggaaggagacttggcagtggatcaacccagtggacttccaggaagaggcatccctgcaggaggccctggtggtagagatgtcctgggacaaaggcgagaggactgagcctctcgagaagacagagctacctgagggtgcccctgagctggccctggatacagagttgtccttggaggatggagacaggtgcaaggccaagatgTAA

Amino acid sequence (SEQ ID NO: 20)

MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCFVCISLMLMVYIRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM

4.4.3 TGFbR2/IL12Rb2

Nucleotide sequence (SEQ ID NO: 21)

AtgggtcgggggctgctcaggggcctgtggccgctgcacatcgtcctgtggacgcgtatcgccagcacgatcccaccgcacgttcagaagtcggatgtggaaatggaggcccagaaagatgaaatcatctgccccagctgtaataggactgcccatccactgagacatattaataacgacatgatagtcactgacaacaacggtgcagtcaagtttccacaactgtgtaaattttgtgatgtgagattttccacctgtgacaaccagaaatcctgcatgagcaactgcagcatcacctccatctgtgagaagccacaggaagtctgtgtggctgtatggagaaagaatgacgagaacataacactagagacagtttgccatgaccccaagctcccctaccatgactttattctggaagatgctgcttctccaaagtgcattatgaaggaaaaaaaaaagcctggtgagactttcttcatgtgttcctgtagctctgatgagtgcaatgacaacatcatcttctcagaagaatataacaccagcaatcctgacttgttgctagtcatatttcaagtgacaggcatcagcctcctgccaccactgggagttgccatatctgtcatcatcatcttctaccagcaaaaggtgtttgttctcctagcagccctcagacctcagtggtgtagcagagaaattccagatccagcaaatagcacttgcgctaagaaatatcccattgcagaggagaagacacagctgcccttggacaggctcctgatagactggcccacgcctgaagatcctgaaccgctggtcatcagtgaagtccttcatcaagtgaccccagttttcagacatcccccctgctccaactggccacaaagggaaaaaggaatccaaggtcatcaggcctctgagaaagacatgatgcacagtgcctcaagcccaccacctccaagagctctccaagctgagagcagacaactggtggatctgtacaaggtgctggagagcaggggctccgacccaaagccagaaaacccagcctgtccctggacggtgctcccagcaggtgaccttcccacccatgatggctacttaccctccaacatagatgacctcccctcacatgaggcacctctcgctgactctctggaagaactggagcctcagcacatctccctttctgttttcccctcaagttctcttcacccactcaccttctcctgtggtgataagctgactctggatcagttaaagatgaggtgtgactccctcatgctctgaTAA

Amino acid sequence (SEQ ID NO: 22)

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML

2. Experimental method

4.5.1 Total RNA extraction, RNA reverse transcription and RT-PCR

The RNA extraction kit (RNeasy Mini Isolation Kit, QIAGEN, cat# 74104) extracts total RNA from cells and then reverse transcribes it into cDNA. The cDNA template, primer (or control gene primer) and reaction solution are mixed in proportion. PCR instrument amplification procedure: 94 ℃ for 5 minutes; 94℃for 20 seconds, 58℃for 20 seconds, 70℃for 20 seconds, 35 cycles; 70 ℃ for 7 minutes and 4 ℃ in infinity. After PCR amplification, the PCR amplified fragments were detected by gel electrophoresis. The reagents and primers used are shown in experimental materials (tables 1.2-1.5).

4.5.2 Construction of TGFbRI/IL12Rb1 and TGFbR2/IL12Rb2 plasmids

Construction of pD-A. TGFbR1/IL12Rb1 and pD-A. TGFbR2/IL12Rb2 (RNA vector) plasmids: according to cDNA sequences of TGFbR1 and IL12Rb1, respectively designing and synthesizing two DNA fragments for encoding an extracellular segment and a transmembrane region of TGFbR1 and an intracellular region of IL12Rb1, assembling the two DNA fragments into TGFbR1/IL12Rb1 by a PCR method, inserting into a pD-A plasmid T7 promoter, and constructing a pD-A.TGFbR1/IL12Rb1 plasmid; the pD-A. TGFbR2/IL12Rb2 plasmid was constructed in the same way.

Construction of MSGV.TGFbR/IL12R plasmid: introducing a T2A sequence (EGRGSLLTCGDVEENPGP) (SEQ ID NO: 34) between TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2 by utilizing a PCR method, connecting TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2 to form TGFbR/IL12R, inserting the TGFbR/IL12R into an MSGV vector through double digestion, transforming competent cells, picking up a monoclonal, extracting plasmids, and carrying out digestion and sequencing to verify that the vector is successfully constructed.

4.5.3 impedance-based real-time cell analysis technique (xcelligence cytotoxicity assay)

The real-time cell analysis technology based on impedance can dynamically detect the in-vitro killing function of the CAR-T cells on the adherent tumor cells under different E:T ratios. The first day E-plate96 was prepared, 50. Mu.l of medium was added to each well and placed on the detection platform for background detection of E-plate 96. Detection of Normal E-plate96 50. Mu.l (1X 10) was added per well ⁴ Individual cells/wells) had been digested into adherent cells in suspension, and placed on the assay platform for overnight incubation at 37 ℃. The next day, T cells were washed and suspended in R10 medium to adjust the cell concentration to 2X 10 ⁶ Each thinThe cells/mL were diluted 2-fold in another 96-well U-shaped plate, and the concentration of T cells after dilution was 1X 10 in order ⁶ /ml、0.5×10 ⁶ /ml、0.25×10 ⁶ /ml、0.125×10 ⁶ /ml、0.06×10 ⁶ /ml、0.03×10 ⁶ Per ml, two replicates per concentration. T cells diluted in a 100. Mu.l-fold ratio were transferred to corresponding positions with E-plate96, and after mixing, T cells and tumor cells were mixed in a T ratio of 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, 0.3:1, and 100. Mu. l R10 in order as negative controls. E-plate96 is placed on the detection platform. The instrument detects the resistance change of the E-plate96 in real time, monitors the growth condition of the adherent cells, and the built-in software of the instrument can directly draw the killing curve of the T cells to the tumor cells.

4.5.4 Luminex assay

T cells were co-cultured with tumor cells in 96-well plates for 16 hours and supernatants were harvested for detection. Human 30 cytokines magnetic plates (magnetic 30-Plex panel, life Technologies LHC 6003M) quantitated the following cytokines: IL-1RA, FGF, MCP-1, G-CSF, IFN-gamma, IL-12, IL-13, IL-7, GM-CSF, TNF-a, IL-1b, IL-2, IL-4, IL-5, IL-6, IFN-alpha, IL-15, IL-10, MIP-1a, IL-17, IL-8, EGF, HGF, VEGF, MIG, RANTES; chemokines include: MIP-1b, IP-10 and IL-2R. Samples were measured on a FlexMAP 3D instrument (Luminex, austin, texas) and analyzed using xPONENT software (Luminex).

Example 5

1. Immunosuppressive molecules highly expressed by tumor cells and their effect on T cell function

5.1.1 immunosuppressive molecules highly expressed by tumor cells

Tumor cells utilize the regulatory mechanisms of the immune system itself to suppress immune cell function by up-regulating expression of some immunosuppressive molecules or secretion of some immunosuppressive molecules, thereby evading immune surveillance. Some tumor cells constitutively express PD-L1, or up-regulate expression of PD-L1 upon stimulation by inflammatory signaling molecules, especially T cell-derived IFN- γ stimulated tumors highly express PD-L1. TGF-beta secreted by tumor cells can induce the formation of Treg, reduce the killing function of T cells, reduce the secretion of cytokines such as IL-12, IFN-gamma and the like, and inhibit the proliferation of T cells.

A549 tumor cells and HepG2 tumor cells were cultured in media containing IFN- γ at different concentrations, and after overnight culture, PD-L1 expression was flow-detected. As shown in FIG. 7A, when IFN-gamma cytokines were not contained in the medium, both A549 and HepG2 tumor cells expressed PD-L1 in low amounts, and the expression increased with increasing IFN-gamma concentration in the medium.

Flow cytometry detected expression of TGF- β in HepG2 tumor cells, and staining results showed that TGF- β staining in HepG2 tumor cells was positive (B of fig. 7).

5.1.2 TGF-beta strongly inhibits proliferation of CAR-T cells

CFSE-labeled resting CD 4T cells were cultured overnight, and the next day were electroporated with 10 μg of verx.bbz or verh.bbz mRNA, and the electroporated T cells were cultured with irradiated HepG2 tumor cells at a ratio of 2:1 in R10 medium containing varying concentrations of TGF- β1. On day 5 of culture, changes in the CFSE fluorescence intensity of the CAR-T cells were detected by flow cytometry. CAR-T cells with reduced CFSE intensity were proliferating cells, and each generation of cells showed a different peak on the histogram (C of fig. 7). Proliferation ratios of verx.bbz and verh.bbz CAR-T cells in TGF- β free medium were 87.4% and 80.6% (right panel); the proliferation ratio of VerX.BBZ and VerH.BBZ CAR-T cells was reduced to 56.6% and 58.1% in a medium containing 2ng/ml TGF- β1 (middle panel); at an increase of 10ng/ml of TGF-. Beta.1 concentration in the medium, the proliferation ratio of VerX.BBZ and VerH.BBZ CAR-T cells was decreased to 21.7% and 23.0% (left panel). Cells co-cultured with HepG2 tumor cells did not proliferate without electrotransferred T cells (NO EP). The decrease in proliferation of verx.bbz and verh.bbz CAR-T cells is linear with an increase in TGF- β1 concentration in the medium, indicating that TGF- β1 strongly inhibits proliferation of CAR-T cells.

2. Construction of TGFbR/IL12R converting receptor and function detection

5.2.1 construction of TGFbR/IL12R converting receptor

TGF-beta receptors (A of FIG. 8) and IL-12 receptors are hetero-tetramers, so this study constructed two switching receptors, TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2.TGFbR1 retains an extracellular domain and a transmembrane domain, and the intracellular domain is replaced by an intracellular domain of IL12Rb1, constituting TGFbR1/IL12Rb1; the same design constitutes the TGFbR2/IL12Rb2 receptor (B of FIG. 8).

The transforming receptor TGFbR1/IL12Rb1 molecules and TGFbR2/IL12Rb2 molecules were cloned into RNAIVT vectors, respectively. TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2 mRNA transcribed separately in vitro were mixed in the same amount and T cells were electrotransformed simultaneously. Electrotransformed T cells co-express TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2 switch receptors, collectively referred to as TGFbR/IL12R. Subsequently, the TGFbR1/IL12Rb1 molecule and the TGFbR2/IL12Rb2 molecule were cloned into an MSGV retroviral vector after being linked by T2A (T2A amino acid sequence: E G R G S L L T C G D V E E N P G P) (SEQ ID NO: 35). The prepared retrovirus expresses TGFbR1/IL12Rb1 and TGFbR2/IL12Rb2 simultaneously after T cell transduction.

As shown in a of fig. 8, TGF- β binds to TGF- β receptor, activating the TGF- β downstream Smad signaling pathway; TGFbR/IL12R converting receptor binds TGF-beta outside cell membrane, and can convert TGF-beta signal into IL-12 signal, activating JAK/STAT signal path.

5.2.2 Detection of TGFbR/IL12R switch receptor function

1) TGFBR/IL 12R-converting receptor can inhibit TGF-beta downstream signaling

TGF-. Beta.binds to its receptor, activating the downstream molecules Smad2 and Smad3 by phosphorylation, thereby forming Smad2/Smad3 heterodimers, which in turn recruits and phosphorylates Smad4 to form Smad2/Smad3/Smad4 heterotrimers, which enter the nucleus to initiate transcription of genes along with co-transcriptional regulators. Activation and transduction of TGF- β signals can thus be detected by staining with pSmad 2/3.

The TGF-beta truncated receptor dnTGFbR2 (dominant negative TGF-beta receptor 2) is used for coexpression with the CAR in clinical experiments, which prove that the TGF-beta truncated receptor dnTGFbR2 can effectively block the inhibition of TGF-beta and enhance the proliferation and killing function of the CAR-T cells, and the experiment uses the dnTGFbR2 as a control for detecting the function of TGFbR/IL 12R. T cells were electrotransformed with 5. Mu.g TGFbR1/IL12Rb 1+5. Mu.g TGFbR2/IL12R2 mRNA or 10. Mu.g dnTGFbR2 mRNA, respectively, and non-electrotransformed T cells (NO EP) were used as controls. After the electrotransformed T cells were cultured overnight, TGF-. Beta.1 was added to the medium for the next day to a final concentration of 10ng/ml, and further cultured at 37℃for 30 minutes, the expression of pSmad2 was detected by flow cytometry (FIG. 9A).

The flow results indicate that: the addition of TGF-beta 1 to the culture medium of NO EP T cells can induce the expression of pSmad2/3, and the addition of TGF-beta 1 does not induce the expression of pSmad 2/3; electrotransformation of dnTGFbR2 can reduce T cell pSmad2/3 expression; electrotransformation of TGFbR/IL12R can significantly reduce the expression of pSmad2/3 of T cells, and the expression of pSmad2/3 is the same as that of NO EP T cells without TGF-beta 1 in a culture medium.

2) TGFBR/IL12R transducer receptor can convert TGF-beta signal into IL-12 signal

IL-12, when bound to the receptor IL12Rb1/IL12Rb2, activates and phosphorylates STAT4, a downstream molecule, to form homodimers, and the phosphorylated STAT4 dimer enters the nucleus and initiates transcription of IFN-gamma and other genes. Thus, whether TGFBR/IL12R conversion receptor can convert TGF-beta signal to IL-12 signal can be achieved by detecting secretion of IFN-gamma.

NK cells were electrotransformed with 5. Mu.g TGFbR1/IL12Rb 1+5. Mu.g TGFbR2/IL12R2 mRNA, 10. Mu.g dnTGFbR2 mRNA, respectively, and non-electrotransformed NK cells (NO EP) were used as controls; the NK cells after electrotransformation were incubated for 2 hours in R10 or 10ng/ml TGF- β1r10 and then co-incubated with K562 cells in the ratio E: t=1:1 for 16 hours, and the supernatants were analyzed for IFN- γ content by ELISA (B of fig. 9). NK cells normally do not secrete IFN-gamma, so secretion of IFN-gamma is not detected when non-electrotransformed NK cells and electrotransformed NK cells of dnTGFbR2 are co-cultured with K562 tumor cells; NK cells electrotransduce TGFbR/IL12R mRNA and provide NK cell IL-12 signals, IFN-gamma expression is started, a large amount of IFN-gamma secretion is detected in the supernatant co-cultured with K562 tumor cells, and the secretion of NK cell IFN-gamma can be further increased by adding TGF-beta 1 into the culture medium.

pSmad2 staining experiments and NK cell IFN-gamma secretion experiments demonstrated that TGFbR/IL12R switch receptors can block reverse TGF-beta inhibition signaling while activating IL-12 signaling.

3) TGFbR/IL12R transducer receptor can relieve TGF-beta inhibition of CAR-T cell proliferation

CFSE-labeled resting CD 4T cells were electrotransformed with 5 μg verh.bbz, 5 μg verh.bbz+10 μg TGFbR/IL12R, 10 μg TGFbR/IL12R mRNA, respectively, and the electrotransformed T cells were cultured with irradiated HepG2 tumor cells in three media (R10, R10 contained 2ng/ml TGF- β1, R10 contained 10ng/ml TGF- β1) at a ratio of E: t=2:1, and CFSE expression was examined by flow cytometry on day 5.

Flow results show that TGF-beta 1 strongly inhibits proliferation of VerH.BBZ CAR-T cells, the proportion of cell proliferation of VerH.BBZ CAR-T cells in R10 medium is 73.4%, the cell proliferation is reduced to 47.4% in medium containing 2ng/ml TGF-beta 1, the cell proliferation is further reduced to 29.7% in medium containing 10ng/ml TGF-beta 1, and the inhibition of the TGF-beta 1 on cell proliferation is positively correlated with concentration. Whereas the proportion of cell proliferation of verh.bbz CAR-T cells co-expressing the TGFbR/IL12R switch receptor in R10 medium was 71.2%, the increase in cell proliferation in 2ng/ml TGF- β1 containing medium was 81.1%, the cell proliferation in 10ng/ml TGF- β1 medium was further increased to 85.7%, TGF- β1 did not inhibit proliferation of CAR-T cells co-expressing the switch receptor but rather promoted cell proliferation, the analysis was that TGF- β binding TGFbR/IL12R switch receptor switched the inhibition signal of TGF- β to the activation signal of IL-12, activating downstream effector molecules and thus stimulating division proliferation of CAR-T cells. T cells electrotransformed TGFbR/IL 12R-transformed receptor mRNA were co-cultured with HepG2 tumor cells without cell proliferation.

3. The conversion receptor significantly enhances the anti-tumor function of GPC3 CAR-T cells

5.3.1 Co-transduced T cells with lentiviruses encoding GPC3 CAR and retroviruses encoding a transduction receptor

TGFbR/IL12R can convert the inhibition signal of TGF-beta into the activation signal of IL-12, so that NK cells which do not secrete IFN-gamma secrete a large amount of IFN-gamma after electrotransformation of the transformation receptor, and CAR-T cells which express the transformation receptor after electrotransformation can not only resist the inhibition of TGF-beta on cell proliferation, but also enhance the proliferation capacity of the CAR-T cells. To further examine the effect of the transducible receptor on GPC3 CAR-T cell function, GPC3 CAR-T cells were intended to co-express the transducible receptor by viral transduction. Cloning into the viral vector of GPC3 CAR reduced lentiviral titers, affecting transduction efficiency, due to the near 2Kb of the switch receptor TGFBR/IL12R molecule. Thus, the molecule encoding the transduction receptor was cloned into a retroviral vector, and T cells were transduced in concert with a lentiviral vector encoding a CAR. Meanwhile, the TGFbR/IL2R conversion receptor is compared with the PD1-CD28 conversion receptor and dnTGFbR2, so that the difference of anti-tumor functions of CAR-T cells respectively expressing the three receptors in vitro and in a mouse animal model is analyzed.

T cells were stimulated with CD3/CD28 magnetic beads, lentivirally transduced on the first day, retrovirus transduced on the second and third days, expanded on day 10, and cells were collected and cryopreserved while flow cytometry detected CAR and switch receptor expression. The transduction efficiency of VerX.BBZ CAR-T cells was 73.0%, and the transduction efficiencies of VerX.BBZ CAR-T cells co-expressing the conversion receptors PD1-CD28, TGFbR/IL12R and dnTGFbR2 were slightly lower than 58.5%, 57.8% and 64.6%, respectively. The transduction efficiency of VerH.BBZ CAR-T cells was 73.7%, and the transduction efficiencies of VerH.BBZ CAR-T cells co-expressing the conversion receptors PD1-CD28, TGFbR/IL12R, dnTGFbR were slightly lower, 67.5%, 68.1%, 65%, respectively (A of FIG. 10). Transduction efficiencies of verx.bbz and verh.bbz CAR-T cells PD1-CD28 were 64.8% and 65%; transduction efficiencies of verx.bbz and verh.bbz CAR-T cell TGFbR/IL12R were 54.9% and 55.8%; transduction efficiencies of verx.bbz and verh.bbz CAR-T cells dnTGFbR2 were 58% and 57.3%.

CAR staining results show that lentiviruses and retroviruses can transduce T cells simultaneously, while transducing retroviruses slightly reduces the transduction efficiency of lentiviruses. When only lentivirus is transduced, the transduction efficiency of VerX.BBZ and VerH.BBZ CAR-T cells is 9% -15% and 5% -8% higher than that of CAR-T cells transduced together with retrovirus. In subsequent experiments of functional assays, the expression of CAR was adjusted to the same ratio for different groups of cells with non-transduced T cells.

5.3.2 Co-expression of the transforming receptor the ability of CAR-T cells to secrete cytokines is markedly enhanced

GPC3 CAR-T cells and CAR-T cells co-expressing the conversion receptor were co-cultured with tumor cells in a ratio of E: T=1:1, and the content of IL-2 and IFN- γ in the culture supernatant was examined by ELISA. The results showed that verx.bbz and verh.bbz CAR-T cells were co-cultured with HepG2 tumor cells that were highly expressed by GPC3, verh.bbz CAR secreted more IL-2 and IFN- γ, as was the case with previous GPC3 CAR mRNA electrotransfer T cells. Co-culture with HepG2-PD-L1 tumor cells, tumor-expressed PD-L1 reduced secretion of VerX.BBZ and VerH.BBZ CAR-T cells IL-2 and IFN-gamma.

The IL-2 generated by co-culturing VerX.BBZ and VerH.BBZ CAR-T cells which co-express PD1-CD28 conversion receptor and HepG2 tumor cells is higher than that of VerX.BBZ and VerH.BBZ CAR-T cells respectively; when the recombinant strain is co-cultured with HepG2-PD-L1 tumor cells, the secreted IL-2 is obviously higher than VerX.BBZ and VerH.BBZ CAR-T cells; since the expression of PD-L1 by HepG2-PD-L1 tumor cells is much higher than that of PD-L1 induced by HepG2, IL-2 produced by co-culturing verx.bbz and verh.bbz CAR-T cells co-expressing PD1-CD28 switch receptors with HepG2-PD-L1 tumor cells is significantly higher than IL-2 produced by co-culturing with HepG2 tumor cells.

The IFN-gamma generated by co-culturing VerX.BBZ and VerH.BBZ CAR-T cells co-expressing TGFbR/IL12R conversion receptor with HepG2 and HepG2-PD-L1 tumor cells is significantly higher than that of VerX.BBZ and VerH.BBZ CAR-T cells.

VerX.BBZ and VerH.BBZ CAR-T cells co-expressing the dnTGFbR2 receptor do not differ significantly from VerX.BBZ and VerH.BBZ CAR-T cells compared to cytokines IL-2 and IFN-gamma produced by co-culture of HepG2 or HepG2-PD-L1 tumor cells.

A549 and MDA231 tumor cells did not express GPC3, so no cytokine secretion was detected by co-culture with both CAR-T cells and CAR-T cells co-expressing the conversion receptor. Neither cytokine secretion was detected by co-culture of untransduced T cells with GPC3 positive or negative tumor cells.

5.3.3 conversion receptor enhancing GPC3 CAR-T cell in vitro killing function

The real-time cell analysis technology (RealTimeCellAnalysis, RTCA) based on impedance can dynamically detect the killing function of VerH.BBZ CAR-T cells and VerH.BBZ CAR-T cells which co-express a conversion receptor on HepG2 tumor cells under different E:T ratios. Compared with a killing experiment based on fluorescence, RTCA can dynamically monitor the long-term behavior of the CAR-T cells in real time, and more accurately predict the CAR-T cells in animal models and tumor patients Antitumor activity in humans.

Dynamic killer curve is time (in hours) on the abscissa, T cell co-culture with tumor cells is initiated at 0, and normalized cell index (no units) on the ordinate. Tumor dynamic killing experiments showed that none of the untransduced T cells inhibited tumor growth at 6 different E:T ratios (E of FIG. 11); verH.BBZ CAR-T cells can effectively control tumor growth at a high E:T ratio of 10:1, and the killing function of VerH.BBZ decreases with decreasing E:T ratio (A of FIG. 11). The killing curves of CAR-T cells co-expressing the conversion receptors PD1-CD28, dnTGFbR2 and TGFbR/IL12R are very similar at high E:T ratios, indicating very close killing functions; at E: t=10:1 (f in B, C, D of fig. 11), all three CAR-T cells co-expressing the switch receptor can kill tumor cells rapidly in a short time, and due to the rapid decrease in tumor cells, CAR-T cells lose sufficient tumor cell stimulation in a short time without massive expansion, so tumor cells recur after 5 hours of co-culture, but then CAR-T cells proliferate again in a massive amount to control tumor cells, and a dynamic killing process occurs between CAR-T cells and tumor cells that negates each other; the T ratio is reduced from 10:1 to 1.25:1, the killing function of the CAR-T cells of the coexpression conversion receptor is slightly reduced, the T ratio is further reduced, the killing function of the CAR-T cells of the coexpression conversion receptor is obviously reduced, but all three CAR-T cells can completely kill tumor cells after being cocultured with the tumor cells for about 35 hours; when E: t=0.3:1, CAR-T cells co-expressing the switch receptor TGFbR/IL12R exhibited stronger tumor killing activity than CAR-T cells expressing PD1-CD28 and dnTGFbR2 over a time frame of 10-20 hours (F of fig. 11).

4. Mechanism study of conversion receptor to enhance GPC3 CAR-T anti-tumor function

CAR-T cells co-expressing the receptor for translation, in particular CAR-T cells expressing the TGFbR/IL2R receptor for translation, show a stronger antitumor activity in the early stages of co-culture with tumor cells. To gain insight into the mechanism by which CAR-T cells expressing the transforming receptor effectively kill tumor cells in vitro, we used irradiated HepG2 tumor cells to stimulate T cells in vitro in two rounds and examined the proliferation, differentiation, cytokine secretion and changes in T cell transcript levels of T cells.

5.4.1 flow cytometry analysis of molecular markers on the surface of CAR-T cells

Verh.bbz CAR-T cells, CAR-T cells co-expressing the conversion receptor (PD 1-CD28, TGFbR/IL12R, dnTGFbR 2), and non-transduced T cells were co-cultured with irradiated HepG2 tumor cells in a 24 well plate at a ratio of E: t=2:1. The co-cultured cells were transferred to 12-well plates and 6-well plates on

days

3 and 5, respectively, and the corresponding volumes of fresh medium were replenished. T cells were collected and sorted on day 7 and a second round of stimulation was performed with the irradiated HepG2 at the ratio E: t=2:1. The following day the supernatants were collected and Luminex analyzed for the content of 30 cytokines in the cell culture supernatants. Flow cytometry detects the expression of CD62L, CD45RA, CD27, CD69 molecules in cultured cells. Meanwhile, after a part of cells are separated by CD45 magnetic beads, RNA is extracted and analyzed by RNA sequencing.

The detection result of the flow cytometry shows that the ratio of VerH.BBZ CAR-T cells to the central memory T cells (CD62L+CD45RA-) expressed by the VerH.BBZ CAR-T cells and the coexpression converting receptor PD1-CD28 and TGFbR/IL12R, dnTGFbR2 is 33.6%, 45.4%, 47.9% and 44.8% respectively; the proportion of all CAR-T cell central memory T cells expressing the conversion receptor was higher than verh.bbz CAR-T cells (p < 0.01). Whereas the ratio of effector memory T cells (CD 62L-CD45 RA-T) expressed in verh.bbz CAR-T cells and CAR-T cells co-expressing the transforming receptors PD1-CD28, TGFbR-IL12R, dnTGFbR was 64.9%, 51.9%, 47.5%, 52.9%, respectively; the proportion of effector memory T cells of verh.bbz CAR-T cells is higher than CAR-T cells co-expressing the switch receptor (p < 0.01).

CD69 is an early molecular marker for T cell activation, and the proportion of CD69+ cells in VerH.BBZ and dnTGFbR+VerH.BBZ CAR-T cells is 48.9% and 48.6%, respectively, which is higher than the proportion of CD69+ cells in PD1-CD28+VerH.BBZ CAR-T cells (42.8%), which is significantly higher than the proportion of CD69+ cells in TGFbR/IL12R+VerH.BBZ (36.1%, p < 0.05). Cd27+ cells expressed up to 38.4% in TGFbR/il12r+verh.bbz CAR-T cells, followed by PD1-cd28+verh.bbz CAR-T (29.7%); the ratio of CD27+ cells in VerH.BBZ and dnTGFbR2+VerH.BBZ CAR-T cells was 25.2% and 24.8%, respectively, with a significant difference (p < 0.01) compared to TGFbR/IL12 R+VerH.BBZ.

5.4.2 converting receptors increase secretion of CAR-T cell TH1/TH2 cytokines and chemokines

Luminex results showed that verh.bbz CAR-T cells and CAR-T cells expressing three different switch receptors exhibited different patterns of secretion of cytokines and chemokines after 16 hours of second round stimulation of tumor cells (cytokine profiles). Compared with VerH.BBZ CAR-T, TGFbR/IL12R+VerH.BBZ significantly increases secretion of IFN-gamma, IL-4, IL-5, IL-6, IL-10, CCL4, CXCL8, CXCL10, dnTGFbR2+VerH.BBZ significantly increases secretion of IL-4, IL-5, CCL4, CXCL8, and PD1-CD28+VerH.BBZ significantly increases secretion of IL-2, IL-10, IL-17A, CCL4, CXCL8, CXCL 10. The profile of TGFbR/il12r+verh.bbz is relatively close to that of dntgfbr2+verh.bbz cytokine secretion, but since TGFbR/IL12R switch receptors provide IL-12 signal while blocking TGF- β signal, secretion of IFN- γ, as well as secretion of cytokines and chemokines regulated by IFN- γ, is significantly increased. The PD1-CD28 switch receptor converts the PD1 inhibitory signal to a CD28 activating signal, significantly increasing secretion of IL-2 and IL-17A compared to verh.bbz CAR-T cells.

5.4.3 RNA-seq reveals that genes differentially expressed by CAR-T cells expressing a switch receptor are involved in biological pathways associated with T cell function

IL-12 cytokines are produced very early during infection or immune response, regulate innate immune responses, determine the type of adoptive immune response, shape immune responses and influence the differentiation of naive T cells. To understand the molecular changes that occur inside early cells of co-culture of CAR-T cells co-expressing the transduction receptor with tumor cells, T cells were sorted by CD45 magnetic beads 16 hours after the second round of tumor stimulation, RNA was extracted, and mRNA sequencing (RNA-seq) was performed.

PCA analysis (Principal Component Analysis) visually evaluates the RNA-seq data, identifies sample characteristics, and reveals correlations between samples, based on multidimensional functional analysis of gene reads obtained after sequencing each RNA sample. Each spot on PCA (a of fig. 12) represents one RNA sample, from 5 groups of 13 RNA samples clustered at different positions (1 RNA sample failed mass detection in both verh.bbz CAR-T group and CAR-T group expressing PD1-CD28 switch receptor). Three non-transduced (NO TD) T cell RNA samples were clustered away from other RNA samples. Three samples of the verh.bbz CAR-T group that co-expressed TGFbR/IL12R switch receptor were also clustered close to each other, away from RNA samples of the other three groups of CAR-T cells. The 7 RNA samples of the verh.bbz CAR-T group and CAR-T group expressing PD1-CD28, dnTGFbR2 receptor were close to each other, but the same group of RNA samples could be divided into the same cluster.

The PCA analysis result confirms the experimental grouping of the samples, and the NO TD group has larger difference with other four groups in RNA transcription level; RNA samples of the CAR-T cells expressing the conversion receptor TGFbR/IL12R are also different from those of the RNA samples of the VerH.BBZ and the CAR-T cells expressing the PD1-CD28 and the dnTGFbR2, and are relatively similar to those of the RNA samples expressing the dnTGFbR group; RNA samples expressing PD1-CD28 switch receptors were significantly different from those expressing dnTGFbR and TGFbR/IL12R switch receptors. The results of the PCA analysis showed that the results of RNA sequencing can be used to further analyze genes differentially expressed between the experimental and control groups, as well as between the experimental groups.

Volcanic plot (B of fig. 13) shows genes differentially expressed for each CAR-T group compared to the NO TD control group. The verh.bbz CAR-T group had 12636 differentially expressed genes, up-regulated 6544 genes, down-regulated 6092 genes; 12540 differentially expressed genes in PD1-CD28 CAR-T group up-regulated 6599 and down-regulated 5941; the TGFbR/IL12R CAR-T group has 12373 differentially expressed genes, up-regulated gene 6332 and down-regulated gene 6041; the dnTGFbR2 CAR-T group had 12554 differentially expressed genes, up-regulated gene 6607 and down-regulated gene 5947. The number of genes differentially expressed for each CAR-T group was around 12500, and 8502 genes differentially expressed for the 4 CAR-T experimental groups, compared to the NO TD control group, enriched in 143 related signaling pathways; venn diagram (E of FIG. 13) shows the results of Meta analysis, indicating that there are also some differential expressed genes in common between the three groups and between the two groups of the experimental group of 4 CAR-T; meanwhile, each CAR-T experimental group has unique differential expression genes, 296 in the VerH.BBZ group, 117 in the CAR-T group expressing PD1-CD28, 134 in the CAR-T group expressing TGFbR/IL12R and 230 in the CAR-T group expressing dnTGFbR 2. The differentially expressed genes of the CAR-T group that expressed the conversion receptor TGFbR/IL12R were enriched in 11 signaling pathways associated with macromolecular synthesis (tables 2-3).

Volcanic plot (C of FIG. 13) shows 178, 364 and 390 genes differentially expressed by three CAR-T groups expressing the switch receptors PD1-CD28, TGFbR/IL12R and dnTGFbR2, respectively, compared to the VerH.BBZ CAR-T group. The morphology of the differential gene distribution on the volcanic image shows that the variation of the CAR-T group gene expressing TGFbR/IL12R is more pronounced. Volcanic FIG. 11D is a graph of TGFbR-IL12R CAR-T group part differentially expressed genes, and log of the differentially expressed genes are shown in tables 2-1 and 2-2, respectively ₂ Up-regulating genes and down-regulating genes at the first 50 absolute values of FC. These differentially expressed genes are associated with cell division, cell adhesion, intercellular signaling, cell migration, and other functions. The Venn diagram (F of FIG. 13) shows the results of the Meta analysis, with only 18 genes differentially expressed in common for the three groups, most of the genes differentially expressed being unique genes for each group. Wherein there are 8 signaling pathways enriched for 283 specific differentially expressed genes in the dnTGFbR2 group and 14 signaling pathways enriched for 231 specific differentially expressed genes in the TGFbR/IL12R group (tables 2-4), including T cell activation, PI3K pathway, cell adhesion, intercellular signaling, cell response to stimulus, etc.

TABLE 2-1

Symbol	ID	LogFC	p-value
				DRD2	1813	3.272	4.57E-04
DAB2IP	153090	2.724	0.023
				CA12	771	2.701	0.009
TMEM47	83604	2.672	5.88E-04
				MRGPR×2	117194	2.491	0.006
NPHP1	4867	2.396	9.02E-05
				NKAPL	222698	2.295	0.042
FOXH1	8928	2.295	0.042
				ARHGEF26	26084	2.294	0.043
IL10	3586	2.109	0.006
				TNFRSF19	55504	2.056	0.016
MLK7-AS1	339751	2.054	0.015
				TRNP1	388610	2.052	0.015
AHSG	197	2.049	0.04
				TBX2	6909	2.015	0.001
FUT7	2529	1.995	6.76E-04
				ZNF713	349075	1.989	6.14E-04
PTCHD2	57540	1.966	0.004
				BOC	91653	1.949	2.59E-05
ITGA2B	3674	1.917	0.026
				EFHD1	80303	1.908	0.015
FGFR3	2261	1.908	0.015
				CEBPD	1052	1.908	0.011
HIST1H4I	8294	1.905	0.037
				RBM11	54033	1.902	0.015
LOC100379224	100379224	1.894	0.021
				CFI	3426	1.807	0.015
ITGBL1	9358	1.797	0.026
				SNED1	25992	1.793	0.025
ARHGAP44	9912	1.762	0.003
				GREM2	64388	1.728	0.002
ICAM5	7087	1.709	0.007
				FRMD4A	55691	1.704	0.048
CADM4	199731	1.669	0.04
				C6orf112	154442	1.61	0.043
SNORA13	654322	1.604	0.014
				TMEM121	80757	1.604	0.037
PTK2	5747	1.562	0.033
				LOC401127	401127	1.56	0.011
TLX2	3196	1.558	2.17E-04
				TMEM30B	161291	1.551	0.032
TGFBR2	7048	1.541	1.00E-06
				TMCC3	57458	1.519	2.65E-04
C10orf55	414236	1.512	0.034
				CAMK1G	57172	1.489	0.026
C19orf20	91978	1.472	0.014
				IL12RB1	3594	1.464	1.00E-06
AMH	268	1.459	0.049
				RBPJL	11317	1.454	0.048
C1orf53	388722	1.432	0.041

TABLE 2-2

Symbol	ID	LogFC	p-value
				DGCR5	26220	-2.684	0.002
CLSTN2	64084	-2.592	8.36E-06
				SYNPO2	171024	-2.527	0.007
KAZN	23254	-2.495	0.009
				SPINK2	6691	-2.293	0.004
NOS3	4846	-2.237	0.004
				TJP1	7082	-2.191	0.025
FN3K	64122	-2.185	0.025
				WDFY4	57705	-2.148	0.015
SMC1B	27127	-2.131	9.96E-05
				AS3MT	57412	-2.079	1.94E-05
CSNK1A1P1	161635	-2.071	0.043
				C9orf24	84688	-2.067	0.042
TRPV3	162514	-2.066	0.006
				RASEF	158158	-2.065	0.01
SIGLEC5	8778	-2.052	0.045
				ACTG2	72	-1.967	0.036
GPR141	353345	-1.88	0.026
				RSPH4A	345895	-1.768	0.04
FAM83E	54854	-1.765	0.04
				PRAME	23532	-1.762	0.001
HTRA1	5654	-1.744	0.002
				CDKN2B-AS	100048912	-1.694	0.004
FOSB	2354	-1.675	4.62E-04
				RHOU	58480	-1.61	0.042
B4GALT6	9331	-1.604	2.39E-06
				SRGAP3	9901	-1.589	9.64E-06
CHMP4C	92421	-1.585	0.007
				CYP2B6	1555	-1.58	0.001
CCL22	6367	-1.553	1.00E-06
				SLC4A4	8671	-1.538	0.002
ETv1	2115	-1.521	2.62E-04
				IGFBP2	3485	-1.489	5.85E-04
RASD2	23551	-1.478	1.00E-06
				TCP10L	140290	-1.475	0.01
C10orf25	220979	-1.438	0.003
				RAI14	26064	-1.411	0.014
SNORD77	692197	-1.393	0.028
				FAM20C	56975	-1.391	0.038
GNAO1	2775	-1.374	1.00E-06
				PLCH1	23007	-1.346	0.01
ZSCAN23	222696	-1.336	0.024
				RHPN2	85415	-1.261	0.003
CAMK2N1	55450	-1.232	9.87E-04
				QPCT	25797	-1.22	0.013
LPCAT2	54947	-1.209	1.25E-05
				PROCR	10544	-1.209	0.017
BCAR3	8412	-1.171	0.011
				PLXDC2	84898	-1.154	0.016
PLA1A	51365	-1.144	0.005

Tables 2 to 3

Tables 2 to 4

Irradiated HepG2 tumor cells were co-cultured with verh.bbz CAR-T and verh.bbz CAR-T cells co-expressing the conversion receptor according to E: t=2:1, and after 7 days, the collected T cells were subjected to a second round of stimulation under the same conditions. After 16 hours, cells were collected, T cells were sorted by CD45 magnetic beads, and RNA was extracted and analyzed for RNA sequence. A cDNA library was constructed using Illumina truSeq stranded mRNA kit and Hiseq4000 sequenced to a sequence of 100bp at one end of the cDNA library. The raw data from RNA sequencing was compared to the hg19 genomic reference sequence by STAR software, leaving only transcripts identical to the reference genomic sequence. Transcripts were assembled by software Cufflinks Version.2.1 and the unit of gene reading was FPKM. Tables 2-4 list the signaling pathways enriched for the differential expressed genes of the TGFbR/IL12R+VerH.BBZ group compared to the VerH.BBZ group.

5. GPC3 CAR-T cells co-expressing the conversion receptor rapidly inhibit and eliminate hepatoma cells in mice

The CAR-T cells co-expressing the conversion receptors PD1-CD28, TGFbR/IL12R and dnTGFbR have significantly enhanced in vitro tumor killing function, increased cytokine and chemokine secretion, central memory cell proliferation, and RNA-seq analysis revealed differentially expressed genes involved in T cell activation, cell proliferation, cell adhesion, and cell-to-cell signaling and cell response to stimuli, among other biological pathways, as compared to CAR-T cells. To further verify the anti-tumor function in vivo of CAR-T cells expressing the conversion receptor, verh.bbz CAR-T and CAR-T cells expressing the conversion receptor were compared on a HepG2 liver cancer model of mice.

Animal experiments were divided into 5 groups, respectively: verh.bbz, PD1-cd28+verh.bbz (verh.bbz CAR-T cells co-expressing PD1-CD28 switch receptor), TGFBR/il12r+verh.bbz (verh.bbz CAR-T cells co-expressing TGFBR/IL12R switch receptor), dntgfbr2+verh.bbz (verh.bbz CAR-T cells co-expressing truncated receptor dnTGFbR 2), and control NO TD (non-transduced T cells). 5 mice per group, mice were subcutaneously injected 1X 10 ⁶ HepG2-CBG tumor cells, vaccinated on day 10 tail intravenous injection 1X 10 ⁶ CAR positive T cells or naiveTransduced T cells.

Tumor size was detected by fluorescence imaging at day 9, day 17, day 25 and day 32 post tumor inoculation (a of fig. 14). Mice were grouped according to the results of day 9 fluorescence imaging. On day 17 (one week after treatment), the fluorescence imaging results showed an increase in tumor in all mice in the treatment and control groups over the treatment. There was no statistical difference between the treatment group and the control group. On day 25, the results of fluorescence imaging show that tumors of the treated mice began to shrink (B of fig. 14). The tumors of the mice in the VerH.BBZ group and the dnTGFbR2+VerH.BBZ group are obviously different from those of the control group (p is less than 0.05); the tumors of mice in the PD1-CD28+VerH.BBZ, TGFBR/IL12R+VerH.BBZ groups are very different from those in the NO TD group (p < 0.01); since the TGFBR/il12r+verh.bbz group had a rapidly shrinking tumor in the second week of treatment, the differences from the verh.bbz, PD1-cd28+verh.bbz and dntgfbr2+verh.bbz groups were significant (p < 0.05). The results of the fluorescent imaging on day 32 showed that the tumor continued to grow in the control mice and further shrink in the treated mice. The tumors of the mice in the PD1-CD28+VerH.BBZ group rapidly shrink in the third week of treatment, and the tumors of 5 mice completely disappear; the tumor of 4 mice in the TGFBR/IL12R+VerH.BBZ group disappears, and the tumor of one mouse is obviously reduced compared with the previous week; two mice had tumor disappeared in both the verh.bbz and dntgfbr2+verh.bbz groups. Statistical analysis of the results of the fluorescent imaging on day 32 showed that all treatment groups of mice had very significant differences in tumor versus NO TD group (p < 0.01); the difference between the PD1-CD28+VerH.BBZ, TGFBR/IL12R+VerH.BBZ and the VerH.BBZ groups was significant (p < 0.05), and the difference between the dnTGFbR2+VerH.BBZ and the VerH.BBZ groups was not significant (C of FIG. 14).

The experimental results of the mice HepG2 liver cancer animal show that: the TGFbR/IL12R conversion receptor and the PD1-CD28 conversion receptor significantly enhance the function of GPC3 CAR-T cells in inhibiting tumors in mice, and can completely eliminate the tumors of most tumor-bearing mice. CAR-T cells expressing TGFBR/IL12R switch receptor can rapidly inhibit tumor growth, and PD1-CD28 switch receptor can cause CATR-T cells to exhibit the strongest tumor controlling function over a long period of time.

While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

Claims

1. A CAR molecule targeting GPC3, wherein the CAR molecule comprises an extracellular domain, a transmembrane region, and an intracellular region that specifically binds GPC3, and wherein the extracellular domain that specifically binds GPC3 comprises an amino acid sequence as set forth in SEQ ID No. 2 or SEQ ID No. 4.

2. The GPC 3-targeting CAR molecule of claim 1, wherein the transmembrane region is a CD8 transmembrane region, the intracellular region comprising a 4-1BB costimulatory signal region and a CD3 zeta signaling domain, the CD8 transmembrane region and the extracellular domain that specifically binds GPC3 being linked by a CD8 hinge region;

Preferably, the amino acid sequences of the CD8 hinge region, CD8 transmembrane region, 4-BB costimulatory signal region, and CD3 zeta signaling domain are shown as SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively;

more preferably, the amino acid sequence of the CAR molecule is as shown in SEQ ID NO. 15 or SEQ ID NO. 16;

even more preferably, the nucleotide sequence encoding the CAR molecule is set forth in SEQ ID NO. 1 or SEQ ID NO. 3.

3. A CAR molecule in combination with a switch receptor, wherein the CAR molecule is a GPC 3-targeting CAR molecule of claim 1 or 2, the switch receptor comprising a first polypeptide associated with a negative signal and a second polypeptide associated with a positive signal; the first polypeptide is selected from CTLA4, PD-1, BTLA, TIM-3, and TGF beta R, and the second polypeptide is selected from CD28, ICOS, 4-1BB, and IL-12R;

preferably, the amino acid sequence of the switch receptor is as shown in SEQ ID NO. 18, SEQ ID NO. 20 or SEQ ID NO. 22 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity thereto;

more preferably, the amino acid sequence of the switch receptor is shown as SEQ ID NO. 18 or SEQ ID NO. 20;

even more preferably, the nucleotide sequence encoding the switch receptor is set forth in SEQ ID NO. 17 or SEQ ID NO. 19.

4. A nucleic acid construct or combination thereof encoding a GPC 3-targeting CAR molecule according to claim 1 or 2; or encodes a CAR molecule according to claim 3 in combination with a switch receptor.

5. The nucleic acid construct or combination thereof of claim 4, wherein the nucleic acid construct comprises a nucleotide sequence as set forth in SEQ ID No. 1 or SEQ ID No. 3.

6. The nucleic acid construct or combination thereof of claim 4 or 5, wherein the nucleotide sequence encoding the switch receptor is set forth in SEQ ID No. 17, SEQ ID No. 19 or SEQ ID No. 21;

preferably, the nucleotide sequence encoding the GPC 3-targeting CAR molecule and the nucleotide sequence encoding the switch receptor are located in the same nucleic acid construct or separate nucleic acid constructs.

7. A recombinant expression vector comprising the nucleic acid construct of any one of claims 4-6 or a combination thereof;

preferably, the starting vector of the recombinant expression vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors and viral vectors of adeno-associated viral vectors; preferably a retroviral vector and/or a lentiviral vector.

8. An immune response cell comprising a GPC 3-targeting CAR molecule according to claim 1 or 2.

9. The immunoresponsive cell of claim 8, further comprising a switch receptor having an amino acid sequence as set forth in SEQ ID No. 18, SEQ ID No. 20, or SEQ ID No. 22;

preferably, the nucleotide sequence encoding the switch receptor is shown as SEQ ID NO. 17, SEQ ID NO. 19 or SEQ ID NO. 21.

10. The immune response cell of claim 8 or 9, wherein said immune response cell is a T cell, a natural killer cell, a hematopoietic stem cell, or a hematopoietic progenitor cell;

preferably, the immune response cell is a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, or a regulatory T cell;

more preferably, the immune response cells are from a human.

11. Kit comprising one or more of the CAR molecule targeting GPC3 according to claim 1 or 2, the CAR molecule and switch receptor combination according to claim 3, the nucleic acid construct according to any one of claims 4 to 6 or a combination thereof, the recombinant expression vector according to claim 7 and the immunoresponsive cell according to any one of claims 8 to 10.

12. A pharmaceutical composition comprising an immunoresponsive cell according to any one of claims 8 to 10; and a pharmaceutically acceptable carrier or excipient.

13. A kit of parts comprising a kit a and a kit B, wherein the kit a comprises an immune response cell comprising a CAR molecule according to claim 1 or 2 that targets GPC3, and the kit B comprises an immune response cell comprising a switch receptor as defined by the CAR molecule and switch receptor combination of claim 3.

14. Use of a CAR molecule targeting GPC3 according to claim 1 or 2, a CAR molecule and a switch receptor combination according to claim 3, a nucleic acid construct according to any one of claims 4 to 6 or a combination thereof, a recombinant expression vector according to claim 7 and an immune response cell according to any one of claims 8 to 10 for the manufacture of a medicament for the treatment of a tumor;

preferably, the tumor is selected from liver cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, leukemia and lymphoma, preferably liver cancer.

15. Use of an immunoresponsive cell according to any one of claims 8-10 or a pharmaceutical composition according to claim 12 for the manufacture of a medicament for the treatment of a tumor;

Preferably, the medicament further comprises a lymphocyte scavenger chemotherapeutic agent, such as cyclophosphamide and/or fludarabine;