Pharmaceutical composition and use thereof

Abstract

The present invention relates to a pharmaceutical composition and the use thereof, in particular to the use of a combination of a dual-target CAR-T targeting TNFRSF10B / ASGR1 and a formula for strengthening vital qi, removing toxins and removing food retention in the preparation of a composition for treating liver cancer. A pharmaceutical composition comprising a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cell and a formula for strengthening vital qi, removing toxins and removing food retention, wherein the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor is expressed on the membrane surface of the dual-target chimeric antigen receptor T cell. It is unexpectedly found that the combined use of the formula for strengthening vital qi, removing toxins and removing food retention with CAR-T can effectively inhibit the activation of the STAT3 pathway, promote the chemotaxis of a CAR-T cell as an effector T cell, improve the infiltration and cytotoxicity, inhibit the depletion of CD8+T cells, and effectively improve the anti-liver cancer capability of the CAR-T cell.

Description

A pharmaceutical composition and application TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, belongs to the IPC classification A61K, and particularly relates to a pharmaceutical composition and application, in particular to a construction method of a third-generation chimeric antigen receptor T cell (CAR-T) for a double-target of TNFRSF10B / ASGR1 on the surface of a primary liver cancer cell and application of the third-generation chimeric antigen receptor T cell (CAR-T) in combination with a Fuzheng Jiedu Xiaoshi prescription (FZJD) to improve the treatment effect of primary liver cancer. BACKGROUND

[0002] Liver cancer, namely liver malignant tumor, is the sixth most common cancer in the world and the fourth leading cause of cancer-related death, and the 5-year survival rate is 18%. Hepatocellular carcinoma (HCC) accounts for 90%, which belongs to primary liver cancer and is a malignant tumor originating from hepatocytes. The incidence and mortality of liver cancer in China rank in the forefront of the world, which has brought a heavy medical burden to the country. The traditional treatment methods of liver cancer include surgery, transcatheter arterial chemoembolization, local ablation, targeted therapy, etc., but the efficacy of these methods is limited, therefore, the importance of tumor immunotherapy is increasingly prominent.

[0003] CD8 + Effector T cells differentiated from T cells can selectively kill tumor cells. Maintaining or restoring the activity of effector T cells is a key link of tumor immunotherapy. The currently widely used immune checkpoint inhibitors (ICIs) such as PD-1 / PD-L1 can partially restore the anti-tumor activity of effector T cells, but the stimulation of chronic inflammation inside the tumor and the immunosuppressive environment will induce effector T cells to become low-reactive T cells, which will lose activity and gradually exhaust. At the same time, about 30% of liver cancer tissues lack T cell and inflammatory cell infiltration, so the expression of antigen target of ICIs is low, which is naturally resistant to ICIs. In summary, the efficacy of ICIs in liver cancer is not satisfactory enough.

[0004] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T cell therapy) as one of the emerging means of tumor immunotherapy is the focus of current tumor treatment research field. Chimeric Antigen Receptor (CAR) is a kind of artificial chimeric protein which is obtained by fusing single-chain antibody recognizing cell surface antigen of cancer cell and signal transduction region inducing T cell activation. By introducing CAR gene into T lymphocyte which does not have tumor reactivity, a large number of CAR-expressing T cells (CAR-T cells) capable of expressing CAR can be prepared. In recent years, CD8 + T cell technology has made a major breakthrough in the field of immunotherapy for tumors. CD8 + T lymphocytes modified by CAR gene can recognize tumor cells positive for tumor-associated antigen (TAA) with high efficiency and specificity through antigen-antibody interaction without major histocompatibility complex (MHC) restriction, and can self-replicate and activate, thus having strong tumor killing ability, and is considered as one of the hopes for completely curing tumors in the future.

[0005] Multiple multi-center clinical trials have treated various B-cell tumors including B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (B-NHL), chronic lymphocytic leukemia (CLL), and Hodgkin lymphoma (HL) by using CAR-T cells targeting CD19, CD20, or CD30. In the treatment of solid tumors, various CAR therapies targeting tumor cell surface associated antigens have entered phase 1-2 clinical trials, such as CAR-T therapies targeting mesothelin (MSLN) expressed by ovarian cancer, mesothelioma, pancreatic cancer, targeting epidermal growth factor type III mutant (EGFR vIII) expressed by neuroblastoma, targeting human epidermal growth factor receptor 2 (HER2) expressed by colorectal cancer and breast cancer, carcinoembryonic antigen (CEA), and targeting prostate-specific membrane antigen (PSMA) for prostate cancer. WO2020017479A1 discloses a single-chain antibody-containing CAR capable of specifically binding to GPC3 on the cell membrane, and by introducing the gene encoding the CAR into immune active cells, an immune active cell expressing the CAR with excellent cancer cell toxicity and IFN-γ production capacity is constructed, but the recognition and killing of tumor cells by single target CAR is limited, which may lead to partial tumor cells escaping the attack of CAR-T cells, increasing the risk of tumor recurrence. US2024043490A1 discloses a targeted cytokine construct for engineered cell therapy, wherein the targeted cytokine construct selectively activates engineered cells with 10-fold or greater potency, thereby achieving the purpose of treating cancer. The main mechanisms of recurrence after CAR-T cell therapy are the limited persistence of CAR-T cells, inhibition of CAR-T cell function, and antigen escape. In order to minimize the risk of recurrence due to target escape, the strategy of dual-target CAR-T recognizing more than one tumor-associated antigen is actively explored in clinical trials. In preclinical models and clinical trials of hematological malignancies, dual-target CAR-T cell therapy has at least three combinations of antigens: CD19 / CD20, CD19 / CD22, and BCMA / CD38. In liver cancer, CAR-T targeting asialoglycoprotein receptor 1 (ASGR1) and glypican-3 (GPC3) is under development. "Off-target effect" is a widespread problem in CAR-T applications. In solid tumors, the recognition of CAR-T cells to non-tumor tissues expressing target antigens often leads to clinically severe non-tumor targeted toxicity. The existing GPC3 single-target CAR-T has low sensitivity and specificity in recognizing liver cancer, and GPC3 single-target CAR-T has occurred in clinical application with a higher level of cytokine release syndrome (grade 5), and its safety needs to be further improved.There is also a GPC3 double-target CAR-T therapy at present, but its killing effect has not been significantly improved compared with GPC3 single-target CAR-T. SUMMARY

[0006] The targets of hematological tumors and gliomas have good specificity, but there are fewer types of antigens on the surface of hepatocellular carcinoma cells that can be recognized by CAR-T as targets. For example, the GPC3 positive rate in the tissues of hepatocellular carcinoma patients is about 60%, which means that the hepatocellular carcinoma cells of about 40% of patients cannot be recognized by CAR-T targeting GPC3. Therefore, it is urgent to develop CAR-T with other targets, higher sensitivity and specificity. In the treatment of hepatocellular carcinoma, the specific chemotaxis of CAR-T to tumors is poor, the infiltration ability of CAR-T to the interior of solid tumors is not strong, and the defects such as the sustained inflammatory environment in the tumor and the T cell exhaustion induced by the high expression of PD-L1 in hepatocellular carcinoma cells affect the exertion of cytotoxicity. On the other hand, most of the tumor-associated antigens expressed on the surface of hepatocellular carcinoma cells are non-tumor-specific antigens, which can be expressed in other normal tissues, so that the normal tissues are easily attacked by CAR-T cells, forming a systemic "cytokine storm" mainly with elevated interleukin 6 (IL-6), which has the risk of developing into multiple organ failure and death.

[0007] GPC3 is a member of the heparan sulfate proteoglycan family and is highly expressed in HCC but not expressed or lowly expressed in normal liver tissue. Studies have shown that the expression of GPC3 is related to the progression of HCC, and about 60-70% of HCC patients express GPC3, and the serum GPC3 expression level of 53% of HCC patients is significantly elevated. The prior art discloses technical solutions of GPC3 single-target CAR-T and GPC3 / ASGR1 double-target CAR-T for treating hepatocellular carcinoma. The immunohistochemical results of clinical samples show that among the 75 hepatocellular carcinoma samples detected, 50 samples have positive GPC3 expression, and the positive rate is 66.7%; while ASGR1 is highly expressed in all normal liver tissues (90.7%). According to statistics, among the 75 liver cancer tissue samples, a total of 41 samples (54.7%) have co-expression of GPC3 and ASGR1; researchers have carried out two consecutive explorations of GPC3 single-target CAR-T therapy in HCC, and the study included 13 patients with advanced HCC, and the 3-year, 1-year and 6-month overall survival (OS) rates were 10.5%, 42.0% and 50.3% respectively, and one patient was still alive after 44.2 months. However, the existing technical solutions have significant defects in the improvement of cytotoxicity and side effects. Specifically, in terms of experimental research efficiency, whether it is against parental MHCC-97L and MHCC-97L -GPC3+ , MHCC-97L -ASGR1+ and MHCC-97L -GPC3+ASGR1+The overexpression cell line, or for Huh-7, HepG2 naturally expressing GPC3 and ASGR1, the double-targeted alpha GPC3-Z+alpha ASGR1-28BB modified T cells did not exhibit stronger cytotoxicity compared with the single-targeted first-generation CAR-T alpha GPC3-Z modified T cells, and the cytotoxicity percentages of the two were about 30% and 60% at the effector-to-target ratio of 3:1 and 1:1, with no significant difference between the two. In addition, by comparing the therapeutic effect of the third-generation CAR-T cells (GPC3-28BBZ) and the double-targeted CAR-T cells on Huh-7 subcutaneous transplanted tumors, it was found that the tumor killing ability of the double-targeted CAR-T cells in vivo was significantly worse than that of the third-generation CAR-T. In summary, it is considered that the abnormal polymerization of the ASGR1 single-chain antibody and the adjacent GPC3 chimeric antigen antibody may occur due to some special structure domains, and continuous phosphorylation may occur, which ultimately leads to the unsatisfactory anti-cancer effect. Therefore, there are still technical problems to be solved in selecting GPC3 and ASGR1 as double targets for combined application in CAR-T. In terms of clinical research safety, cytokine storm (CRS) is a common adverse event in CAR-T immunotherapy, with a high incidence rate, and the clinical incidence rate is 80%-90%. However, the incidence rate of severe CRS, i.e., CRS of more than three degrees, is relatively low, about 10%-30%. The current phase I study of GPC3 single-target CAR-T includes 13 patients, and 9 patients have cytokine release syndrome (CRS), of which 8 patients are of grade 1-2, and 1 patient is of grade 5. Four patients received high-dose hormone shock therapy, and two of them also received monoclonal antibody tocilizumab therapy for interleukin-6 receptor. One patient had grade 5 CRS when 20.0x10 8 The safety of the clinical application of single-target GPC3 CAR-T cell therapy still needs to be further improved, and the cytokine release needs to be further effectively controlled.

[0008] In view of the deficiencies of the prior art, the present application provides a pharmaceutical composition comprising TNFRSF10B / ASGR1 double-target chimeric antigen receptor T cells and a Fuzheng Jiedu Xiaojifang (FZJD), wherein the double-target chimeric antigen receptor T cells express TNFRSF10B / ASGR1 double-target chimeric antigen receptors on the cell membrane surface.

[0009] The Fuzheng Jiedu Xiaojifang combined with CAR-T can effectively inhibit the activation of the STAT3 pathway, promote the chemotaxis of CAR-T cells as effector T cells, improve the infiltration and cytotoxicity, inhibit the CD8 +T cell exhaustion, effectively enhance the anti-liver cancer ability of CAR-T cells. At the same time, the present application inhibits each link of STAT3 downstream through multiple targets and multiple links, and suppresses the "inflammatory factor storm" caused by excessive release of IL-6, thereby reducing inflammation-related damage during treatment.

[0010] According to a preferred embodiment, the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor comprises a signal peptide, a first antigen binding region targeting TNFRSF10B (anti-TNFRSF10B scFv), a second antigen binding region targeting ASGR1 (anti-ASGR1 scFv), a hinge region, a transmembrane region, and an intracellular region.

[0011] According to a preferred embodiment, the intracellular region comprises a costimulatory signal factor, CD3 zeta and 4-1BB (CD137), CD28.

[0012] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B comprises an anti-TNFRSF10B antibody light chain variable region (VL) and an anti-TNFRSF10B antibody heavy chain variable region (VH). Preferably, the anti-TNFRSF10B antibody light chain variable region and the anti-TNFRSF10B antibody heavy chain variable region are connected by a linker or a connecting peptide. Specifically, the amino acid sequence of the linker is shown in SEQ ID NO: 10. Preferably, the anti-TNFRSF10B antibody light chain variable region and the anti-TNFRSF10B antibody heavy chain variable region are connected by one or more linkers, and the arrangement can be VL-linker-VH or VH-linker-VL.

[0013] According to a preferred embodiment, the amino acid sequence of the anti-TNFRSF10B antibody light chain variable region is shown in SEQ ID NO: 1.

[0014] According to a preferred embodiment, the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is shown in SEQ ID NO: 2.

[0015] According to a preferred embodiment, the second antigen binding region targeting ASGR1 comprises an anti-ASGR1 antibody heavy chain variable region.

[0016] According to a preferred embodiment, the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed for tumor cell surface antigen ASGR1, which can specifically bind to ASGR1 on the surface of tumor cells. Preferably, the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed for liver cancer cell surface antigen ASGR1, which can specifically bind to ASGR1 on the surface of liver cancer cells.

[0017] According to a preferred embodiment, the amino acid sequence of the variable region of the heavy chain of the anti-ASGR1 antibody is shown in SEQ ID NO: 3.

[0018] According to a preferred embodiment, the formula for strengthening the body, detoxifying, and eliminating stagnation includes: Codonopsis pilosula, Astragalus membranaceus, Atractylodes macrocephala, Poria cocos, Adenophora stricta, Ophiopogon japonicus, Angelica sinensis, Rehmannia glutinosa, Paris polyphylla, Curcuma zedoaria, and Pinellia ternata. Specifically, the formula is: Codonopsis pilosula 15g, Astragalus membranaceus 15g, Atractylodes macrocephala 15g, Poria cocos 15g, Adenophora stricta 15g, Ophiopogon japonicus 15g, Angelica sinensis 15g, Rehmannia glutinosa 15g, Paris polyphylla 15g, Curcuma zedoaria 9g, and Pinellia ternata 9g.

[0019] Another aspect of the present invention provides the use of the aforementioned pharmaceutical composition in the prevention and / or treatment of tumors.

[0020] Another aspect of the present invention provides the use of the aforementioned pharmaceutical composition in the prevention and / or treatment of liver cancer.

[0021] In another aspect, the present invention provides the use of the aforementioned pharmaceutical composition in improving the therapeutic effect of liver cancer.

[0022] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji formula enhances the inhibitory effect of TNFRSF10B / ASGR1 dual-target CAR-T cells on liver cancer cells, thereby improving the therapeutic effect of the aforementioned drug composition on tumors.

[0023] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji formula enhances the inhibitory effect of TNFRSF10B / ASGR1 dual-target CAR-T cells on liver cancer cells, thereby improving the therapeutic effect of the aforementioned drug composition on liver cancer.

[0024] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji formula enhances the therapeutic effect of liver cancer by promoting the release of CD107a and Granzym B in TNFRSF10B / ASGR1 dual-target CAR-T cells.

[0025] According to a preferred embodiment, the formula for strengthening the body, detoxifying, and eliminating stagnation increases CD4 levels. + Naïve T cells in CAR-T cells ( The ratio of T cells (cells) to central memory T cells (TCM) enables the aforementioned drug composition to improve the treatment effect of liver cancer.

[0026] According to a preferred embodiment, the formula for strengthening the body, detoxifying, and eliminating stagnation increases CD8 levels. + Naïve T cells in CAR-T cells ( The ratio of T cells (cells) to central memory T cells (TCM) enables the aforementioned drug composition to improve the treatment effect of liver cancer.

[0027] According to a preferred embodiment, the Fuzheng Jiedu Xiaojie Formula can improve the therapeutic effect of the aforementioned pharmaceutical composition by increasing the expression of the immune stimulatory receptor on the surface of the TNFRSF10B / ASGR1 double-target CAR-T cell.

[0028] According to a preferred embodiment, the Fuzheng Jiedu Xiaojie Formula can improve the therapeutic effect of the aforementioned pharmaceutical composition by reducing the expression of the immune inhibitory receptor TIGIT on the surface of the CAR-T cell.

[0029] According to a preferred embodiment, the Fuzheng Jiedu Xiaojie Formula can improve the therapeutic effect of the aforementioned pharmaceutical composition by reducing the expression of PD-L1 on the surface of the tumor cell.

[0030] According to a preferred embodiment, the Fuzheng Jiedu Xiaojie Formula can improve the therapeutic effect of the aforementioned pharmaceutical composition by reducing the expression of the pro-inflammatory factor IL-6.

[0031] According to a preferred embodiment, the Fuzheng Jiedu Xiaojie Formula can improve the therapeutic effect of the aforementioned pharmaceutical composition by improving the ability of the TNFRSF10B / ASGR1 double-target CAR-T cell to secrete IFN-γ, thereby improving its killing function.

[0032] According to a preferred embodiment, the Fuzheng Jiedu Xiaojie Formula can improve the therapeutic effect of the aforementioned pharmaceutical composition by inhibiting the STAT3 signaling pathway.

[0033] According to a preferred embodiment, the Fuzheng Jiedu Xiaojie Formula can improve the therapeutic effect of the aforementioned pharmaceutical composition by improving the release level of T cell chemotactic factor from the liver cancer cell.

[0034] Another aspect of the present application provides a double-target chimeric antigen receptor (CAR) comprising a signal peptide, a first antigen binding region targeting TNFRSF10B, a second antigen binding region targeting ASGR1, a hinge region, a transmembrane region, and an intracellular region.

[0035] According to a preferred embodiment, the structure of the first antigen binding region targeting TNFRSF10B is a specific antigen binding domain designed for the tumor cell surface antigen TNFRSF10B, which can specifically bind to TNFRSF10B on the surface of the tumor cell. Preferably, the first antigen binding region targeting TNFRSF10B is a specific antigen binding domain designed for the liver cancer cell surface antigen TNFRSF10B, which can specifically bind to TNFRSF10B on the surface of the liver cancer cell.

[0036] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B comprises an anti-TNFRSF10B antibody light chain variable region (VL) and an anti-TNFRSF10B antibody heavy chain variable region (VH). Preferably, the anti-TNFRSF10B antibody light chain variable region and the anti-TNFRSF10B antibody heavy chain variable region are connected by a linker or a connecting peptide. Preferably, the anti-TNFRSF10B antibody light chain variable region and the anti-TNFRSF10B antibody heavy chain variable region are connected by one or more linkers in the order of VL-linker-VH or VH-linker-VL.

[0037] According to a preferred embodiment, the amino acid sequence of the anti-TNFRSF10B antibody light chain variable region is shown as SEQ ID NO: 1.

[0038] According to a preferred embodiment, the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is shown as SEQ ID NO: 2.

[0039] According to a preferred embodiment, the second antigen binding region targeting ASGR1 comprises an anti-ASGR1 antibody heavy chain variable region.

[0040] According to a preferred embodiment, the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed against the tumor cell surface antigen ASGR1, which can specifically bind to the ASGR1 on the surface of tumor cells. Preferably, the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed against the liver cancer cell surface antigen ASGR1, which can specifically bind to the ASGR1 on the surface of liver cancer cells.

[0041] According to a preferred embodiment, the amino acid sequence of the anti-ASGR1 antibody heavy chain variable region is shown as SEQ ID NO: 3.

[0042] According to a preferred embodiment, the dual-targeting chimeric antigen is connected in parallel, and the dual-targeting chimeric antigen comprises a first CAR and a second CAR, the first CAR comprising: a first antigen binding region targeting TNFRSF10B; a first hinge region; a first transmembrane binding region; and a CD3 zeta.

[0043] The second CAR comprises: a signal peptide; a second antigen binding region targeting ASGR1; a second hinge region; a second transmembrane binding region; and a co-stimulatory signal binding region.

[0044] According to a preferred embodiment, the first CAR can be represented as TNFRSF10B ScFv-Hinge-TM-CD3 zeta.

[0045] According to a preferred embodiment, a tag protein for labeling, sorting, eliminating host cells can be present on the dual-targeting CAR, which can be linked to the dual-targeting CAR through a cleavable domain or another promoter.

[0046] According to a preferred embodiment, a tag protein is included on the first CAR. Preferably, the tag protein is a red fluorescent tag (dsRed).

[0047] According to a preferred embodiment, the signal domain of the first CAR includes CD8a(signal peptide).

[0048] According to a preferred embodiment, the first hinge region of the first CAR includes CD8aH (which can be denoted as Hinge).

[0049] According to a preferred embodiment, the first transmembrane binding region can anchor to the cell membrane.

[0050] According to a preferred embodiment, the second transmembrane binding region can anchor to the cell membrane.

[0051] According to a preferred embodiment, the first transmembrane binding region includes CD8aTM.

[0052] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B can be linked to the first transmembrane binding region through the first hinge region.

[0053] According to a preferred embodiment, CD3zeta is the intracellular signaling domain.

[0054] According to a preferred embodiment, the first CAR can be denoted as CD8a signal peptide-TNFRSF10B VH-linker-TNFRSF10B VL-CD8aH-CD8aTM-CD3zeta.

[0055] According to a preferred embodiment, the second CAR can be denoted as CD8a signal peptide-ASGR1VH-CD8aH-CD8aTM-CD137-CD28.

[0056] According to a preferred embodiment, a tag protein is included on the second CAR. Preferably, the tag protein is a green fluorescent tag (eGFP).

[0057] According to a preferred embodiment, the signal domain of the second CAR includes CD8a(signal peptide).

[0058] According to a preferred embodiment, the second hinge region of the second CAR comprises CD8aH (which can be denoted as Hinge).

[0059] According to a preferred embodiment, the second transmembrane binding region comprises CD8aTM.

[0060] According to a preferred embodiment, the co-stimulatory signaling region comprises a 4-1BB (CD137) co-stimulatory signaling domain and a CD28 co-stimulatory signaling domain.

[0061] According to a preferred embodiment, the intracellular co-stimulatory signaling domain can comprise a CD3 zeta intracellular signaling domain capable of activating an immune response cell.

[0062] According to a preferred embodiment, the second CAR can be denoted as CD8a signal peptide-ASGR1 VH-CD8aH-CD8aTM-CD137-CD28.

[0063] According to a preferred embodiment, the immune response cell comprises a T lymphocyte, an NK cell, an NKT cell.

[0064] According to a preferred embodiment, the signal peptide can be a human CD8a signal peptide, a human GM-CSF signal peptide, a human insulin signal peptide, a human IL-2 signal peptide, a human trypsinogen signal peptide. Preferably, the signal peptide can be a human CD8a signal peptide, the amino acid sequence of which is shown as SEQ ID NO: 4.

[0065] According to a preferred embodiment, the amino acid sequence of the CD8a hinge region is shown as SEQ ID NO: 5.

[0066] According to a preferred embodiment, the amino acid sequence of the CD8aTM transmembrane binding region is shown as SEQ ID NO: 6.

[0067] According to a preferred embodiment, the amino acid sequence of the CD28 co-stimulatory signaling domain is shown as SEQ ID NO: 7.

[0068] According to a preferred embodiment, the amino acid sequence of the 4-1BB (CD137) co-stimulatory signaling domain is shown as SEQ ID NO: 8.

[0069] According to a preferred embodiment, the CD3 zeta is an intracellular signaling domain, the amino acid sequence of which is shown as SEQ ID NO: 9.

[0070] According to a preferred embodiment, the first CAR is a primary recognition site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0071] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0072] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0073] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0074] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0075] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0076] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0077] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0078] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0079] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0080] According to a preferred embodiment, the second CAR is a costimulatory site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0081] According to one preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VL-linker-ASGR1 VH-linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

[0082] According to one preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VH-linker-ASGR1 VL-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0083] According to one preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VH-linker-ASGR1 VL-linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

[0084] According to one preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VH-linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

[0085] According to one preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0086] According to one preferred embodiment, the tandem chimeric antigen receptor can be A VH-linker-A VL-Linker-B VL-Linker-B VH or A VHH-linker-B VHH.

[0087] According to one preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VH.

[0088] According to one preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VL-ASGR1 VH.

[0089] According to one preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VH-ASGR1 VL.

[0090] According to one preferred embodiment, the dual-target chimeric antigen receptor comprises: a CD8a signal peptide, an ASGR1 scFv, a G4S linker, a DR5 scFv, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB, a CD28 co-stimulatory molecule, and a CD3 zeta signal transduction molecule.

[0091] According to one preferred embodiment, the TNFRSF10B / ASGR1 dual-target tandem CAR is connected with a mCherry gene sequence through a T2A.

[0092] According to one preferred embodiment, the nucleotide sequence of the TNFRSF10B / ASGR1 dual-target tandem CAR is shown in SEQ ID NO: 13.

[0093] According to one preferred embodiment, the amino acid sequence of the TNFRSF10B / ASGR1 dual-target tandem CAR is shown in SEQ ID NO: 14.

[0094] The present application reserves the right to modify the heavy chain variable domain VH of the scFv or humanized heavy chain antibody into a camelid antibody heavy chain variable domain VHH.

[0095] Another aspect of the present application provides a host cell, which comprises the aforementioned vector or chromosome with the aforementioned nucleic acid molecule integrated therein.

[0096] According to one preferred embodiment, the host cell expresses the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0097] Another aspect of the present application provides the use of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, the nucleic acid molecule encoding the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, the vector containing the nucleic acid molecule encoding the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, and the aforementioned host cell in the preparation of a medicament for preventing and / or treating cancer.

[0098] According to one preferred embodiment, the cancer is liver cancer.

[0099] Another aspect of the present application provides a kit for preparing a host cell, which comprises a nucleic acid molecule and / or a vector encoding the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0100] Another aspect of the present application provides a method for preparing an immune cell expressing the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, wherein the method comprises the following steps:

[0101] The immune cells to be modified are provided; a nucleic acid molecule encoding a TNFRSF10B / ASGR1 double-target chimeric antigen receptor or a vector containing the nucleic acid molecule encoding the TNFRSF10B / ASGR1 double-target chimeric antigen receptor is transduced into the immune cells, so as to obtain the modified immune cells.

[0102] The present application has the following beneficial effects: The present application designs and successfully constructs a double-target CAR-T cell which can be fully activated only when two targeted antigens are recognized at the same time. In addition to TNFRSF10B as a target point which can mediate the transmission of the first signal (CAR) after being recognized, ASGR1 as a target molecule recognized by a chimeric antigen co-stimulatory receptor (CCR) is a stable and specific receptor protein expressed on the surface of hepatocytes, so that the double-target CAR-T cell of TNFRSF10B / ASGR1 can specifically obtain more co-stimulatory signals in the liver. Through the combination of the CAR and the CCR, the sensitivity and specificity of the killing of liver cancer cells can be maximized, and the "off-target effect" can be effectively avoided. TNFRSF10B is selected as the main target point, and TNFRSF10B is expressed in various tumor tissues (pan-cancer). The expression of TNFRSF10B in pan-cancer and liver cancer is shown in FIG. 20: FIG. 20A is the expression of DR5 protein in human liver cancer tissue detected by immunohistochemistry and immunofluorescence; FIG. 20B is the correlation between the expression of TNFRSF10B in liver cancer and prognosis queried from the TCGA database; and FIG. 20C is the expression of TNFRSF10B in pan-cancer pathological tissues queried from The Human Protein Atlas database. The expression amount of TNFRSF10B in liver cancer tissue is greater than 75%, which is higher than the average expression amount of GPC3; at the same time, the expression amount of TNFRSF10B in normal tissues is extremely low. TNFRSF10B is related to the prognosis of liver cancer patients, which means that the target point is more widely distributed in liver cancer tissues, and the sensitivity and specificity of CAR-T which recognizes the target are higher. BRIEF DESCRIPTION OF DRAWINGS

[0103] FIG. 1 is a schematic diagram of the lentivirus skeleton vector GV400 and GV401 plasmid structure provided by the present application;

[0104] FIG. 2 is the enzyme digestion result of the enzyme digestion site BamH I / EcoR I provided by the present application;

[0105] FIG. 3 is the enzyme digestion result of the enzyme digestion site BamH I / BamH I provided by the present application;

[0106] FIG. 4 is the PCR result of the target fragment with the cloning site BamH I / EcoR I provided by the present application;

[0107] Figure 5 is the PCR result of the target fragment with the cloning site of BamH I / BamH I provided by the application;

[0108] Figure 6 is the identification result of the target fragment with the cloning site of BamH I / EcoR I exchanged into the linear expression vector provided by the application;

[0109] Figure 7 is the identification result of the target fragment with the cloning site of BamH I / BamH I exchanged into the linear expression vector provided by the application;

[0110] Figure 8 is a dilution schematic diagram for detecting virus titer by fluorescence, fluorescence microscope observation of plasmid transfection and counting provided by the application;

[0111] Figure 9 is the result of flow cytometry for detecting the transfection efficiency of CAR-T provided by the application;

[0112] Figure 10 is the comparison result of the killing effect of CAR-T cells on hepatoma cells at different effector-target ratios provided by the application;

[0113] Figure 11 is the apoptosis of hepatoma cells detected by flow cytometry when the effector-target ratio is 2:1 provided by the application;

[0114] Figure 12 is the release level of CD107a and Granzym B detected by flow cytometry after the co-incubation of TNFRSF10B (DR5) single-target and double-target CAR-T cells with hepatoma cells provided by the application;

[0115] Figure 13 is the result of CCK8 method for detecting the inhibition of tumor cell proliferation by the combination of FZJD drug-containing serum and DR5 / ASGR1 double-target CAR-T cells provided by the application;

[0116] Figure 14 is the experimental result of FZJD drug-containing serum enhancing the apoptosis of hepatoma cells by DR5 / ASGR1 double-target CAR-T cells provided by the application;

[0117] Figure 15 is the release of CD107a and Granzym B after the combination of FZJD drug-containing serum and DR5 / ASGR1 double-target CAR-T cells provided by the application;

[0118] Figure 16 is the experimental result of flow cytometry for detecting the phenotype change of FZJD combined with DR5 / ASGR1 double-target CAR-T cells (Figures A-C) and FZJD acting on hepatoma cells HepG2 (Figure D) provided by the application;

[0119] Figure 17 is the result of ELISA for detecting the expression amount of cytokine IL-6 after the co-culture of double-target CAR-T and HepG2 provided by the application;

[0120] Figure 18 is the result of an ELISA test provided by the present application to detect the level of interferon release when FZJD drug-containing serum increases the killing of liver cancer cells by DR5 / ASGR1 double-target CAR-T cells;

[0121] Figure 19 is the test result provided by the present application that FZJD drug-containing serum increases the release level of T cell chemotactic factor of liver cancer cells (Stattic is a STAT3 small molecule inhibitor);

[0122] Figure 20 is the expression of TNFRSF10B protein in human liver cancer tissue provided by the present application and the related expression of TNFRSF10B in TCGA database and The Human Protein Atlas database.

[0123] Figure 21 is the detection result of residual tumor cells of lung cancer calu-3 cells killed by TNFRSF10B / ASGR1 double-target tandem CAR-T detected by flow cytometry provided by the present application;

[0124] Figure 22 is the statistical result of residual tumor cells of lung cancer calu-3 cells killed by TNFRSF10B / ASGR1 double-target tandem CAR-T detected by flow cytometry provided by the present application;

[0125] Figure 23 is the tumor cell fluorescence expression intensity (%) of lung cancer calu-3 cells killed by different groups of cells detected by luciferase fluorescence provided by the present application;

[0126] Figure 24 is the statistical result of LDH release of tumor cells killed by lung cancer calu-3 cells killed by different groups of cells detected by LDH release test provided by the present application;

[0127] Figure 25 is the statistical result of interferon release of lung cancer calu-3 cells killed by different groups of cells detected by ELISA provided by the present application;

[0128] Figure 26 is the detection result of residual tumor cells of liver cancer HepG2 cells killed by TNFRSF10B / ASGR1 double-target tandem CAR-T detected by flow cytometry provided by the present application;

[0129] Figure 27 is the statistical result of residual tumor cells of liver cancer HepG2 cells killed by TNFRSF10B / ASGR1 double-target tandem CAR-T detected by flow cytometry provided by the present application;

[0130] Figure 28 is the statistical result of interferon (IFN-γ) release amount of liver cancer HepG2 cells killed by different groups of cells detected by ELISA provided by the present application;

[0131] Figure 29 is a statistical result of the release of LDH by tumor cells killed by different groups of cells provided by the present application in the LDH release test for killing liver cancer HepG2 cells;

[0132] Figure 30.1 is a comparison result of the killing effect of liver cancer HepG2 cells by TNFRSF10B-28z single-target CAR-T, TNFRSF10B / ASGR1 double-target tandem CAR-T and TNFRSF10B / ASGR1 double-target parallel CAR-T at different effector-to-target ratios provided by the present application;

[0133] Figure 30.2 is a statistical result of the killing effect of liver cancer HepG2 cells by TNFRSF10B-28z single-target CAR-T, TNFRSF10B / ASGR1 double-target tandem CAR-T and TNFRSF10B / ASGR1 double-target parallel CAR-T provided by the present application;

[0134] Figure 31 is a schematic diagram of TNFRSF10B / ASGR1 double-target tandem CAR-T provided by the present application;

[0135] Figure 32 is a schematic diagram of the structure of the chimeric antigen receptor of TNFRSF10B / ASGR1 double-target tandem provided by the present application;

[0136] Figure 33 is a lentiviral vector of TNFRSF10B / ASGR1 double-target tandem CAR provided by the present application. DETAILED DESCRIPTION

[0137] The following will be described in detail with reference to the accompanying drawings.

[0138] TNFRSF10B is also referred to as DR5 in the present application. TNFRSF10B / ASGR1 double-target CAR-T cell is equivalent to DR5 / ASGR1 double-target CAR-T cell.

[0139] Fuzheng Jiedu Xiaojifang is also referred to as FZJD in the present application.

[0140] In the present application, untransfected T cells are also referred to as NT cells, which are represented by NT in the drawings.

[0141] In the present application, control (blank) refers to a control without any treatment or without any control drug, which is used to observe whether the experiment is in a normal state.

[0142] In the present application, unless otherwise specified, the biological materials, reagents and detection kits involved can be obtained from commercial channels.

[0143] It is to be understood that all technical and scientific terms used herein have meanings commonly used in the fields of the disclosure. The term "antibody" can refer to an intact antibody molecule, or to fragments of antibody molecules that retain the ability to bind an immunogen, which can be routinely used in vivo or in vitro. The term "antibody" is a specific binding antigen and comprises interlinked heavy (H) chains and light (L) chains, or antigen binding portions thereof, which can comprise two heavy (H) chains and two light (L) chains. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each L chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. scFv is a single-chain antibody fragment of VH and VL combined by a connecting peptide. The variable regions of H chains and L chains contain the binding domain that interacts with the antigen. The VHH mentioned in the present application is the heavy chain variable domain of camelid heavy chain antibody, also known as nanobody, which can be modified from the traditional VH.

[0144] The TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor of the disclosed subject matter and the immune response cells expressing it can be applied to a subject systemically, directly to a subject for treating cancer or improving the efficacy of tumor. The immune response cells refer to cells that play a role in the immune response, or their progenitor cells, or their progeny cells. The TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor and the immune response cells expressing it can be administered by any physiologically acceptable means. In some embodiments, in the treatment of liver cancer, an effective dose of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor and the immune response cells expressing it is directly injected into the target organ (such as the organ affected by tumor formation), or an effective dose of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor and the immune response cells expressing it is administered to the circulatory system to indirectly provide the target organ. An effective dose of FZJD can be administered in conjunction with the administration of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor and the immune response cells expressing it before, during or after the administration of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor and the immune response cells expressing it to improve the efficacy of liver cancer treatment. The immune response cells expressing the TNFRSF10B / ASGR1 specific chimeric antigen receptor and the compositions comprising it can be obtained from one subject and administered to the same subject or a different compatible subject. The peripheral blood-derived T cells and their progeny of the disclosed subject matter can be administered by systemic injection, local injection, intravenous injection or parenteral administration. For example: at least 1 x 10 5 cells, eventually reaching 1 x 10 12 or more.

[0145] When administering the pharmaceutical composition disclosed in this application, it can be formulated as a unit-dose injectable form, such as a solution or suspension. Additives such as chelating agents, buffers, and antioxidants can be added to enhance the stability of the pharmaceutical composition. For the purposes of this application, any additives, carriers, or other diluents used must be compatible with immune-responding cells expressing the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor disclosed in this application. The amount of cells to be administered can be varied according to individual factors of the subject, including age, sex, weight, and other specific circumstances. In some embodiments, approximately 1 × 10⁻⁶ cells will be administered. 5 To approximately 1×10 12 Approximately 1×10 6 To approximately 1×10 11 Approximately 1×10 7 To approximately 1×10 10 Approximately 1×10 8 To approximately 1×10 9 The immune response cells disclosed in this application are administered to the subject. In some embodiments, a smaller or larger number of the immune response cells disclosed in this application may be administered to the subject. In some embodiments, approximately 1 × 103 4 Approximately 1×10 5 Approximately 2×10 6 Approximately 3×10 6 Approximately 4×10 6 Approximately 5×10 6 Approximately 2×10 7 Approximately 3×10 7 Approximately 4×10 7 Approximately 5×10 7 Approximately 2×10 8 Approximately 3×10 8 Approximately 4×10 8 Approximately 5×10 8 The immune response cells disclosed in this application are administered to a subject.

[0146] The skilled artisan can determine the cells and optional additional substances or vehicle substances in the pharmaceutical composition and the specific mode of administration. For example: the additives (in addition to the active cells and / or active substances) are present in an amount of a solution of about 0.001 wt% to about 50 wt% in phosphate buffered saline, and the active ingredients therein are present in the order of micrograms to milligrams, such as the active ingredients are present in an amount of about 0.0001 wt% to about 0.05 wt%, about 0.0001 wt% to about 0.01 wt%, about 0.0001 wt% to about 1 wt%, about 0.0001 wt% to about 5 wt%, about 0.0001 wt% to about 10 wt%, about 0.01 wt% to about 1 wt%, about 0.01 wt% to about 5 wt%, about 0.01 wt% to about 10 wt%, about 0.05 wt% to about 1 wt%, about 0.05 wt% to about 5 wt%, about 0.05 wt% to about 20 wt%.

[0147] An "effective dose" of a drug or pharmaceutical composition referred to in this application is a dose sufficient to effect any one or more of the beneficial or intended results. For therapeutic use, beneficial or intended results include eliminating or reducing the risk of tumor progression, improving efficacy, such as reducing the incidence of one or more symptoms of a condition (e.g., liver cancer) or causing symptoms to improve, one drug in a pharmaceutical composition is able to enhance the effect of another drug, thereby improving the overall therapeutic effect. An effective dose can be given in one administration or in a plurality of administrations. In this application, an effective dose of a drug or pharmaceutical composition refers to an amount sufficient to directly or indirectly accomplish a purposeful treatment. Depending on the context of the clinical treatment, an effective dose of a drug or pharmaceutical composition can or can not be used in conjunction with another drug or pharmaceutical composition. Thus, an "effective dose" can be considered in the context of administering one or more therapeutic agents, and if used in conjunction with one or more other drugs, the desired result can be achieved or realized.

[0148] Example 1

[0149] The present embodiment provides a pharmaceutical composition comprising TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells and FZJD, wherein the dual-target chimeric antigen receptor T cells express TNFRSF10B / ASGR1 dual-target chimeric antigen receptors on the membrane surface. Specifically, the FZJD is: Dangshen 15g, Shenghuangqi 15g, Baishu 15g, Fuling 15g, Shashen 15g, Maidou 15g, Danggui 15g, Shudi 15g, Qiyexianhua 15g, E'zhu 9g, Banxia 9g.

[0150] Example 2

[0151] The embodiment provides a chimeric antigen receptor, comprising a signal peptide, a first antigen binding region targeting TNFRSF10B, a second antigen binding region targeting ASGR1, a hinge region, a transmembrane region and an intracellular region. According to a preferred embodiment, the structure of the first antigen binding region targeting TNFRSF10B is a specific antigen binding domain designed for tumor cell surface antigen TNFRSF10B, which can specifically bind to TNFRSF10B on the surface of tumor cells. Preferably, the first antigen binding region targeting TNFRSF10B is a specific antigen binding domain designed for liver cancer cell surface antigen TNFRSF10B, which can specifically bind to TNFRSF10B on the surface of liver cancer cells. According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B comprises an anti-TNFRSF10B antibody light chain variable region and an anti-TNFRSF10B antibody heavy chain variable region. Preferably, the anti-TNFRSF10B antibody light chain variable region and the anti-TNFRSF10B antibody heavy chain variable region are connected by a linker or a connecting peptide. Preferably, the anti-TNFRSF10B antibody light chain variable region and the anti-TNFRSF10B antibody heavy chain variable region are connected by one or more linkers, which are arranged in the order of VL-linker-VH or VH-linker-VL. The chimeric antigen receptor is a parallel chimeric antigen receptor.

[0152] According to a preferred embodiment, the amino acid sequence of the anti-TNFRSF10B antibody light chain variable region is shown in SEQ ID NO: 1.

[0153] According to a preferred embodiment, the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is shown in SEQ ID NO: 2.

[0154] According to a preferred embodiment, the second antigen binding region targeting ASGR1 comprises an anti-ASGR1 antibody heavy chain variable region.

[0155] According to a preferred embodiment, the structure of the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed for tumor cell surface antigen ASGR1, which can specifically bind to ASGR1 on the surface of tumor cells. Preferably, the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed for liver cancer cell surface antigen ASGR1, which can specifically bind to ASGR1 on the surface of liver cancer cells.

[0156] According to a preferred embodiment, the amino acid sequence of the anti-ASGR1 antibody heavy chain variable region is shown in SEQ ID NO: 3.

[0157] According to a preferred embodiment, the dual-target chimeric antigen receptor is in parallel connection, and the dual-target chimeric antigen receptor comprises a first CAR and a second CAR, the first CAR comprises, from carboxyl terminus to amino terminus, a signal peptide, a first antigen binding region targeting TNFRSF10B, a first hinge region, a first transmembrane binding region, and a CD3 zeta, and the second CAR comprises, from carboxyl terminus to amino terminus, a signal peptide, a second antigen binding region targeting ASGR1, a second hinge region, a second transmembrane binding region, and a co-stimulatory signal binding region.

[0158] According to a preferred embodiment, the first CAR can be represented as TNFRSF10B ScFv-Hinge-TM-CD3 zeta.

[0159] According to a preferred embodiment, a tag protein for labeling, sorting, eliminating host cells can be present on the dual-target chimeric antigen receptor, and the tag protein is connected to the dual-target chimeric antigen receptor through a cleavable domain or another promoter.

[0160] According to a preferred embodiment, the first CAR comprises a tag protein. Preferably, the tag protein is a red fluorescent tag (dsRED). According to a preferred embodiment, the first hinge region of the first CAR comprises CD8aH (which can be represented as Hinge).

[0161] According to a preferred embodiment, the first transmembrane binding region can be anchored to the cell membrane. The second transmembrane binding region can be anchored to the cell membrane. According to a preferred embodiment, the first transmembrane binding region comprises CD8aTM.

[0162] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B is connected to the first transmembrane binding region through the first hinge region.

[0163] According to a preferred embodiment, the CD3 zeta is an intracellular signal transduction domain.

[0164] Specifically, the first CAR can be represented as CD8a signal peptide-TNFRSF10B VH-linker-TNFRSF10B VL-CD8aH-CD8aTM-CD3 zeta.

[0165] According to a preferred embodiment, the second CAR can be represented as ASGR1 VH-Hinge-TM-CD137-CD28.

[0166] According to a preferred embodiment, the second CAR can be represented as CD8a signal peptide-ASGR1 VH-CD8aH-CD8aTM-CD137 (4-1BB)-CD28.

[0167] According to a preferred embodiment, the second CAR comprises a tag protein. Preferably, the tag protein is a green fluorescent tag (eGFP). The second hinge region of the second CAR comprises CD8aH (which can be denoted as Hinge).

[0168] According to a preferred embodiment, the second transmembrane binding region comprises CD8TM.

[0169] According to a preferred embodiment, the co-stimulatory signaling region comprises a 4-1BB (CD137) co-stimulatory signaling domain and a CD28 co-stimulatory signaling domain.

[0170] According to a preferred embodiment, the intracellular co-stimulatory signaling domain can comprise a CD3 zeta intracellular signaling domain capable of activating an immune response cell.

[0171] According to a preferred embodiment, the immune response cell comprises a T lymphocyte, an NK cell, an NKT cell.

[0172] According to a preferred embodiment, the signal peptide can be a human CD8a signal peptide, a human GM-CSF signal peptide, a human insulin signal peptide, a human IL-2 signal peptide, a human trypsinogen signal peptide. Preferably, the signal peptide can be a human CD8a signal peptide, the amino acid sequence of which is shown as SEQ ID NO: 4.

[0173] According to a preferred embodiment, the amino acid sequence of the CD8a hinge region is shown as SEQ ID NO: 5.

[0174] According to a preferred embodiment, the amino acid sequence of the CD8aTM transmembrane binding region is shown as SEQ ID NO: 6.

[0175] According to a preferred embodiment, the amino acid sequence of the CD28 co-stimulatory signaling domain is shown as SEQ ID NO: 7.

[0176] According to a preferred embodiment, the amino acid sequence of the 4-1BB (CD137) co-stimulatory signaling domain is shown as SEQ ID NO: 8.

[0177] According to a preferred embodiment, the CD3 zeta is an intracellular signaling domain, the amino acid sequence of which is shown as SEQ ID NO: 9.

[0178] According to a preferred embodiment, the binding site of the first CAR-T is the primary recognition site of the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor.

[0179] According to a preferred embodiment, the binding site of the second CAR-T is the synergistic recognition site of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0180] Embodiment 3

[0181] The present embodiment provides a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0182] Embodiment 4

[0183] The present embodiment provides a vector containing a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. According to a preferred embodiment, the vector can be one or several of an adenovirus, a lentivirus, an adenoviral-associated virus or a retrovirus.

[0184] Embodiment 5

[0185] The present embodiment provides a host cell containing a vector or a chromosome as previously described, or a foreign nucleic acid molecule integrated in the aforementioned nucleic acid molecule. According to a preferred embodiment, the host cell expresses a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0186] Embodiment 6

[0187] The present embodiment provides a kit for preparing a host cell, which contains a nucleic acid molecule and / or a vector encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0188] Embodiment 7

[0189] The present embodiment provides a method for preparing an immune cell expressing a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, wherein the method comprises the following steps:

[0190] providing an immune cell to be modified; transducing a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor or a vector containing a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor into the immune cell, thereby obtaining a modified immune cell.

[0191] Embodiment 8

[0192] The present embodiment provides specific steps for constructing a CAR plasmid. The construction of the CAR plasmid includes: vector digestion, acquisition of the target gene fragment, exchange of the PCR product and the vector, transformation, colony PCR identification, sequencing, plasmid transformation, single colony picking, plasmid extraction, lentivirus packaging, lentivirus concentration, and fluorescence method for determining the titer of lentivirus.

[0193] Figure 1 is a schematic diagram of lentiviral backbone vector GV400 and GV401 plasmid structure, Figure 1A is a schematic diagram of GV400 plasmid structure, Figure 1B is a schematic diagram of GV401 plasmid structure. The element sequence of GV400 plasmid vector is EF1a-ScFv, and the cloning site is BamH I / EcoR I. The element sequence of GV401 plasmid vector is EF1a-ScFv-CarT-2A-EGFP, and the cloning site is BamH I / BamH I.

[0194] 1. Vector digestion

[0195] GV400 plasmid vector was digested with BamH I and EcoR I, and GV401 plasmid vector was digested with BamH I. A 50 μl digestion system was configured as shown in Table 1 and Table 2. According to the order in Table 1, various reagents were added in turn, mixed gently with a pipette, and placed at 37°C for 3h or overnight.

[0196] Table 1

[0197] Table 2

[0198] After the reaction was completed, the vector digestion products were subjected to agarose gel electrophoresis. The digestion results of the BamH I / EcoR I site are shown in Figure 2. In Figure 2, 10 kb Marker was used for electrophoresis, Lane 1 is Marker, and the bands from top to bottom are: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp. Lane 2 is the vector digestion product. Lane 3 is the uncut vector. The digestion results of the BamH I / BamH I site are shown in Figure 3. In Figure 3, 10 kb Marker was used for electrophoresis, Lane 1 is Marker, and the bands from top to bottom are: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp. Lane 2 is the vector digestion product. Lane 3 is the uncut vector.

[0199] GV400 plasmid vector was digested with BamH I / EcoR I, and GV401 plasmid vector was digested with BamH I / BamH I, both purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.

[0200] Recovery of target bands. The recovery process includes:

[0201] (1) Under long-wave ultraviolet lamp, cut the DNA band to be recovered with a clean blade, and try to remove the gel without DNA as much as possible, so as to obtain smaller gel volume;

[0202] (2) Put the cut gel containing DNA band into a 1.5 ml centrifuge tube and weigh it;

[0203] First, weigh an empty 1.5 ml centrifuge tube, then put the gel block into it and weigh it again. Subtract the two weights to get the weight of the gel;

[0204] (3) Add 3 times the volume of gel solution DD;

[0205] If the gel weighs 100 mg, its volume can be considered as 100 μl, and 300 μl of gel solution should be added. If the gel concentration is greater than 2%, 6 times the volume of gel solution should be added;

[0206] (4) Place in a 56°C water bath for 10 minutes (or until the gel is completely dissolved), and vortex every 2-3 minutes to help accelerate dissolution;

[0207] (5) Optionally, generally not needed, add 150 μl of isopropanol per 100 mg of initial gel weight, and mix well;

[0208] Sometimes adding isopropanol can improve recovery. Do not centrifuge after adding isopropanol. When recovering fragments greater than 4Kb, do not add isopropanol, as adding it may sometimes reduce recovery efficiency.

[0209] Equilibrium liquid pretreatment of the adsorption column:

[0210] It is necessary to pretreat the silica gel membrane adsorption column with equilibrium liquid. The specific method is as follows: take a new silica gel membrane adsorption column and place it in the collection tube, and then suck 100 μl of equilibrium liquid into the column, centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and then place the adsorption column back into the collection tube. At this time, the pretreatment of the column with equilibrium liquid is complete.

[0211] (6) Add the solution obtained in the previous step to the adsorption column EC (the adsorption column is placed in the collection tube), and let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube;

[0212] If the total volume exceeds 750 μl, the solution can be added to the same adsorption column EC in two portions.

[0213] After the filtrate is mixed with the residual strong alkaline equilibrium liquid in the collection tube, the sol solution may change from yellow to orange red or even purple, which is the normal color change of phenol red PH indicator under alkaline conditions.

[0214] (7) Add 600 μl of rinse solution WB (check if anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid;

[0215] (8) Add 600 μl of rinse solution WB, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid;

[0216] (9) Put the adsorption column EC back into the empty collection tube, centrifuge at 12,000 rpm for 2 minutes, try to remove the rinse solution as much as possible to avoid inhibition of the downstream reaction by residual ethanol in the rinse solution;

[0217] (10) Take out the adsorption column EC and put it into a clean centrifuge tube, add 50 μl of elution buffer EB (the elution buffer is better heated in advance in a water bath at 65-70°C) to the middle part of the adsorption membrane, stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute. If more DNA is needed, the obtained solution can be re-added to the adsorption column and centrifuged for 1 minute;

[0218] The larger the elution volume, the higher the elution efficiency. If a higher DNA concentration is needed, the elution volume can be appropriately reduced, but the minimum volume should not be less than 25 μl. Too small volume reduces the DNA elution efficiency and reduces the yield.

[0219] 2. Acquisition of the target gene fragment

[0220] The primers and plasmid templates used in this process were synthesized by Shanghai Jikai Gene Medical Technology Co., Ltd. The primer sequences of the target fragment corresponding to the cloning site BamH I / EcoR I are shown in the following table.

[0221] Primer description: contains exchange pairing bases, enzyme cutting site, and contains part of the sequence at the 5' end of the target gene for PCR fishing of the target gene.

[0222] The primer sequences of the target fragment corresponding to the cloning site BamH I / BamH I are shown in the following table.

[0223] The acquisition steps include: PCR amplification of the target gene fragment, configuration of the reaction system, as shown in Table 3, various reagents are added in the order shown in Table 3, gently blown and mixed with a pipette, and then centrifuged briefly and placed in a PCR instrument for reaction. The reaction conditions are shown in Table 4. The results of the target fragment corresponding to the cloning site BamH I / EcoR I are shown in Figure 4, and the size of the obtained PCR product is 2129 bp. In Figure 4, the markers from top to bottom are: 5 kb, 3 kb, 2 kb, 1.5 kb, 1 Kb, 750 bp, 500 bp, 250 bp, and 100 bp.

[0224] The results of the cloning site corresponding to the target fragment of BamH I / BamH I are shown in Figure 5, and the size of the obtained PCR product is 974 bp. In Figure 5, the markers from top to bottom are: 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, and 100 bp.

[0225] Table 3

[0226] Table 4

[0227] 3. Exchange of PCR product and vector

[0228] The reaction system shown in Table 5 was prepared in an ice water bath, mixed thoroughly (to avoid air bubbles), centrifuged quickly, and then reacted at 37°C for 30 min. After cooling in an ice water bath for 5 min, transformation was immediately performed to obtain the exchange reaction product. The PCR identification primers are shown in the following table, and the identification results of the target fragment exchanged into the linear expression vector corresponding to BamH I / EcoR I are shown in Figure 6. In Figure 6, the size of the PCR product of the positive transformant is 997 bp, lane 1 is the negative control (ddH2O), lane 2 is the negative control (empty self-ligation control group), lane 3 is the positive control (GAPDH), lane 4 is the marker, from top to bottom are: 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, and 100 bp, and lanes 5-12 are No. 1-8 transformants, respectively.

[0229] The PCR identification primers are shown in the following table, and the identification results of the target fragment exchanged into the linear expression vector corresponding to BamH I / BamH I are shown in Figure 7. In Figure 7, the size of the PCR product of the positive transformant is 1120 bp, lane 1 is the negative control (ddH2O), lane 2 is the negative control (empty self-ligation control group), lane 3 is the positive control (GAPDH), lane 4 is the marker, from top to bottom are: 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, and 100 bp, and lanes 5-12 are No. 1-8 transformants, respectively.

[0230] Table 5

[0231] 4. Transformation

[0232] Add 10 μl of the exchange reaction product to a centrifuge tube containing 100 μl of competent cells, shake well, and place on ice for 30 min. Heat shock at 42°C for 90 s, and immediately incubate in an ice water bath for 2 min. Add 500 μl of LB liquid medium to the mixture, and place on a 37°C shaker for 1 h. After incubation, take an appropriate amount of bacterial solution and evenly spread it on a plate containing ampicillin (Amp), and incubate in an inverted manner in a constant temperature incubator for 12-16 h.

[0233] 5. Colony PCR identification

[0234] The primers used in this process were synthesized by Shanghai Jikai Gene Medical Technology Co., Ltd.

[0235] Prepare the reaction system according to Table 6, and after preparation, shake the reaction system well, and centrifuge quickly. In a clean bench, pick a single colony into 20 μl of the identification system, mix well, and then place in a PCR instrument for reaction. The PCR reaction conditions are shown in Table 7.

[0236] Table 6

[0237] Table 7

[0238] 6. Sequencing

[0239] After identification, inoculate the positive clone transformant in an appropriate amount of LB liquid medium containing Amp, and incubate at 37°C for 12-16 h. Take an appropriate amount of bacterial solution for sequencing, and compare and analyze the sequencing results with the sequence of the target gene. The comparison results of the positive clone transformant of GV400 are shown in SEQ ID NO: 11, and SEQ ID NO: 11 is the complete nucleic acid sequence of the first antigen binding region targeting TNFRSF10B, including the nucleic acid sequence of the light chain variable region of the anti-TNFRSF10B antibody and the heavy chain variable region of the anti-TNFRSF10B antibody. The comparison results of the positive clone transformant of GV401 are shown in SEQ ID NO: 12, and SEQ ID NO: 12 is the nucleic acid sequence of the second antigen binding region targeting ASGR1, including the nucleic acid sequence of the heavy chain variable region of the anti-ASGR1 antibody.

[0240] 7. Plasmid transformation

[0241] The specific steps include: turn on the water bath in advance and heat it to 42°C; add 10 μl of plasmid to a centrifuge tube containing 100 μl of competent cells, shake the tube wall several times to mix well, and place on ice for 30 min; after 42°C heat shock for 90 s and ice water bath incubation for 2 min, add 500 μl of LB liquid medium, and place on a 37°C shaker for 1 h; after incubation, take an appropriate amount of bacterial solution and evenly spread it on a plate containing Amp, and incubate in a constant temperature incubator for 12-16 h.

[0242] 8. Pick single clone

[0243] Specific steps include: configuration of LB liquid medium containing Amp, 200 ml of LB liquid medium + 200 μl of Amp, mix well, microwave oven boiling; take autoclaved culture bottle, add 50 ml of LB liquid medium in each culture bottle for standby; take out the plate in the 37℃ constant temperature incubator, find single colony, pick up the colony and put it into the culture bottle; put the culture bottle in the constant temperature shaker, set the speed to 200 rpm, the temperature to 37℃, and shake the bacteria for 16-20h.

[0244] 9. Plasmid extraction

[0245] Specific steps include: transfer the culture medium in the culture bottle to a 50 ml centrifuge tube, centrifuge at 3000g for 10 min, discard the liquid; add 4 ml of Buffer P1 reagent to each centrifuge tube and mix well; add 4 ml of Buffer P2 reagent to each centrifuge tube, mix well by inverting, until the liquid becomes blue and sticky, stand for 3 min; add 4 ml of Buffer S3 reagent to each centrifuge tube, mix well by inverting, until the liquid becomes transparent, filter the liquid in the centrifuge tube through a needle tube, transfer the filtered liquid to a new 50 ml centrifuge tube; add 2 ml of Buffer BB to each centrifuge tube; filter the liquid in the centrifuge tube through a filter plug into the centrifugal column, remove the centrifugal column, centrifuge at 18000g for 1 min; add 0.7 ml of Buffer ETR to the centrifugal column, centrifuge at 18000g for 1 min, discard the liquid; add 0.7 ml of Buffer PE to the centrifugal column, centrifuge at 18000g for 1 min, discard the liquid, repeat once; empty the centrifugal column at 18000g for 1 min, discard the liquid, add 200 μl of Elution Buffer, stand for 1 min, centrifuge at 18000g for 1 min, transfer the collected liquid after centrifugation to a new EP tube; take 2 μl of plasmid to detect the concentration of plasmid.

[0246] 10. Lentivirus packaging

[0247] Specific steps include: resuscitation of 293T cells in advance, 24h before transfection, digestion of logarithmic growth phase 293T cells with trypsin, adjustment of cell density to about 5×10 6Cells / 15ml, re-inoculated in six-well plates, cultured at 37°C, 5% CO2 incubator for 24h, when the cell density reached 70%-80%, it can be used for transfection; according to the purpose of plasmid: psPAX2: pMD2G is 3:2:1 ratio, total mass of 4μg plasmid amount; the required volume of plasmid is added to 400μl of serum-free DMEM medium, 6μl of Turbo is added and mixed, and it is placed at room temperature for 15-20min; the mixed solution is slowly added to the culture dish, shaken, and cultured in a 37°C incubator; after 24h of culture, the liquid is changed, and the virus is collected once every 48h and once every 72h, and the two times of collected virus are mixed and prepared for concentration.

[0248] 11. Lentivirus concentration

[0249] The specific steps include: harvesting virus, transferring cell supernatant to a 50ml centrifuge tube, 3500g, centrifuging for 10min; adding virus concentration agent to a new 50ml centrifuge tube according to the volume ratio of cell supernatant: concentration reagent=3:1, pre-cooling in a 4°C refrigerator; transferring the centrifuged cell supernatant to the pre-cooled virus concentration reagent, mixing thoroughly, and low-speed shaking overnight in a 4°C refrigerator; taking the centrifuge tube after overnight, 3500g, 4°C, centrifuging for 1h; discarding the supernatant, 3500g, 4°C, centrifuging for 1min; preparing virus resuspension protection solution, wherein the volume ratio of virus resuspension solution: virus protection reagent=9:1, taking 9ml of virus resuspension solution and 1ml of virus protection reagent into a 15ml centrifuge tube for standby; taking out the centrifuged virus, discarding the supernatant, adding virus resuspension protection solution with a volume of 1%-10% of the virus stock solution, slow blowing for 20-30 times, and standing for 10min; 12000g, 4°C, centrifuging for 3-5min, and sucking the supernatant as concentrated virus; transferring the concentrated virus to a 1.5ml centrifuge tube and storing in a-80°C refrigerator.

[0250] 12. Fluorescent method for determining lentivirus titer

[0251] As shown in Figure 8, Figure 8A is a schematic diagram of virus gradient dilution; Figure 8B is a fluorescence microscope observation of plasmid transfection; and Figure 8C is a fluorescence microscope counting. The specific determination steps include: one day before determination, 293T adherent cells were plated in a 96-well plate, 4×10 4A cell, 100 μl of culture medium; according to the expected titer of the virus, 7-10 sterile EP tubes are prepared, 90 μl of serum-free medium is added to each tube; 10 μl of virus stock solution to be determined is added to the first tube, mixed well, and recorded as 1E+1 μl, the second EP tube is diluted by ten times, and the resulting virus stock solution is 1 / 10 of the first EP tube, recorded as 1E+0 μl, and so on, the eighth EP tube is diluted by seven times, recorded as 1E-6 μl; select the required cell hole, discard 90 μl of culture medium, add 90 μl of diluted virus solution, and incubate in a 37°C, 5% CO2 incubator; 24 h later, 100 ml of complete culture medium is added to the cell hole, and the process avoids blowing up the cells; 4 days later, observe the fluorescence expression, and the number of fluorescent cells decreases with the increase of the dilution multiple; according to the observation results of the fluorescence microscope, the virus titer is calculated, wherein the virus titer = the number of fluorescent cells / the amount of virus stock solution, for example: 1E-6 μl of virus stock solution infects a hole and 1 fluorescent cell is observed in the hole, which indicates that at least 1 virus particle has infected the cell, i.e. 1 / (1E-6) = 1E+6 (TU / μl), that is, the virus titer is 1x10 9 TU / ml.

[0252] Example 9

[0253] The present embodiment provides a method for preparing CAR-T cells. The preparation process includes: determining that the T cells are in good condition, setting the rotation speed to 1200 rpm, centrifuging for 5 min, resuspending with 1640 culture medium, and counting the cells; after counting, the cells are diluted to 1x10 6 / ml; take a six-well plate, add 500 μl of cell suspension, 500 μl of concentrated virus, and 40 μl of transduction promoting reagent to each well, and mix well; after mixing, place it in a 37°C incubator for culture, and after 8-12 h, add fresh culture medium and observe the cell state; after 3-4 days of infection, observe the fluorescence expression, and for cells that grow slowly or metabolize slowly, the infection time can be appropriately extended for observation, and the medium can be changed in the middle to maintain the growth activity of the cells.

[0254] Example 10

[0255] The embodiment is a method for detecting the transfection efficiency of CAR-T by flow cytometry and the detection result. The method for detecting the transfection efficiency of CAR-T comprises the following steps: taking a flow tube, labeling the tube, taking 1 ml of ASGR1 CAR-T cells, DR5 CAR-T cells and DR5 / ASGR1 CAR-T cells from each hole of a six-hole plate respectively, setting the rotation speed to 1200 rpm, centrifuging for 5 min, and discarding the liquid; adding 2 ml of PBS to resuspend the cells, uniformly oscillating, setting the rotation speed to 1200 rpm, centrifuging for 5 min, and discarding the liquid; adding 100 μl of PBS to resuspend the cells, and uniformly oscillating to machine; adjusting the voltage of the flow machine, selecting the FITC channel to observe ASGR1, selecting the PE channel to observe DR5, and observing the transfection efficiency of the two kinds of CAR-T cells.

[0256] The results are shown in FIG. 9. The CAR positive rate of DR5 CAR-T is 50.22%; the CAR positive rate of ASGR1 CAR-T is 54.33%; the single DR5z CAR positive rate of double-target DR5+ASGR1 CAR-T is 26.75%; the single ASGR1 CAR positive rate is 53.80%; and the DR5+ASGR1 double-target CAR positive rate is 20.59%.

[0257] Embodiment 11

[0258] The embodiment is a comparison result of the killing function of CAR-T cells with different effector-to-target ratios on hepatoma cells (LDH release test). LDH (lactate dehydrogenase) is an enzyme stably present in the cytoplasm of cells. In a normal state, it only exists in cells. When cells are stimulated to death, the plasma membrane is broken, and LDH is rapidly released to the outside of the cells (in this experiment, LDH is rapidly released to the cell culture solution). Therefore, the number of dead cells can be determined by the amount of LDH released by the damaged cell membrane detected in the LDH release test. The detection result of the LDH release test directly reflects the death rate of cells. The higher the absorbance measured, the stronger the cytotoxicity of the detected substance.

[0259] The specific operation steps include: target cell plating, resuspending the target cells (HepG2 cells) after digestion to 2×10 5 / ml, 50 μl / well, inoculating the target cells into a 96-well plate, and setting 3 parallel sub-wells for each group; effector cell plating, resuspending the effector cells (double-target CAR-T, single-target CAR-T or untransfected T cells) to 3.2×10 6 / ml, 1.6×10 6 / ml, 8×10 5 / ml, 4×10 5 / ml, 50 μl / well, mixing with the target cells, and incubating at 37°C, 5% CO2, with a killing time of 16-18 h.

[0260] Specifically, the present embodiment quantitatively detects the toxic killing effect on cells by detecting the LDH release in the cell culture supernatant. The test grouping provided by the present embodiment is as follows: ① experimental group: LDH released by target cells + effector cells; ② spontaneous control group: LDH spontaneously released by target cells; ③ maximum release group: LDH released after complete lysis of target cells; ④ effector cell spontaneous group: LDH spontaneously released by effector T cells; ⑤ volume control group: background value generated by blank medium, wherein the effector cells are untransfected T cells (NT in Figure 10, equivalent to untransfected T cells in the present embodiment), DR5 single-target CAR-T cells, and DR5 / ASGR1 double-target CAR-T cells. The detection method is carried out according to the instructions.

[0261] After the killing test, 10 μl of Lysis Buffer was added to the high control well, and then incubated at 37°C in a CO2 incubator for 30 min. Then, 100 μl of Working Solution was added to each well, and the entire culture plate was wrapped with aluminum foil and incubated at room temperature in the dark. Then, 50 μL of Stop Solution was added to each well, and immediately placed in a microplate reader to measure the absorbance at 490 nm.

[0262] The present embodiment provides a cytotoxicity calculation formula, which is: cytotoxicity (%) = (experimental group - spontaneous control group - effector cell spontaneous group) / (maximum release group - effector cell spontaneous group - volume control group) x 100%.

[0263] The comparison results of the killing effects of CAR-T cells with different effector-to-target ratios on liver cancer cells are shown in Figure 10. Specifically, the LDH release test compared the damage to tumor cells Huh7 after co-culturing the two CAR-T cells with the tumor cells at effector-to-target ratios of 2:1, 4:1, 8:1, and 16:1. The results of Figures 10A-10E show that the damage to tumor cells Huh7 by DR5 single-target CAR-T cells was higher than that by untransfected T cells at each effector-to-target ratio, and the damage to tumor cells Huh7 by DR5 / ASGR1 double-target CAR-T cells was higher than that by DR5 single-target CAR-T cells at each effector-to-target ratio.

[0264] Example 12

[0265] The present embodiment is the comparison results of the killing function of CAR-T cells on liver cancer cells (apoptosis detection of liver cancer cells).

[0266] Figure 11 is the flow cytometry detection of liver cancer cell apoptosis at an effector-to-target ratio of 2:1.

[0267] Flow cytometry was used to detect the apoptosis of tumor cells Huh7 and HepG2 after co-culture with DR5 single-target CAR-T cells and DR5 / ASGR1 double-target CAR-T cells. As shown in FIGS. 11A and 11B, the DR5 single-target CAR-T cells promoted the apoptosis of Huh7 cells better than the untransfected T cells (P<0.05), and the DR5 single-target CAR-T cells promoted the apoptosis of HepG2 cells better than the untransfected T cells (P<0.0001). The DR5 / ASGR1 double-target CAR-T cells promoted the apoptosis of Huh7 cells better than the DR5 single-target CAR-T cells, and the result was statistically significant (P<0.001). The DR5 / ASGR1 double-target CAR-T cells promoted the apoptosis of HepG2 cells better than the DR5 single-target CAR-T cells, and the result was statistically significant (P<0.01).

[0268] Example 13

[0269] This example provides the operating method of the cytotoxicity test (Granzym B release test). The specific operation steps include: taking the Huh7 / HepG2 cells grown to 90% from the incubator, discarding the supernatant, and washing once with 5 ml of PBS; adding 2 ml of trypsin, digesting in a 37°C incubator for 3 min, and neutralizing in 3 ml of MEM / DMEM complete medium; transferring the cells to a 15 ml centrifuge tube, setting the speed to 1200 rpm, and centrifuging for 5 min, discarding the supernatant; adding 3 ml of MEM / DMEM complete medium to resuspend the cells, taking 10 μl of the cell suspension for cell counting, and adjusting the tumor cell density to 1×10 6 / ml; taking 24-well plates, adding 500 μl of the cell suspension per well, and culturing; taking the untransfected T cells and CAR-T cells to a 15 ml centrifuge tube, setting the speed to 1200 rpm, and centrifuging for 5 min; adding 3 ml of 1640 medium to resuspend the cells, taking 10 μl of the cell suspension for counting, and adjusting the cell concentration to 1×10 6 / ml; adding 500 μl of the untransfected T cell suspension or CAR-T cell suspension per well to the 24-well plate, and incubating in the incubator for 18 h; taking 1 ml of the CAR-T cells per well from the 24-well plate to a flow tube, setting the speed to 1200 rpm, and centrifuging for 5 min, discarding the liquid, adding 1 μl of CD3 antibody, mixing well, and incubating in the dark for 30 min; washing once with PBS, adding the corresponding reagents (calcein AM (live cell indicator), SYTOX TMDeep Red nucleic acid stain (dead cell indicator), mix well, avoid light incubation for 30 min, PBS wash once; add Fix / perm buffer, mix well, avoid light incubation for 50 min; add Perm wash buffer and wash once, set the speed to 1200 rpm and centrifuge for 5 min; add the appropriate amount of Granzym B according to the number of cells, avoid light incubation for 40 min; wash once and discard the supernatant.

[0270] Example 14

[0271] This example provides the operation method and test results of the cytotoxicity test (CD107a expression experiment). CD107a is a highly glycosylated transmembrane protein present in lysosomes, and is one of the most abundant proteins in cytolytic granules, which can be used to evaluate NK cell or T cell activity. Specifically, granules reach the plasma membrane of NK / T cells, the granule membrane fuses with the cell membrane, and CD107a is exposed on the cell membrane surface. The membrane expression of CD107a molecules can directly reflect the degranulation process of NK cells or cytotoxic T cells (CTLs), and is directly related to the cytotoxic activity of NK cells or CTLs. After specific antibody labeling, flow cytometry detection can be performed.

[0272] The specific operation method includes: opening the biological safety cabinet 30 min in advance and irradiating with ultraviolet light for 30 min for standby; adjusting the cell density of HepG2 / Huh7 cells to 1×10 6 / ml, taking 1 ml per well and inoculating in a 24-well plate, and culturing in a 37℃ incubator; adjusting the concentration of untransfected T cells and three kinds of CAR-T cells to 1×10 6 / ml, respectively; dividing the cells into three groups, namely untransfected T cell group, DR5 CAR-T cell group and DR5 / ASGR1 CAR-T cell group, respectively adding 1 ml of corresponding CAR-T cell suspension into the corresponding cell group, and adding Golgi inhibitors monensin and Brefeldin a into the culture medium, and co-culturing in a 37℃ incubator for 18 h; taking the cells from the incubator, transferring the cells to a flow tube, setting the speed to 1200 rpm and centrifuging for 5 min, and discarding the liquid; adding 1 ml of PBS to resuspend the cells, setting the speed to 1200 rpm and centrifuging for 5 min, and discarding the liquid; adding CD107a antibody and avoiding light incubation for 30 min; adding 1 ml of PBS to resuspend the cells, setting the speed to 1200 rpm and centrifuging for 5 min, and discarding the liquid; adding 100 μl of PBS dropwise and detecting on the machine.

[0273] Figure 12A is the flow cytometry detection of CD107a expression of three T cells (untransfected T cells, DR5 single-target T cells, and DR5 / ASGR1 double-target T cells) after co-incubation with liver cancer cells Huh-7 and HepG2, respectively; Figure 12B is the flow cytometry detection of Granzym B expression of three T cells after co-incubation with liver cancer cells Huh-7 and HepG2, respectively. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3.

[0274] Figure 12 shows that the amount of CD107a released after co-culturing DR5 single-target CAR-T with Huh7 cells is higher than that of untransfected T cells (P<0.01); the amount of CD107a released after co-culturing DR5 / ASGR1 double-target CAR-T with Huh7 cells is higher than that of DR5 single-target CAR-T (P<0.001). The amount of CD107a released after co-culturing DR5 single-target CAR-T with HepG2 cells is higher than that of untransfected T cells (P<0.001); the amount of CD107a released after co-culturing DR5 / ASGR1 double-target CAR-T with HepG2 cells is higher than that of DR5 single-target CAR-T (P<0.01) (Figure 12A). The amount of Granzym B released after co-culturing DR5 single-target CAR-T with Huh7 cells is higher than that of untransfected T cells (P<0.001); the amount of Granzym B released after co-culturing DR5 / ASGR1 double-target CAR-T with Huh7 cells is higher than that of DR5 single-target CAR-T (P<0.001). The amount of Granzym B released after co-culturing DR5 single-target CAR-T with HepG2 cells is higher than that of untransfected T cells (P<0.01); the amount of Granzym B released after co-culturing DR5 / ASGR1 double-target CAR-T with HepG2 cells is higher than that of DR5 single-target CAR-T (P<0.01) (Figure 12B).

[0275] Example 15

[0276] This example provides the test results of FZJD drug-containing serum enhancing the inhibitory effect of DR5 / ASGR1 double-target CAR-T cells on liver cancer cells.

[0277] The present example detects the inhibitory effect of FZJD drug-containing serum combined with DR5 / ASGR1 double-target CAR-T cells on tumor cell proliferation by CCK8 method. The results are shown in Figure 13. When the serum concentration is 30%, FZJD drug-containing serum can effectively increase the effect of DR5 / ASGR1 double-target CAR-T cells on inhibiting the growth of Huh7 cells, and the result is statistically significant (P<0.05) (Figure 13A). When the serum concentration is 20% and 30%, FZJD drug-containing serum can effectively increase the effect of CAR-T cells on inhibiting the growth of HepG2 cells (Figure 13B), and the result is statistically significant.

[0278] Example 16

[0279] The present example provides the test results of FZJD drug-containing serum enhancing the apoptosis effect of DR5 / ASGR1 double-target CAR-T cells on hepatoma cells, and the results are shown in Figure 14.

[0280] The present example detects the apoptosis effect of FZJD drug-containing serum on increasing DR5 / ASGR1 double-target CAR-T cells on tumor cells Huh7 and HepG2 by flow cytometry. As shown in Figures 14A and B, the apoptosis-promoting effect of DR5 / ASGR1 double-target CAR-T cells on Huh7 cells is better than that of untransfected T cells (P<0.0001). The apoptosis-promoting effect of DR5 / ASGR1 double-target CAR-T cells on HepG2 cells is better than that of untransfected T cells (P<0.001). FZJD drug-containing serum can effectively increase the apoptosis-promoting effect of DR5 / ASGR1 double-target CAR-T cells on Huh7 cells, and the result is statistically significant (P<0.01). FZJD drug-containing serum can effectively increase the apoptosis-promoting effect of DR5 / ASGR1 double-target CAR-T cells on HepG2 cells, and the result is statistically significant (P<0.05).

[0281] Example 17

[0282] The present example provides the release of Granzym B after the combined action of FZJD drug-containing serum and DR5 / ASGR1 double-target CAR-T cells.

[0283] The specific operation method for detecting the release of Granzym B after the combined action of FZJD drug-containing serum and DR5 / ASGR1 double-target CAR-T cells includes: digesting Huh7 / HepG2 cells growing to 90%, and adjusting the tumor cell density to 1×10 6 / ml; take 24-well plates, add 500 μl of cell suspension to each well, and incubate in a 37°C incubator; take untransfected T cells and double-target CAR-T cells into 15 ml centrifuge tubes, respectively, and adjust the cell concentration to 1×10 6 / ml; the cells were divided into three groups, namely the untransfected T cell group, the DR5 / ASGR1 double-target CAR-T cell group and the FZJD combined with DR5 / ASGR1 double-target CAR-T cell group, 500 μl of untransfected T cell suspension was added to the untransfected T cell group, 500 μl of CAR-T cell suspension was added to the DR5 / ASGR1 double-target CAR-T cell group and the FZJD combined with DR5 / ASGR1 double-target CAR-T cell group, and 500 μl of culture medium containing normal rat serum was added to the untransfected T cell group and the DR5 / ASGR1 double-target CAR-T cell group, and the FZJD combined with DR5 / ASGR1 double-target CAR-T cell group was added with FZJD drug-containing serum, so that the serum concentration of each group was 30%, and the culture was carried out in a 37°C incubator for 18h; 1ml of CAR-T cell suspension was taken from each well of the 24-well plate into a flow tube, the rotation speed was set to 1200rpm, and centrifugation was carried out for 5min, the liquid was discarded, 1 μl of CD3 antibody was added, and mixing was carried out, and incubation was carried out for 30min in the dark; PBS was washed once, live / dead was added and mixed, and incubation was carried out for 30min in the dark, and PBS was washed once; Fix perm buffer was added, mixed, and incubated for 50min in the dark; Perm wash buffer was added and washed once, the rotation speed was set to 1200rpm, and centrifugation was carried out for 5min; the appropriate amount of granzyme B antibody was added according to the cell number, and incubation was carried out for 40min in the dark; it was washed once, the supernatant was discarded, and it was put on the machine.

[0284] Example 18

[0285] This example provides the detection of CD107a expression after the combined action of FZJD drug-containing serum and DR5 / ASGR1 double-target CAR-T cells and the test results, and the results are shown in Figure 15.

[0286] The specific operation method for detecting the CD107a expression after the combined action of FZJD drug-containing serum and DR5 / ASGR1 double-target CAR-T cells includes: taking Huh7 / HepG2 cells growing to 90% from the incubator, washing once with 5ml of PBS; adding 2ml of trypsin, incubating in a 37°C incubator for 3min, and neutralizing in 3ml of MEM / DMEM complete culture medium; transferring the cells to a 15ml centrifuge tube, setting the rotation speed to 1200rpm, and centrifuging for 5min, and discarding the supernatant; adding 3ml of MEM / DMEM complete culture medium to resuspend the cells, taking 10 μl of cell suspension for counting, adjusting the tumor cell density to 1×10 6 / ml; take 24-well plates, 500 μl of cell suspension per well, 37°C incubator culture; take the untransfected T cells and DR5 / ASGR1 double-target CAR-T cells into 15-ml centrifuge tubes, set the rotation speed to 1200 rpm, centrifuge for 5 min; add 3 ml of 1640 culture medium to resuspend the cells, take 10 μl of cell suspension for counting, adjust the cell concentration to 1 x 10 6 / ml; the cells are divided into three groups, namely the untransfected T cell group, the DR5 / ASGR1 double-target CAR-T cell group and the Fuzheng Jiedu Xiaoshi Decoction combined with DR5 / ASGR1 double-target CAR-T cell group, 500 μl of untransfected T cell suspension is added to the untransfected T cell group, 500 μl of CAR-T cell suspension is added to the DR5 / ASGR1 double-target CAR-T cell group and the Fuzheng Jiedu Xiaoshi Decoction combined with DR5 / ASGR1 double-target CAR-T cell group, and 500 μl of culture medium containing normal rat serum is added to the untransfected T cell group and the DR5 / ASGR1 double-target CAR-T cell group, the Fuzheng Jiedu Xiaoshi Decoction combined with DR5 / ASGR1 double-target CAR-T cell group is added with Fuzheng Jiedu Xiaoshi Decoction drug-containing serum, so that the serum concentration of each group is 30%, and Golgi inhibitors monensin and brefeldin a are added to the culture medium, and cultured in a 37°C incubator for 18 h; remove the cells from the incubator, transfer the cells to flow tubes, set the rotation speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add 1 ml of PBS to resuspend the cells, set the rotation speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add CD107a antibody, avoid light incubation for 30 min; add 1 ml of PBS to resuspend the cells, set the rotation speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add 100 μl of PBS dropwise, and detect on the machine.

[0287] Figure 15A is a flow cytometry detection of T cell CD107a expression; Figure 15B is a flow cytometry detection of T cell Granzym B expression, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3. After co-culturing DR5 / ASGR1 double-target CAR-T cells with Huh7 cells, the CD107a release amount was higher than that of untransfected T cells (P<0.001), and FZJD drug-containing serum could significantly increase the CD107a release amount after co-culturing DR5 / ASGR1 double-target CAR-T cells with Huh7 cells (P<0.01). After co-culturing DR5 / ASGR1 double-target CAR-T cells with HepG2 cells, the CD107a release amount was higher than that of untransfected T cells (P<0.0001), and FZJD drug-containing serum could significantly increase the CD107a release amount after co-culturing DR5 / ASGR1 double-target CAR-T cells with HepG2 cells (P<0.05) (Figure 15A). After co-culturing DR5 / ASGR1 double-target CAR-T cells with Huh7 cells, the Granzym B release amount was higher than that of untransfected T cells (P<0.0001), and FZJD drug-containing serum could significantly increase the Granzym B release amount after co-culturing DR5 / ASGR1 double-target CAR-T cells with Huh7 cells (P<0.001). After co-culturing DR5 / ASGR1 double-target CAR-T cells with HepG2 cells, the Granzym B release amount was higher than that of untransfected T cells (P<0.001), and FZJD drug-containing serum could significantly increase the Granzym B release amount after co-culturing DR5 / ASGR1 double-target CAR-T cells with HepG2 cells, and the Granzym B release amount was higher than that of DR5 / ASGR1 double-target CAR-T cells (P<0.05) (Figure 15B).

[0288] Example 19

[0289] This example provides a method for detecting the phenotype changes of FZJD combined DR5 / ASGR1 double-target CAR-T cells by flow cytometry and the test results, and the results are shown in Figure 16.

[0290] Studies have shown that STAT3 signal activation can cause tumor cell PD-L1 expression to increase, causing T cells inside the tumor to be exhausted, and the synergistic expression of IL-6 and PD-L1 can significantly inhibit the infiltration ability of CD8 + T cells in tumors.

[0291] The specific procedure for flow cytometry detection of phenotypic changes in FZJD combined with DR5 / ASGR1 dual-target CAR-T cells includes: preparing a mixed antibody against CD3 / CD4 / CD8 / CD45RA / CCR7 / CD38 / HLA4 / PD1 / TIGIT / TIM3 / CTLA4; transferring CAR-T cells from a T25 cell culture flask to a 15ml centrifuge tube, centrifuging at 1200rpm for 5min, resuspending the cells in 1640 medium, counting the cells, and adjusting the cell concentration to 1×10⁻⁶. 6 / ml; Transfer the cell suspension to 24-well plates and divide them into FZJD-containing serum group and normal rat serum group; Add FZJD-containing serum and normal rat serum to the corresponding wells and incubate at 37℃ for 24h; Remove the cells, centrifuge at 1200rpm for 5min and discard the liquid; Add 1ml of PBS to resuspend the cells, centrifuge at 1200rpm for 5min and discard the liquid; Add mixed antibody and incubate in the dark for 30min; Add 1ml of PBS to resuspend the cells, centrifuge at 1200rpm for 5min and discard the liquid; Add 100μl of PBS and perform instrumental analysis.

[0292] The results are shown in Figure 16. Figures 16A and 16B show the results of T cell phenotype detection by flow cytometry; Figure 16C shows the results of CD4 detection by flow cytometry. + T cell proportion; Figure 16D shows the tumor cell phenotype detected by flow cytometry, *P<0.05, **P<0.01, vs Control, n=3. FZJD-containing serum can increase CD4+. + Naïve T cells in CAR-T cells ( The ratio of naive T cells (Cells) and central memory T cells (TCMs) was compared. Specifically, FZJD-containing serum had no statistically significant effect on increasing naive T cells (P > 0.05), while FZJD-containing serum had a statistically significant effect on increasing central memory T cells (P < 0.01). FZJD-containing serum can increase CD8+. + Naïve T cells in CAR-T cells ( The proportion of naive T cells and central memory T cells (TCM) was increased by FZJD drug-containing serum, and the increase of both naive T cells and central memory T cells was statistically significant (P<0.05) (Figure 16A). The effect of FZJD drug-containing serum on the expression of immune stimulatory receptors and immune inhibitory receptors on the surface of CAR-T cells was detected by flow cytometry in this example. The results showed that FZJD drug-containing serum could significantly increase the expression of immune stimulatory receptors on the surface of CAR-T cells (P<0.01), significantly reduce the expression of immune inhibitory receptor TIGIT on the surface of CAR-T cells (P<0.05), and FZJD drug-containing serum had a trend of reducing the expression of immune inhibitory receptors PD-1, TIM3 and CTLA-4 on the surface of CAR-T cells (Figure 16B). Flow cytometry was used to detect whether FZJD drug-containing serum could change the CD8 + The proportion of T cells was not significantly different (Figure 16C). Flow cytometry was used to detect whether FZJD drug-containing serum could reduce the expression of PD-L1 on the surface of tumor cells, and the results showed that FZJD drug-containing serum had a trend of reducing the expression of PD-L1 on the surface of tumor cells (Figure 16D).

[0293] Example 20

[0294] This example provides the test results of FZJD drug-containing serum reducing the release level of inflammatory cytokines in the process of killing target cells by hepatocellular carcinoma cells and DR5 / ASGR1 double-target CAR-T.

[0295] Cytokine storm (CRS) is a common adverse event related to CAR-T immunotherapy, and its incidence is high. Interleukin 6 (IL-6) is a pro-inflammatory cytokine that is widely involved in various inflammatory responses in the body and plays an important role in the "cytokine storm" that occurs in CAR-T immunotherapy. IL-6 is both an activator of STAT3 and a downstream product of the STAT3 pathway. Activation of STAT3 can promote hepatocellular carcinoma cells to secrete IL-6, forming a positive feedback effect, exacerbating cytokine storm, and creating a persistent chronic inflammatory environment in the tumor, promoting tumor progression through multiple pathways. In the phase I study of GPC3 single-target CAR-T in CRS, 70% of patients developed CRS. Four patients received high-dose hormone shock therapy, and two of them also received monoclonal antibody tocilizumab (tocilizumab) treatment against IL-6 receptor. Therefore, inhibiting IL-6 can effectively inhibit cytokine storm and reduce adverse reactions during treatment. In addition, activation of STAT3 can reduce the expression of chemokine receptor CXCR3 on the surface of CD8 + T cells, and reduce the secretion of IFN-γ by NK, CD4 + T cells, and reduce the secretion of IFN-γ by NK, CD4 +The level of T chemokine CXCL10 leads to CD8 + Cells have difficulty chemotactically attracting tumor tissue.

[0296] This embodiment used ELISA to detect the expression of IL-6 in control serum, FZJD-containing serum, DR5 / ASGR1 dual-target CAR-T cells, and the cell supernatant after co-culturing FZJD combined with DR5 / ASGR1 dual-target CAR-T cells and target cells HepG2, and observed the changes in IL-6 release caused by FZJD-containing serum. As shown in Figure 17, co-culturing DR5 / ASGR1 dual-target CAR-T cells with target cells significantly increased the expression of the pro-inflammatory factor IL-6 (P < 0.01), while FZJD-containing serum effectively reduced the release of the pro-inflammatory factor IL-6 after co-culturing DR5 / ASGR1 dual-target CAR-T cells with target cells, which may effectively inhibit the formation of cytokine storm during CAR-T therapy (P < 0.01).

[0297] Example 21

[0298] This embodiment provides experimental results showing that FZJD-containing serum increases the level of interferon release in DR5 / ASGR1 dual-target CAR-T cells when killing liver cancer cells.

[0299] In this embodiment, the expression of interferon-γ (IFN-γ) in the cell supernatant after co-culturing FZJD-containing serum with DR5 / ASGR1 dual-target CAR-T cells and target cells HepG2 was detected by ELISA, demonstrating the promoting effect of FZJD-containing serum on the killing of target cells by DR5 / ASGR1 dual-target CAR-T cells. The results are shown in Figure 18.

[0300] The results are shown in Figures 18A (target cells are HepG2) and 18B (target cells are HepG3B). FZJD-containing serum can effectively enhance the ability of co-cultured DR5 / ASGR1 dual-target CAR-T cells to secrete IFN-γ and improve their killing function (P < 0.05).

[0301] Example 22

[0302] This embodiment provides experimental results showing that FZJD-containing serum increases the release level of T-cell chemokines in liver cancer cells, as shown in Figure 19.

[0303] The CXCR3 / CXCL10 chemokine axis is an important signaling pathway for T cell migration, and CXCL9 / 10 / 11 can recruit CD8+ cells. +Various immune cells of T cells directly kill tumor cells or play an anti-tumor effect, thereby improving the overall survival rate of patients. The expression defect of CXCL9 / 10 / 11 at the tumor site may be one of the mechanisms causing tumor immune escape. This embodiment detects the release level of T cell chemotactic factor of hepatocellular carcinoma cells by FZJD drug-containing serum by ELISA, and the results are shown in FIGS. 19A to C, *P<0.05, **P<0.01, ***P<0.001, n=3. The results show that the contents of CXCL9 / 10 / 11 in the FZJD group and the STAT3 inhibitor (stattic) group are significantly increased (p<0.05).

[0304] Embodiment 23

[0305] This embodiment provides a tandem chimeric antigen receptor. According to a preferred embodiment, the tandem chimeric antigen receptor can be A VH-linker-A VL-Linker-B VL-Linker-B VH or A VHH-linker-B VHH.

[0306] A and B can be TNFRSF10B and ASGR1, and the positions can be interchanged; the positions of VH and VL of the same protein can be interchanged.

[0307] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-linker-ASGR1 VHH.

[0308] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-linker-ASGR1 VH.

[0309] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-linker-ASGR1 VL-ASGR1 VH.

[0310] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-linker-ASGR1 VH-ASGR1 VL.

[0311] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can be TNFRSF10B VH-linker-TNFRSF10B VL-Linker-ASGR1 VHH.

[0312] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be TNFRSF10B VL-linker-TNFRSF10B VH-Linker-ASGR1 VHH.

[0313] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0314] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0315] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0316] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0317] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0318] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0319] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0320] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0321] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0322] Example 24

[0323] The embodiment provides a TNFRSF10B / ASGR1 double-target tandem CAR-T, and FIG. 31 is a schematic diagram of the TNFRSF10B / ASGR1 double-target tandem CAR-T. The structure of the TNFRSF10B / ASGR1 double-target tandem chimeric antigen receptor is shown in FIG. 32. Specifically, the TNFRSF10B / ASGR1 double-target tandem CAR comprises: a CD8a signal peptide, an ASGR1 scFv, a G4S linker, a DR5 scFv, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB, a CD28 costimulatory molecule, and a CD3 zeta signal transduction molecule. Meanwhile, the TNFRSF10B / ASGR1 double-target tandem CAR is connected with an mCherry gene sequence through a T2A. After synthesizing the ASGR1 scFv molecule sequence gene in the embodiment, the ASGR1 scFv molecule sequence gene is connected to a DR5 scfv CD28 Costi CD3 zeta CAR lentivirus vector, as shown in FIG. 33. According to the embodiment, the nucleotide sequence of the TNFRSF10B / ASGR1 double-target tandem CAR is shown as SEQ ID NO: 13. The amino acid sequence of the TNFRSF10B / ASGR1 double-target tandem CAR is shown as SEQ ID NO: 14.

[0324] The present embodiment provides the detection results of residual tumor cells of lung cancer calu-3 cells killed by the TNFRSF10B / ASGR1 double-target tandem CAR-T detected by flow cytometry, as shown in Figures 21-25. In Figure 21, the effector-target ratios of lung cancer calu-3 cells killed by the double-target tandem CAR-T are 1:1, 2:1 and 5:1, the killing time is 24 hours, and the control group is T cells transfected with a plasmid without scfv (indicated as Mock T in Figure 21). In Figure 21, the abscissa (FITC-H) represents the intensity of the fluorescence signal, which reflects the intensity of the binding of the cell surface or the inside with the FITC-labeled antibody (such as tumor-specific antigen antibody), which is used to distinguish tumor cells (positive signal) from non-tumor cells (negative or low signal), and the ordinate (SSC-H) is the intensity of the side scatter light, which is used to distinguish different cell types; in P5 / E7, P5 refers to the tumor cell gate circled by SSC-H and FITC-H, and E7 represents the number of events (cell number) in the gate, indicating that specific residual tumor cells are detected. When the effector-target ratio is 1:1, the residual calu-3 cells of the Mock T control group are 40.01%, while the residual calu-3 cells of the Tan DR5z-ASGR1 CAR T group are 25.29%; when the effector-target ratio is 2:1, the residual calu-3 cells of the Mock T control group are 32.79%, while the residual calu-3 cells of the Tan DR5z-ASGR1 CAR T group are 11.57%; when the effector-target ratio is 5:1, the residual calu-3 cells of the Mock T control group are 16.08%, while the residual calu-3 cells of the Tan DR5z-ASGR1 CAR T group are 4.79%.

[0325] Figure 22 is the statistical results of residual tumor cells of lung cancer calu-3 cells killed by the TNFRSF10B / ASGR1 double-target tandem CAR-T detected by flow cytometry in Figure 21, wherein the abscissa is the effector-target ratio of the control group (indicated as Mock T+calu-3 in Figure 22) and the TNFRSF10B / ASGR1 double-target tandem CAR-T group (indicated as CART+calu-3 in Figure 22), and the ordinate is the proportion of residual tumor cells (%), and the killing time is 24 hours. When the effector-target ratio (E:T) is 1:1, 2:1 and 5:1, the proportion of residual tumor cells of the control group is significantly greater than that of the TNFRSF10B / ASGR1 double-target tandem CAR-T group (all with statistical significance).

[0326] Figure 23 is a graph showing the luciferase fluorescence expression intensity of tumor cells killed by different groups of cells against lung cancer calu-3 cells, wherein the abscissa is the effector-to-target ratio of the control group (Mock T+calu-3 in Figure 23) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (CAR-T+calu-3 in Figure 23), and the ordinate is the luciferase activity (%), and the killing time is 24 hours. When the effector-to-target ratio (E:T) is 1:1, 2:1 and 5:1, the tumor cell death rate of the control group is significantly lower than that of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0327] Figure 24 is a statistical result of LDH release test for tumor cells killed by different groups of cells against lung cancer calu-3 cells, wherein the abscissa is the effector-to-target ratio of the control group (Mock T+calu-3 in Figure 24) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (CAR-T+calu-3 in Figure 24), and the ordinate is the LDH release rate (%), and the killing time is 24 hours. When the effector-to-target ratio (E:T) is 1:1, 2:1 and 5:1, the tumor cell death rate of the control group is significantly lower than that of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0328] Figure 25 is a statistical result of interferon (IFN-γ) release detected by ELISA for tumor cells killed by different groups of cells against lung cancer calu-3 cells, wherein the abscissa is the effector-to-target ratio of the control group (Mock T+calu-3 in Figure 25) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (CAR-T+calu-3 in Figure 25), and the ordinate is the release amount of IFN-γ (pg / ml), and the killing time is 24 hours. When the effector-to-target ratio (E:T) is 1:1, 2:1 and 5:1, the release amount of IFN-γ of the control group is significantly lower than that of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0329] The results of FIGS. 21-25 show that the TNFRSF10B / ASGR1 dual-target tandem CAR-T group has a better killing effect on lung cancer cells than the Mock T control group. Specifically, the residual tumor cell proportion of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group is significantly reduced, the tumor cell death rate is significantly increased, the LDH release amount is significantly increased, and the IFN-γ release amount is significantly increased. That is, the TNFRSF10B / ASGR1 dual-target tandem CAR-T cells can significantly improve the killing effect of tumor cells compared with T cells.

[0330] Example 25

[0331] This example provides the detection results of flow cytometry for detecting the residual tumor cells of TNFRSF10B / ASGR1 dual-target tandem CAR-T killing hepatocarcinoma hepG2 cells, as shown in FIGS. 26-29. In FIG. 26, the effector-target ratio of the dual-target tandem CAR-T killing hepatocarcinoma hepG2 cells is 1:1, 2:1 and 5:1, the killing time is 24 hours, and the control group is T cells transfected with a plasmid without scfv (indicated as Mock T in FIG. 26). When the effector-target ratio is 1:1, the residual hepG2 cells of the Mock T control group are 47.36%, while the residual hepG2 cells of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group are 28.89%; when the effector-target ratio is 2:1, the residual hepG2 cells of the Mock T control group are 26.08%, while the residual hepG2 cells of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group are 22.33%; when the effector-target ratio is 5:1, the residual hepG2 cells of the Mock T control group are 17.92%, while the residual hepG2 cells of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group are 8.23%. The statistical results of flow cytometry for detecting residual tumor cells corresponding to FIG. 26 are shown in FIG. 27. In FIG. 27, the abscissa is the effector-target ratio of the control group (indicated as Mock T+HepG2 in FIG. 27) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (indicated as CART+HepG2 in FIG. 27), and the ordinate is the residual tumor cell proportion (%), the killing time is 24 hours. When the effector-target ratio is 1:1, 2:1 and 5:1, the residual tumor cell proportion of the control group is significantly greater than that of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group, and the difference is statistically significant when the effector-target ratio is 1:1 and 5:1.

[0332] Figure 28 shows the statistical results of the interferon (IFN-γ) release amount of different groups of cells killing liver cancer HepG2 cells detected by ELISA, wherein the abscissa is the effector-to-target ratio of the control group (Mock T+HepG2 in Figure 28) and the TNFRSF10B / ASGR1 double-target tandem CAR-T group (CART+HepG2 in Figure 28), and the ordinate is the release amount of IFN-γ (pg / ml). The release amount of IFN-γ of the control group was significantly lower than that of the TNFRSF10B / ASGR1 double-target tandem CAR-T group (all with statistical significance) when the effector-to-target ratio (E:T) was 1:1, 2:1 and 5:1, and the killing time was 24 hours.

[0333] Figure 29 shows the statistical results of the release of LDH of tumor cells killed by different groups of cells killing liver cancer HepG2 cells detected by LDH release test, wherein the abscissa is the effector-to-target ratio of the control group (Mock T+HepG2 in Figure 29) and the TNFRSF10B / ASGR1 double-target tandem CAR-T group (CART+HepG2 in Figure 29), and the ordinate is the release rate of LDH (%). The tumor cell death rate of the control group was significantly lower than that of the TNFRSF10B / ASGR1 double-target tandem CAR-T group when the effector-to-target ratio (E:T) was 1:1, 2:1 and 5:1, and the killing time was 24 hours.

[0334] Figures 26-29 show that the TNFRSF10B / ASGR1 double-target tandem CAR-T group has better killing effect on liver cancer cells than the Mock T control group. Specifically, the residual tumor cell proportion of the TNFRSF10B / ASGR1 double-target tandem CAR-T group is significantly reduced, the tumor cell death rate is significantly increased, the release amount of IFN-γ is significantly increased, and the release amount of LDH is significantly increased. That is, the TNFRSF10B / ASGR1 double-target tandem CAR-T cells can significantly improve the killing effect of tumor cells compared with T cells.

[0335] Example 26

[0336] This example provides a comparison of the liver cancer HepG2 cell killing effect of single-target CAR-T (TNFRSF10B-28z single-target CAR-T), TNFRSF10B / ASGR1 double-target tandem CAR-T and TNFRSF10B / ASGR1 double-target parallel CAR-T, and the results are shown in Figures 30.1 and 30.2.

[0337] Figure 30.1 is a flow cytometry detection result of residual tumor cells of each group of CAR-T cells killing liver cancer HepG2 cells at different effector to target ratios.

[0338] Figure 30.2A is a statistical result of the proportion of dead tumor cells of each group of cells killing liver cancer HepG2 cells at different effector to target ratios. In Figure 30.2B, the horizontal axis is the effector to target ratio, and the vertical axis is the proportion of dead tumor cells (%). When the effector to target ratio is 1:1 and 2:1, the killing effect of TNFRSF10B / ASGR1 double-target parallel CAR-T cells and TNFRSF10B / ASGR1 double-target series CAR-T cells on liver cancer HepG2 cells is better than that of single-target CAR-T, and the killing effect of TNFRSF10B / ASGR1 double-target parallel CAR-T cells and TNFRSF10B / ASGR1 double-target series CAR-T cells is similar.

[0339] Figure 30.2C is a statistical result of the IFN-γ release amount of each group of cells killing liver cancer HepG2 cells, wherein the horizontal axis is the effector to target ratio, and the vertical axis is the IFN-γ release amount (pg / ml). When the effector to target ratio is 1:1 and 2:1, the IFN-γ release amount of the TNFRSF10B / ASGR1 double-target parallel CAR-T group and the TNFRSF10B / ASGR1 double-target series CAR-T group is significantly higher than that of the TNFRSF10B-28z single-target CAR-T group. When the effector to target ratio is 1:1, the IFN-γ release amount of the TNFRSF10B / ASGR1 double-target parallel CAR-T group and the TNFRSF10B / ASGR1 double-target series CAR-T group is similar, while when the effector to target ratio is 2:1, the IFN-γ release amount of the TNFRSF10B / ASGR1 double-target series CAR-T group is significantly higher than that of the TNFRSF10B / ASGR1 double-target parallel CAR-T group, that is, the ability of TNFRSF10B / ASGR1 double-target series CAR-T cells to release IFN-γ is better than that of TNFRSF10B / ASGR1 double-target parallel CAR-T cells.

[0340] The present application reserves the right to modify the heavy chain variable domain VH of scFv or humanized heavy chain antibody into a camel antibody heavy chain variable domain VHH.

Claims

1. A pharmaceutical composition, characterized by, The TNFRSF10B / ASGR1 double-target chimeric antigen receptor T cell and the Fuzheng Jiedu Xiaojijiang prescription, The double-target chimeric antigen receptor of the TNFRSF10B / ASGR1 double-target chimeric antigen receptor T cell comprises a signal peptide, a first antigen binding region targeting TNFRSF10B, a second antigen binding region targeting ASGR1, a hinge region, a transmembrane region, and an intracellular region.

2. The pharmaceutical composition of claim 1, wherein, The first antigen binding region targeting TNFRSF10B comprises an anti-TNFRSF10B antibody light chain variable region and an anti-TNFRSF10B antibody heavy chain variable region, wherein the amino acid sequence of the anti-TNFRSF10B antibody light chain variable region is shown in SEQ ID NO: 1, and the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is shown in SEQ ID NO: 2; The second antigen binding region targeting ASGR1 comprises an anti-ASGR1 antibody heavy chain variable region, wherein the amino acid sequence of the anti-ASGR1 antibody heavy chain variable region is shown in SEQ ID NO:

3.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The double-target chimeric antigen receptor of the TNFRSF10B / ASGR1 double-target chimeric antigen receptor T cell is in parallel type.

4. The pharmaceutical composition according to claim 1 or 2, characterized by, The TNFRSF10B / ASGR1 double-target chimeric antigen receptor comprises a first CAR and a second CAR, The first CAR comprises, from the carboxyl end to the amino end, a first antigen binding region targeting TNFRSF10B, a first hinge region, a first transmembrane binding region, and CD3 zeta; and the second CAR comprises, from the carboxyl end to the amino end, a signal peptide, a second antigen binding region targeting ASGR1, a second hinge region, a second transmembrane binding region, and a co-stimulatory signal binding region.

5. The pharmaceutical composition of claim 1, wherein, The double-target chimeric antigen receptor of the TNFRSF10B / ASGR1 double-target chimeric antigen receptor T cell is in tandem type.

6. The pharmaceutical composition of claim 5, wherein, The double-target chimeric antigen receptor comprises a CD8a signal peptide, an ASGR1 scFv, a G4S linker, a DR5 scFv, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB, a CD28 co-stimulatory molecule, and a CD3 zeta signal transduction molecule.

7. The pharmaceutical composition of claim 1, wherein, The Fuzheng Jiedu Xiaojijiang prescription comprises 15g of Dangshen, 15g of Shenghuangqi, 15g of Baishu, 15g of Fuling, 15g of Shashen, 15g of Maidong, 15g of Danggui, 15g of Shudi, 15g of Qiyexizihua, 9g of E'zhu, and 9g of Banxia.

8. A cell expressing a TNFRSF10B / ASGRl dual-targeting chimeric antigen receptor, characterized in that, The double-target chimeric antigen receptor of the TNFRSF10B / ASGR1 double-target chimeric antigen receptor T cell comprises a signal peptide, a first antigen binding region targeting TNFRSF10B, a second antigen binding region targeting ASGR1, a hinge region, a transmembrane region, and an intracellular region.

9. The cell of claim 8, wherein, The signal peptide can be a human CD8a signal peptide, a human GM-CSF signal peptide, a human insulin signal peptide, a human IL-2 signal peptide, or a human trypsinogen signal peptide.

10. The cell of claim 8, wherein The first antigen binding region targeting TNFRSF10B comprises an anti-TNFRSF10B antibody light chain variable region and an anti-TNFRSF10B antibody heavy chain variable region, wherein the amino acid sequence of the anti-TNFRSF10B antibody light chain variable region is shown as SEQ ID NO: 1, and the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is shown as SEQ ID NO:

2.

11. The cell of claim 8, wherein The second antigen binding region targeting ASGR1 comprises an anti-ASGR1 antibody heavy chain variable region, wherein the amino acid sequence of the anti-ASGR1 antibody heavy chain variable region is shown as SEQ ID NO:

3.

12. The cell of claim 8, wherein The TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor is in parallel type, and the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor comprises a first CAR and a second CAR, The first CAR comprises, sequentially from the carboxyl end to the amino end, a first antigen binding region targeting TNFRSF10B; a first hinge region; a first transmembrane binding region; and CD3 zeta, The first antigen binding region targeting TNFRSF10B comprises an anti-TNFRSF10B antibody light chain variable region and an anti-TNFRSF10B antibody heavy chain variable region, wherein the amino acid sequence of the anti-TNFRSF10B antibody light chain variable region is shown as SEQ ID NO: 1, and the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is shown as SEQ ID NO:

2. The second CAR comprises, sequentially from the carboxyl end to the amino end, a signal peptide; a second antigen binding region targeting ASGR1; a second hinge region; a second transmembrane binding region; and a co-stimulatory signal binding region, The second antigen binding region targeting ASGR1 comprises an anti-ASGR1 antibody heavy chain variable region, wherein the amino acid sequence of the anti-ASGR1 antibody heavy chain variable region is shown as SEQ ID NO:

3.

13. The cells of claim 8, wherein, The dual-targeting chimeric antigen receptor of the cell expressing the TNFRSF10B / ASGR1 dual-targeting chimeric antigen receptor is in tandem type.

14. The cell of claim 13, wherein, The dual-targeting chimeric antigen receptor comprises a CD8a signal peptide, an ASGR1 scFv, a G4S linker, a DR5 scFv, a CD8a hinge region, a CD8a transmembrane region, a 4-1BB, a CD28 co-stimulatory molecule, and a CD3 zeta signal transduction molecule.

15. Use of the pharmaceutical composition according to any one of claims 1-7 and the cell expressing the dual-targeting chimeric antigen receptor according to any one of claims 8-14 in the preparation of a medicament for treating cancer.

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