A pharmaceutical composition and application

Abstract

The present invention relates to a pharmaceutical composition and application, and in particular to an application of a dual-target CAR-T targeting TNFRSF10B / ASGR1 in combination with a Fuzheng Jiedu Xiaoji recipe for preparing a liver cancer treatment composition. The pharmaceutical composition comprises a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cell and a Fuzheng Jiedu Xiaoji recipe, wherein the dual-target chimeric antigen receptor T cell membrane surface expresses the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. The present invention unexpectedly found that the Fuzheng Jiedu Xiaoji recipe combined with CAR-T can effectively inhibit the activation of the STAT3 pathway, promote the chemotaxis of CAR-T cells as effector T cells, increase infiltration and cytotoxicity, and inhibit CD8 + T cell depletion effectively enhances the anti-liver cancer ability of CAR-T cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, belongs to IPC classification number A61K, and specifically relates to a pharmaceutical composition and application, and in particular to a method for constructing a third-generation chimeric antigen receptor T cell (CAR-T) with dual targets of TNFRSF10B / ASGR1 on the surface of primary liver cancer cells, and an application thereof in improving the efficacy of treating primary liver cancer after being used in combination with Fuzheng Jiedu Xiaoji Recipe (FZJD). Background Art

[0002] Liver cancer, or malignant tumors of the liver, is the sixth most common cancer in the world and the fourth leading cause of cancer-related death, with a 5-year survival rate of 18%. Hepatocellular carcinoma (HCC) accounts for 90% of cases and is a primary liver cancer, a malignant tumor that originates from liver cells. Traditional treatments for liver cancer include surgery, transcatheter arterial chemoembolization, local ablation, targeted therapy, etc., but the efficacy of these methods has been limited. Therefore, the importance of combined tumor immunotherapy is becoming 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 in tumor immunotherapy. Currently, immune checkpoint inhibitors (ICIs) such as PD-1 / PD-L1, which are widely used in clinical practice, can partially restore the anti-tumor activity of effector T cells, but the stimulation of chronic inflammation and the immunosuppressive environment inside the tumor will induce effector T cells to become low-responsive T cells, which will lose their activity and gradually become exhausted. At the same time, about 30% of liver cancer tissues lack infiltration of T cells and inflammatory cells, so the antigen target expression of ICIs is low and they are inherently resistant to them. In short, the current efficacy of ICIs in liver cancer is not satisfactory.

[0004] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T cell therapy) is one of the emerging methods of tumor immunotherapy and is currently the focus of tumor treatment research. Chimeric Antigen Receptor (CAR) is an artificial chimeric protein obtained by fusing a single-chain antibody that recognizes the cell surface antigen of cancer cells with a signal transduction region that induces T cell activation. By introducing the gene encoding CAR into T lymphocytes that do not have tumor reactivity, CAR-expressing T cells (CAR-T cells) that can express CAR are prepared in large quantities. In recent years, CD8 +T cell technology has made significant breakthroughs in the field of immuno-oncology therapy. + T lymphocytes are not restricted by the Major Histocompatibility Complex (MHC) and can directly and efficiently and specifically identify tumor-associated antigen (TAA)-positive tumor cells through antigen-antibody interaction. They are also able to self-proliferate and activate and have powerful tumor-killing ability.

[0005] Multiple multicenter clinical trials have used CAR-T cells targeting CD19, CD20 or CD30 to treat a variety of B-cell tumors, including B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin's lymphoma (B-NHL), chronic lymphocytic leukemia (CLL) and Hodgkin's lymphoma (HL). In the treatment of solid tumors, there are also a variety of CAR therapies targeting tumor cell surface-related antigens that have entered Phase 1-2 clinical trials, such as CAR-T therapies targeting mesothelin (MSLN) expressed by ovarian cancer, mesothelioma, and 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 prostate-specific membrane antigen (PSMA) targeting prostate cancer. The main mechanisms of relapse after CAR-T cell therapy are the limited persistence of CAR-T cells, CAR-T cell function inhibition and antigen escape. In order to minimize the risk of relapse due to target escape, clinical trials are actively exploring the strategy of dual-target CAR-T recognizing more than one tumor-associated antigen. In preclinical models and clinical trials of hematological malignancies, there are at least three combinations of antigens for dual-target CAR-T cell therapy: CD19 / CD20, CD19 / CD22 and BCMA / CD38. In liver cancer, CAR-T targeting Asialoglycoprotein Receptor 1 (ASGR1) and Glypican-3 (GPC3) are under development. "Off-target effect" is a widespread problem faced in CAR-T applications. In solid tumors, the recognition of non-tumor tissues expressing target antigens by CAR-T cells often leads to clinically serious non-tumor target toxicity. The existing GPC3 single-target CAR-T has low sensitivity and specificity in identifying liver cancer. In clinical applications, GPC3 single-target CAR-T has caused a high level of cytokine release syndrome (grade 5), and its safety needs to be further improved. Currently, there is also a GPC3 dual-target CAR-T therapy, but its killing effect has not been significantly improved compared to GPC3 single-target CAR-T.

[0006] In addition, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making the present invention, but due to space limitations, not all details and contents are listed in detail. However, this does not mean that the present invention does not have the characteristics of these prior arts. On the contrary, the present invention already has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art to the background technology. Summary of the invention

[0007] Abnormal activation of multiple tumor-related signal transduction pathways can cause the malignant progression of tumors, and the signal transducer and activator of transcription 3 (STAT3) pathway is one of the more important pathways. STAT3 is a key signaling molecule that regulates the function of multiple inflammatory factors, cytokines, and growth factors in the tumor microenvironment. It directly participates in the regulation of downstream functional genes by transducing extracellular signals to the cell nucleus. Multiple evidences show that STAT3 is not only present in cancer cells, but also overactivated in a variety of immune cells and fibroblasts in the microenvironment. After activation, STAT3 can be phosphorylated to become p-STAT3, which enters the cell nucleus after dimerization and binds to DNA response elements, regulates the transcription and activity of multiple genes in the cell, promotes tumor cell proliferation, angiogenesis, and inhibits tumor cell apoptosis. Therefore, abnormal activation of STAT3 promotes the progression of liver cancer.

[0008] The targets of hematological tumors and gliomas have good specificity, but there are few types of antigens on the surface of liver cancer cells that can be used as CAR-T recognition targets. For example, in the tissues of liver cancer patients, the GPC3 positive rate is about 60%, which means that the liver cancer cells of about 40% of patients cannot be recognized by CAR-T targeting GPC3. Therefore, it is urgent to develop other CAR-Ts with stronger targeting, sensitivity and specificity. In the treatment of liver cancer, defects such as poor chemotaxis of CAR-T to tumors, weak infiltration into solid tumors, persistent inflammatory environment inside tumors and T cell exhaustion induced by high expression of PD-L1 in liver cancer cells affect its cytotoxicity. On the other hand, most tumor-associated antigens expressed on the surface of liver cancer cells are non-tumor-specific antigens, which can be expressed in other normal tissues, making normal tissues susceptible to attack by CAR-T cells, forming a systemic "cytokine storm" dominated by elevated interleukin 6 (IL-6), with the risk of developing multiple organ failure and death.

[0009] GPC3 is a member of the heparan sulfate proteoglycan family and is highly expressed in HCC, but not expressed or expressed at a low level in normal liver tissue. Studies have confirmed that the expression of GPC3 is associated with the progression of HCC. About 60-70% of HCC patients express GPC3, and 53% of HCC patients have significantly increased serum GPC3 expression levels. The prior art discloses technical solutions for the treatment of liver cancer with GPC3 single-target CAR-T and GPC3 / ASGR1 dual-target CAR-T. The results of immunohistochemistry of clinical samples showed that 50 of the 75 hepatocellular carcinoma samples tested were positive for GPC3 expression, with a positive rate of 66.7%; while ASGR1 was expressed at a high level in all normal liver tissues (90.7%). According to statistics, among 75 liver cancer tissue samples, 41 cases (54.7%) had co-expression of GPC3 and ASGR1. Currently, researchers have conducted two consecutive GPC3 single-target CAR-T therapy applications in HCC. A total of 13 patients with advanced HCC were included in the study. 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 terms of increased cytotoxicity and side effects. Specifically, in terms of experimental research efficacy, whether it is for parentalMHCC-97L or MHCC-97L -GPC3+ , MHCC-97L -ASGR1+ and MHCC-97L -GPC3+ASGR1+For overexpression cell lines, or for Huh-7 and HepG2 that naturally express GPC3 and ASGR1, dual-targeted αGPC3-Z+αASGR1-28BB modified T cells did not show stronger cytotoxicity than single-targeted first-generation CAR-T αGPC3-Z modified T cells. When the effect-target ratio was 3:1 and 1:1, the cytotoxicity percentages of the two were about 30% and 60%, respectively, with no significant difference between the two. In addition, by comparing the therapeutic effects of third-generation CAR-T cells (GPC3-28BBZ) and dual-targeted CAR-T cells on Huh-7 subcutaneous transplanted tumors, it was found that the tumor killing ability of dual-targeted CAR-T cells in vivo was significantly worse than that of third-generation CAR-T. In summary, it is considered that the ASGR1 single-chain antibody and some special domains of the adjacent GPC3 chimeric antigen antibody are abnormally aggregated and continuously phosphorylated, which ultimately leads to its unsatisfactory anti-cancer effect. The selection of GPC3 and ASGR1 as dual targets for combined use in CAR-T still has difficult-to-solve technical problems. In terms of clinical research safety, cytokine storm (CRS) is a common related adverse event in CAR-T immunotherapy, with a high incidence rate of 80% to 90% in clinical cases. However, severe CRS, that is, CRS of more than three degrees, has a low incidence rate of about 10%-30%. The current Phase I study of GPC3 single-target CAR-T included 13 patients, 9 of whom developed cytokine release syndrome (CRS), 8 of whom were grade 1 to 2 and 1 of grade 5. Four patients received high-dose hormone pulse therapy, and two of them also received tocilizumab, a monoclonal antibody against the interleukin-6 receptor. One patient was given 20.0×10 8 In the study, grade 5 CRS occurred when only 10 cells (low-dose group) were used, and this was the first reported case of grade 5 CRS in HCC patients after CAR-T cell therapy. In summary, the safety of clinical application of single-target GPC3 CAR-T cell therapy still needs to be further improved, and cytokine release needs to be further effectively controlled.

[0010] In view of the deficiencies of the prior art, the present invention provides a pharmaceutical composition, including TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells and Fuzheng Jiedu Xiaoji Recipe (FZJD), wherein the dual-target chimeric antigen receptor T cells express TNFRSF10B / ASGR1 dual-target chimeric antigen receptor on their membrane surface.

[0011] The combination of Fuzheng Jiedu Xiaoji prescription and CAR-T can effectively inhibit the activation of STAT3 pathway, promote the chemotaxis of CAR-T cells as effector T cells, increase infiltration and cytotoxicity, and inhibit CD8 +T cell exhaustion effectively improves the anti-liver cancer ability of CAR-T cells. At the same time, the present invention inhibits the downstream links of STAT3 through multiple targets and multiple links, inhibits the "inflammatory factor storm" caused by excessive release of IL-6, and thus reduces the inflammation-related damage during treatment.

[0012] According to a preferred embodiment, the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor includes a signal peptide, a first antigen binding region targeting TNFRSF10B (anti-TNFRSF10BscFv), a second antigen binding region targeting ASGR1 (anti-ASGR1scFv), a hinge region, a transmembrane region and an intracellular region.

[0013] According to a preferred embodiment, the intracellular region includes co-stimulatory signal factors, CD3ζ and 4-1BB (CD137), and CD28.

[0014] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B includes 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.

[0015] According to a preferred embodiment, the amino acid sequence of the light chain variable region of the anti-TNFRSF10B antibody is shown as SEQ ID NO: 1.

[0016] According to a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-TNFRSF10B antibody is shown in SEQ ID NO:2.

[0017] According to a preferred embodiment, the second antigen binding region targeting ASGR1 comprises the heavy chain variable region of an anti-ASGR1 antibody.

[0018] According to a preferred embodiment, the structure of the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed for the 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 the liver cancer cell surface antigen ASGR1, which can specifically bind to ASGR1 on the surface of liver cancer cells.

[0019] According to a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-ASGR1 antibody is shown in SEQ ID NO:3.

[0020] According to a preferred embodiment, the prescription for strengthening the body, detoxifying and eliminating accumulation includes: Codonopsis pilosula, raw astragalus, Atractylodes macrocephala, Poria cocos, Adenophora scutellaria, Ophiopogon japonicus, Angelica sinensis, Rehmannia glutinosa, Seven-leaf parsley, Curcuma zedoaria, and Pinellia ternata. Specifically, the prescription for strengthening the body, detoxifying and eliminating accumulation includes: Codonopsis pilosula 15 g, raw astragalus 15 g, Atractylodes macrocephala 15 g, Poria cocos 15 g, Adenophora scutellaria 15 g, Ophiopogon japonicus 15 g, Angelica sinensis 15 g, Rehmannia glutinosa 15 g, Seven-leaf parsley, Curcuma zedoaria 9 g, and Pinellia ternata 9 g.

[0021] Another aspect of the present invention provides use of the aforementioned pharmaceutical composition in preventing and / or treating tumors.

[0022] Another aspect of the present invention provides use of the aforementioned pharmaceutical composition in preventing and / or treating liver cancer.

[0023] Another aspect of the present invention provides use of the aforementioned pharmaceutical composition in improving the therapeutic effect of liver cancer.

[0024] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the tumor treatment effect by enhancing the inhibitory effect of TNFRSF10B / ASGR1 dual-target CAR-T cells on liver cancer cells.

[0025] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by enhancing the inhibitory effect of TNFRSF10B / ASGR1 dual-target CAR-T cells on liver cancer cells.

[0026] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe promotes the release of CD107a and Granzym B in TNFRSF10B / ASGR1 dual-target CAR-T cells, thereby enabling the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer.

[0027] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription increases CD4 + The aforementioned pharmaceutical composition can improve the therapeutic effect of liver cancer due to the ratio of initial T cells (Naïve cells) and central memory T cells (TCM) in CAR-T cells.

[0028] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription increases CD8 + The aforementioned pharmaceutical composition can improve the therapeutic effect of liver cancer due to the ratio of initial T cells (Naïve cells) and central memory T cells (TCM) in CAR-T cells.

[0029] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by increasing the expression of immunostimulatory receptors on the surface of TNFRSF10B / ASGR1 dual-target CAR-T cells.

[0030] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by reducing the expression of the immunosuppressive receptor TIGIT on the surface of CAR-T cells.

[0031] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by reducing the expression of PD-L1 on the surface of tumor cells.

[0032] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by reducing the expression of the pro-inflammatory factor IL-6.

[0033] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe improves the killing function of TNFRSF10B / ASGR1 dual-target CAR-T cells by enhancing their ability to secrete IFN-γ, thereby enabling the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer.

[0034] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by inhibiting the STAT3 signaling pathway.

[0035] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji recipe enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by increasing the release level of T cell chemokines from liver cancer cells.

[0036] On the other hand, the present invention provides a dual-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.

[0037] 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 tumor cells. 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 liver cancer cells.

[0038] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B includes 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, and the arrangement can be VL-linker-VH or VH-linker-VL.

[0039] According to a preferred embodiment, the amino acid sequence of the light chain variable region of the anti-TNFRSF10B antibody is shown as SEQ ID NO: 1.

[0040] According to a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-TNFRSF10B antibody is shown in SEQ ID NO:2.

[0041] According to a preferred embodiment, the second antigen binding region targeting ASGR1 comprises the heavy chain variable region of an anti-ASGR1 antibody.

[0042] According to a preferred embodiment, the structure of the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed for the 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 the liver cancer cell surface antigen ASGR1, which can specifically bind to ASGR1 on the surface of liver cancer cells.

[0043] According to a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-ASGR1 antibody is shown in SEQ ID NO:3.

[0044] According to a preferred embodiment, the dual-target chimeric antigen is connected in parallel, and the dual-target chimeric antigen includes a first CAR and a second CAR, and the first CAR comprises: a first antigen binding region targeting TNFRSF10B connected sequentially from the carboxyl terminus to the amino terminus; a first hinge region; a first transmembrane binding region and CD3ζ.

[0045] The second CAR comprises: a signal peptide connected sequentially from the carboxyl terminus to the amino terminus; a second antigen binding region targeting ASGR1; a second hinge region; a second transmembrane binding region and a co-stimulatory signal binding region.

[0046] According to a preferred embodiment, the first CAR can be expressed as TNFRSF10BScFv-Hinge-TM-CD3ζ.

[0047] According to a preferred embodiment, a tag protein for marking, sorting, and eliminating host cells can be present on the dual-target chimeric antigen receptor, and the tag protein can be connected to the dual-target chimeric antigen receptor via a cleavable domain or another promoter.

[0048] According to a preferred embodiment, the first CAR includes a tag protein. Preferably, the tag protein is a red fluorescent tag (dsRed).

[0049] According to a preferred embodiment, the signal domain of the first CAR includes CD8α (signal peptide).

[0050] According to a preferred embodiment, the first hinge region of the first CAR includes CD8αH (can be expressed as Hinge).

[0051] According to a preferred embodiment, the first transmembrane binding region is capable of anchoring and binding to the cell membrane.

[0052] According to a preferred embodiment, the second transmembrane binding region is capable of anchoring and binding to the cell membrane.

[0053] According to a preferred embodiment, the first transmembrane binding region comprises CD8α TM.

[0054] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B and the first transmembrane binding region may be connected via a first hinge region.

[0055] According to a preferred embodiment, CD3ζ is an intracellular signaling domain.

[0056] According to a preferred embodiment, the first CAR can be expressed as CD8α signal peptide -TNFRSF10BVH-linker-TNFRSF10B VL-CD8αH-CD8α TM-CD3ζ.

[0057] According to a preferred embodiment, the second CAR can be expressed as CD8α signal peptide-ASGR1 VH-CD8αH-CD8α TM-CD137-CD28.

[0058] According to a preferred embodiment, the second CAR includes a tag protein. Preferably, the tag protein is a green fluorescent tag (eGFP).

[0059] According to a preferred embodiment, the signal domain of the second CAR includes CD8α (signal peptide).

[0060] According to a preferred embodiment, the second hinge region of the second CAR includes CD8αH (can be expressed as Hinge).

[0061] According to a preferred embodiment, the second transmembrane binding region comprises CD8α TM.

[0062] According to a preferred embodiment, the costimulatory signal binding region includes a 4-1BB (CD137) costimulatory signal domain and a CD28 costimulatory signal domain.

[0063] According to a preferred embodiment, the intracellular co-stimulatory signaling domain can comprise a CD3ζ intracellular signaling domain that can activate immune response cells.

[0064] According to a preferred embodiment, the second CAR can be expressed as CD8α signal peptide-ASGR1 VH-CD8αH-CD8αTM-CD137-CD28.

[0065] According to a preferred embodiment, the immune response cells include T lymphocytes, NK cells, and NKT cells.

[0066] According to a preferred embodiment, the signal peptide can be human CD8α signal peptide, human GM-CSF signal peptide, human insulin signal peptide, human IL-2 signal peptide, human trypsinogen signal peptide. Preferably, the signal peptide can be human CD8α signal peptide, and its amino acid sequence is shown in SEQ ID NO:4.

[0067] According to a preferred embodiment, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO:5.

[0068] According to a preferred embodiment, the amino acid sequence of the CD8α TM transmembrane binding region is shown in SEQ ID NO:6.

[0069] According to a preferred embodiment, the amino acid sequence of the CD28 costimulatory signal domain is shown in SEQ ID NO:7.

[0070] According to a preferred embodiment, the amino acid sequence of the 4-1BB (CD137) costimulatory signaling domain is shown as SEQ ID NO: 8.

[0071] According to a preferred embodiment, CD3ζ is an intracellular signaling domain, and its amino acid sequence is shown in SEQ ID NO:9.

[0072] According to a preferred embodiment, the first CAR is the primary recognition site of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

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

[0074] Another aspect of the present invention provides a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0075] Another aspect of the present invention provides a vector, which contains a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0076] According to a preferred embodiment, the vector can be one or more of adenovirus, lentivirus, adeno-associated virus or retrovirus.

[0077] The present invention provides another dual-target chimeric antigen receptor, wherein the dual-target chimeric antigens are connected in series.

[0078] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-linker-ASGR1 VHH.

[0079] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VH-linker-TNFRSF10B VL-Linker-ASGR1 VH.

[0080] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VL-linker-TNFRSF10B VH-Linker-ASGR1 VH.

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

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

[0083] According to a preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VL-linker-ASGR1VH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

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

[0085] According to a preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VH-linker-ASGR1VL-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0086] According to a preferred embodiment, the tandem chimeric antigen receptor can be ASGR1 VH-linker-ASGR1VL-linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

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

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

[0089] According to a preferred embodiment, the tandem chimeric antigen receptors can be A VH-linker-A VL-Linker-B VL-Linker-B VH or A VHH-linker-B VHH.

[0090] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VH.

[0091] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VL-ASGR1 VH.

[0092] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VH-ASGR1 VL.

[0093] The present invention reserves the right to transform the heavy chain variable domain VH of scFv or humanized heavy chain antibody into the heavy chain variable domain VHH of camelid antibody.

[0094] Another aspect of the present invention provides a host cell, wherein the host cell contains the aforementioned vector or the aforementioned exogenous nucleic acid molecule integrated into the chromosome.

[0095] According to a preferred embodiment, the host cell expresses a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0096] On the other hand, the present invention provides a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, a nucleic acid molecule encoding the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, a vector containing a nucleic acid molecule encoding the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, and the use of the aforementioned host cells in the preparation of drugs for preventing and / or treating cancer.

[0097] According to a preferred embodiment, the cancer is liver cancer.

[0098] Another aspect of the present invention provides a kit for preparing host cells, the kit comprising a nucleic acid molecule and / or a vector encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0099] Another aspect of the present invention provides a method for preparing immune cells, wherein the immune cells express a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, wherein the method comprises the following steps:

[0100] Providing immune cells to be modified; transducing nucleic acid molecules encoding TNFRSF10B / ASGR1 dual-target chimeric antigen receptors or vectors containing nucleic acid molecules encoding TNFRSF10B / ASGR1 dual-target chimeric antigen receptors into immune cells, thereby obtaining modified immune cells.

[0101] Beneficial effects of the present invention: The present invention has designed and successfully constructed a dual-target CAR-T cell that can only be fully activated when it recognizes two targeted antigens at the same time. In addition to TNFRSF10B, which is a target that can mediate the transmission of the first signal (CAR) after being recognized, ASGR1, a target molecule recognized by the chimeric antigen co-stimulatory receptor (CCR), is a receptor protein that is stably and specifically expressed on the surface of hepatocytes, enabling the TNFRSF10B / ASGR1 dual-target CAR-T cells to specifically obtain more co-stimulatory signals in the liver. Through this combination of CAR and CCR, the sensitivity and specificity of killing liver cancer cells can be maximized, effectively avoiding the "off-target effect". TNFRSF10B was selected as the main target. TNFRSF10B is expressed in various tumor tissues (pan-cancer). The expression in pan-cancer and liver cancer is shown in the following figure. Fig. 20 As shown: Fig. 20A is the expression of DR5 protein in human liver cancer tissue detected by immunohistochemistry and immunofluorescence; Fig. 20 B is the correlation between the expression of TNFRSF10B in liver cancer and prognosis by querying the TCGA database; Fig. 20 C is the expression of TNFRSF10B in pan-cancer pathological tissues queried in The Human Protein Atlas database. The expression of TNFRSF10B in liver cancer tissues is greater than 75%, which is higher than the average expression of GPC3; at the same time, the expression of TNFRSF10B in normal tissues is extremely low. TNFRSF10B is associated with the prognosis of liver cancer patients, which means that this target is more widely distributed in liver cancer tissues, and the sensitivity and specificity of CAR-T using it as an identification target are higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 It is a schematic diagram of the structure of the lentiviral backbone vector GV400 and GV401 plasmid provided by the present invention;

[0103] Figure 2 The restriction site provided by the present invention is Bm I / EcoR The result of enzyme digestion of I;

[0104] Figure 3 The restriction site provided by the present invention is Bm I / Bm The result of enzyme digestion of I;

[0105] Figure 4 The cloning site provided by the present invention is Bm I / EcoR PCR result of target fragment I;

[0106] Figure 5 The cloning site provided by the present invention is Bm I / Bm PCR result of target fragment I;

[0107] Figure 6 The present invention provides Bm I / EcoR Identification result of the target fragment corresponding to I exchanged into the linear expression vector;

[0108] Figure 7 The present invention provides Bm I / Bm Identification result of the target fragment corresponding to I exchanged into the linear expression vector;

[0109] Figure 8 It is a dilution schematic diagram of the fluorescence detection of virus titer provided by the present invention, and the observation of plasmid transfection and counting under a fluorescence microscope;

[0110] Fig. 9 It is the result of detecting the transfection efficiency of CAR-T by flow cytometry provided by the present invention;

[0111] Fig.10 It is the comparison result of the killing effect of CAR-T cells with different effector-target ratios provided by the present invention on liver cancer cells;

[0112] Fig.11 The apoptosis of liver cancer cells detected by flow cytometry when the effector-target ratio provided by the present invention is 2:1;

[0113] Fig.12 The present invention provides flow cytometry to detect the release levels of CD107a and Granzym B after co-incubation of TNFRSF10B (DR5) single-target and dual-target CAR-T cells with liver cancer cells;

[0114] Fig.13 The CCK8 method provided by the present invention detects the inhibition of tumor cell proliferation by the combined treatment of FZJD drug-containing serum and DR5 / ASGR1 dual-target CAR-T cells;

[0115] Fig.14 This is the experimental result that the FZJD drug-containing serum provided by the present invention enhances the apoptosis effect of DR5 / ASGR1 dual-target CAR-T cells on liver cancer cells;

[0116] Fig.15 It is the release of CD107a and Granzym B after the combined action of FZJD drug-containing serum provided by the present invention and DR5 / ASGR1 dual-target CAR-T cells;

[0117] Fig.16 The experimental results provided by the present invention are the flow cytometry results of detecting the phenotypic changes of FZJD combined with DR5 / ASGR1 dual-target CAR-T cells (Figures AC) and FZJD acting on liver cancer cells HepG2 (Figure D);

[0118] Fig.17 It is the result of an ELISA provided by the present invention to detect the expression of cytokine IL-6 after co-culture of dual-target CAR-T and HepG2;

[0119] Fig.18 The present invention provides an ELISA to detect the result of FZJD drug-containing serum increasing the level of interferon release when DR5 / ASGR1 dual-target CAR-T cells kill liver cancer cells;

[0120] Fig.19It is the test result of the FZJD drug-containing serum provided by the present invention increasing the release level of T cell chemokines in liver cancer cells (Stattic is a STAT3 small molecule inhibitor);

[0121] Fig. 20 The invention provides the expression of TNFRSF10B protein in human liver cancer tissue and the related expression of TNFRSF10B found in the TCGA database and The Human Protein Atlas database. DETAILED DESCRIPTION

[0122] The following is a detailed description with reference to the accompanying drawings.

[0123] TNFRSF10B is also referred to as DR5 in this application. TNFRSF10B / ASGR1 dual-target CAR-T cells are equivalent to DR5 / ASGR1 dual-target CAR-T cells.

[0124] The Fuzheng Jiedu Xiaoji recipe is also represented as FZJD in this application.

[0125] In the present application, non-transfected T cells are also referred to as NT cells and are represented by NT in the accompanying drawings.

[0126] In this application, blank control refers to a control without any treatment or control drug, which is used to observe whether the experiment is in a normal state.

[0127] In this application, unless otherwise specified, the biological materials, reagents, and detection kits involved can be obtained from commercial channels.

[0128] It should be understood that all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. The term "antibody" can refer to a complete antibody molecule, and also refers to an antibody molecule fragment that retains the ability to bind to an immunogen, which can be used routinely in vivo or in vitro. The term "antibody" refers to a heavy (H) chain and a light (L) chain that specifically bind to an antigen and contain disulfide bonds, or an antigen-binding portion thereof, wherein two heavy (H) chains and two light (L) chains may be included. Each H chain includes a heavy chain variable region (abbreviated as VH in this application) and a heavy chain constant region. Each L chain includes a light chain variable region (abbreviated as VL in this application) and a light chain constant region. The variable regions of the H chain and the L chain contain a binding domain that interacts with the antigen. The VHH mentioned in the present invention is the heavy chain variable domain of the heavy chain antibody of camelids, also known as nano antibodies, which can be transformed from traditional VH.

[0129] The TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and immune response cells expressing it of the subject matter disclosed in the present application can be systemically applied to a subject, can be directly applied to a subject, for the treatment of cancer or to improve the efficacy of tumors. Immune response cells refer to cells that play a role in immune responses, or their progenitor cells, or their progeny cells. The TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and immune response cells expressing it can be administered by any means accepted by physiological disciplines. In some embodiments, in the treatment of liver cancer, an effective dose of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and immune response cells expressing it are directly injected into the target organ (such as an organ affected by tumor formation), or an effective dose of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and immune response cells expressing it are administered to the circulatory system and indirectly provided to the target organ. Before, during or after the administration of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and the immune response cells expressing it, an effective dose of FZJD can be administered to improve the treatment effect of liver cancer. The immune response cells expressing the TNFRSF10B / ASGR1-specific chimeric antigen receptor and the composition containing it can be obtained from one subject and administered to the same subject or different compatible subjects. The peripheral blood-derived T cells and their progeny of the subject disclosed in the present application can be administered by systemic injection, local injection, intravenous injection or parenteral administration. For example: administering at least 1×10 5 cells, eventually reaching 1×10 12 or more.

[0130] When administering the pharmaceutical composition disclosed in the present application, it can be configured into a unit dose injectable form, such as a solution, suspension, etc. Additives such as chelating agents, buffers, antioxidants, etc. can be added to enhance the stability of the pharmaceutical composition. According to the purpose of this application, when any additive, carrier or other diluent is used, it must be compatible with the immune response cells expressing the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor disclosed in the present 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, about 1×10 5 About 1×10 12 , about 1×10 6 About 1×10 11 , about 1×10 7 About 1×10 10 , about 1×10 8 About 1×10 9In some embodiments, a smaller or larger number of immune response cells of the disclosed subject matter may be administered to a subject. In some embodiments, about 1×10 4 , about 1×10 5 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 2×10 8 , about 3×10 8 , about 4×10 8 , about 5×10 8 The immune response cells of the presently disclosed subject matter are administered to a subject.

[0131] Those skilled in the art can determine the cells and optional additives or vehicle substances in the pharmaceutical composition and determine the specific mode of administration. For example, the additive (except the active cells and / or active substances) is present in a solution of about 0.001 wt% to about 50 wt% in phosphate buffered saline, and the active ingredient therein is present in the order of micrograms to milligrams, such as the active ingredient is 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%.

[0132] The "effective dose" of the drug or pharmaceutical composition involved in this application is a dose sufficient to produce any one or more beneficial or expected effects. For therapeutic use, beneficial or expected results include eliminating or reducing the risk of tumor deterioration, improving the efficacy, such as reducing the incidence of one or more symptoms of a disease (such as liver cancer) or improving the symptoms. In a pharmaceutical composition, one drug can enhance the effect of another drug, thereby improving the overall therapeutic effect. The effective dose can be administered once or multiple times. In this application, the effective dose of a drug or pharmaceutical composition refers to an amount sufficient to directly or indirectly complete the therapeutic effect. According to the background of clinical treatment, the effective dose of a drug or pharmaceutical composition may or may not be used in combination with another drug or pharmaceutical composition. Therefore, the "effective dose" can be considered in the case of administering one or more therapeutic agents. If used in combination with one or more other drugs, it can be considered to administer a single drug at an effective dose, and the ideal result may be achieved or realized. Example 1

[0133] This embodiment provides a pharmaceutical composition, including TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells and FZJD, wherein the dual-target chimeric antigen receptor T cell membrane surface expresses TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. Specifically, FZJD is: 15 g of Codonopsis pilosula, 15 g of raw Astragalus, 15 g of Atractylodes macrocephala, 15 g of Poria, 15 g of Adenophora adenophora, 15 g of Ophiopogon japonicus, 15 g of Angelica sinensis, 15 g of Rehmannia glutinosa, 15 g of Polygonum multiflorum, 15 g of Curcuma zedoaria, and 9 g of Pinellia ternata. Example 2

[0134] This embodiment provides a chimeric antigen receptor, including 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 the 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 the 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 includes 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 light chain variable region of the anti-TNFRSF10B antibody and the heavy chain variable region of the anti-TNFRSF10B antibody are connected by one or more linkers, and the arrangement can be VL-linker-VH or VH-linker-VL. The chimeric antigen receptor is a parallel chimeric antigen receptor.

[0135] According to a preferred embodiment, the amino acid sequence of the light chain variable region of the anti-TNFRSF10B antibody is shown as SEQ ID NO: 1.

[0136] According to a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-TNFRSF10B antibody is shown in SEQ ID NO:2.

[0137] According to a preferred embodiment, the second antigen binding region targeting ASGR1 comprises the heavy chain variable region of an anti-ASGR1 antibody.

[0138] According to a preferred embodiment, the structure of the second antigen binding region targeting ASGR1 is a specific antigen binding domain designed for the 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 the liver cancer cell surface antigen ASGR1, which can specifically bind to ASGR1 on the surface of liver cancer cells.

[0139] According to a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-ASGR1 antibody is shown in SEQ ID NO:3.

[0140] According to a preferred embodiment, the connection mode of the dual-target chimeric antigen is a parallel type, and the dual-target chimeric antigen includes a first CAR and a second CAR, the first CAR comprises: a signal peptide connected in sequence from the carboxyl terminus to the amino terminus; a first antigen binding region targeting TNFRSF10B; a first hinge region; a first transmembrane binding region and CD3ζ, and the second CAR comprises: a signal peptide connected in sequence from the carboxyl terminus to the amino terminus; a second antigen binding region targeting ASGR1; a second hinge region; a second transmembrane binding region and a co-stimulatory signal binding region.

[0141] According to a preferred embodiment, the first CAR can be expressed as TNFRSF10B ScFv-Hinge-TM-CD3ζ.

[0142] According to a preferred embodiment, a tag protein for marking, sorting, and eliminating host cells can be present on the dual-target chimeric antigen receptor, and the tag protein can be connected to the dual-target chimeric antigen receptor via a cleavable domain or another promoter.

[0143] According to a preferred embodiment, the first CAR includes 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 includes CD8αH (which can be expressed as Hinge).

[0144] According to a preferred embodiment, the first transmembrane binding region is capable of anchoring and binding to a cell membrane. The second transmembrane binding region is capable of anchoring and binding to a cell membrane. According to a preferred embodiment, the first transmembrane binding region comprises CD8α TM.

[0145] According to a preferred embodiment, the first antigen binding region targeting TNFRSF10B and the first transmembrane binding region may be connected via a first hinge region.

[0146] According to a preferred embodiment, CD3ζ is an intracellular signaling domain.

[0147] Specifically, the first CAR can be expressed as CD8α signal peptide-TNFRSF10B VH-linker-TNFRSF10B VL-CD8αH-CD8α TM-CD3ζ.

[0148] According to a preferred embodiment, the second CAR can be expressed as ASGR1 VH-Hinge-TM-CD137-CD28.

[0149] According to a preferred embodiment, the second CAR may be expressed as CD8αsignal peptide-ASGR1 VH-CD8αH-CD8α TM-CD137 (4-1BB)-CD28.

[0150] According to a preferred embodiment, the second CAR includes a tag protein. Preferably, the tag protein is a green fluorescent tag (eGFP). The second hinge region of the second CAR includes CD8αH (which can be expressed as Hinge).

[0151] According to a preferred embodiment, the second transmembrane binding region comprises CD8 TM.

[0152] According to a preferred embodiment, the costimulatory signal binding region includes a 4-1BB (CD137) costimulatory signal domain and a CD28 costimulatory signal domain.

[0153] According to a preferred embodiment, the intracellular co-stimulatory signaling domain can comprise a CD3ζ intracellular signaling domain that can activate immune response cells.

[0154] According to a preferred embodiment, the immune response cells include T lymphocytes, NK cells, and NKT cells.

[0155] According to a preferred embodiment, the signal peptide can be human CD8α signal peptide, human GM-CSF signal peptide, human insulin signal peptide, human IL-2 signal peptide, human trypsinogen signal peptide. Preferably, the signal peptide can be human CD8α signal peptide, and its amino acid sequence is shown in SEQ ID NO:4.

[0156] According to a preferred embodiment, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO:5.

[0157] According to a preferred embodiment, the amino acid sequence of the CD8α TM transmembrane binding region is shown in SEQ ID NO:6.

[0158] According to a preferred embodiment, the amino acid sequence of the CD28 costimulatory signal domain is shown in SEQ ID NO:7.

[0159] According to a preferred embodiment, the amino acid sequence of the 4-1BB (CD137) costimulatory signaling domain is shown as SEQ ID NO: 8.

[0160] According to a preferred embodiment, CD3ζ is an intracellular signaling domain, and the amino acid sequence is shown in SEQ ID NO:9.

[0161] According to a preferred embodiment, the binding site of the first CAR-T is the primary recognition site of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0162] According to a preferred embodiment, the binding site of the second CAR-T is the cooperative recognition site of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. Example 3

[0163] This embodiment provides a nucleic acid molecule, which encodes a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. Example 4

[0164] This embodiment provides a vector, which contains a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. According to a preferred embodiment, the vector can be one or more of adenovirus, lentivirus, adeno-associated virus or retrovirus. Example 5

[0165] This embodiment provides a host cell, wherein the host cell contains the aforementioned vector or the aforementioned nucleic acid molecule integrated into the chromosome. According to a preferred embodiment, the host cell expresses a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. Example 6

[0166] This embodiment provides a kit for preparing host cells, wherein the kit comprises a nucleic acid molecule and / or a vector encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor. Example 7

[0167] This embodiment provides a method for preparing an immune cell, wherein the immune cell expresses a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor, wherein the method comprises the following steps:

[0168] Providing immune cells to be modified; transducing nucleic acid molecules encoding TNFRSF10B / ASGR1 dual-target chimeric antigen receptors or vectors containing nucleic acid molecules encoding TNFRSF10B / ASGR1 dual-target chimeric antigen receptors into immune cells, thereby obtaining modified immune cells. Example 8

[0169] This example provides specific steps for constructing a CAR plasmid. The construction of the CAR plasmid includes: vector restriction enzyme digestion, acquisition of target gene fragments, exchange of PCR products with vectors, transformation, colony PCR identification, sequencing, plasmid transformation, single clone selection, plasmid midi extraction, lentivirus packaging, lentivirus concentration, and determination of lentivirus titer by fluorescence method.

[0170] Figure 1 This is a schematic diagram of the structure of the lentiviral backbone vector GV400 and GV401 plasmid. Figure 1 A is a schematic diagram of the GV400 plasmid structure. Figure 1B is a schematic diagram of the GV401 plasmid structure. The element sequence of the GV400 plasmid vector is EF1a-ScFv, and the cloning site is Bm I / EcoR I. The element sequence of the GV401 plasmid vector is EF1a-ScFv-CarT-2A-EGFP, and the cloning site is Bm I / Bm I.

[0171] 1. Vector digestion

[0172] Bm I and EcoR I double restriction enzyme vector GV400, Bm I digested vector GV401. Prepare 50 µl of the digestion system as shown in Table 1 and Table 2. Add various reagents in the order in Table 1, mix them by gently pipetting, and place at 37°C for 3 h or overnight.

[0173]

[0174] After the reaction was completed, the vector digestion products were subjected to agarose gel electrophoresis. Bm I / EcoR The results of restriction digestion at site I are as follows Figure 2 shown. Figure 2 In the electrophoresis, a 10 kb marker was used. Lane 1 is the 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 undigested vector. Bm I / Bm The results of restriction digestion at site I are as follows Figure 3 shown. Figure 3 In the electrophoresis, a 10 kb marker was used. Lane 1 is the 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 undigested vector.

[0175] GV400 plasmid vector Bm I / EcoR I was used for restriction digestion, and the GV400 plasmid vector was purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd. The GV401 plasmid vector was Bm I / Bm I was purchased from Shanghai Jikai Gene Chemistry Technology Co., Ltd.

[0176] Recycle the target band. The recycling process includes:

[0177] (1) Under long-wave ultraviolet light, use a clean blade to cut out the DNA band to be recovered, and try to remove the gel that does not contain DNA. The smaller the gel volume, the better;

[0178] (2) Place the excised gel containing the DNA band into a 1.5 ml centrifuge tube and weigh it;

[0179] Weigh an empty 1.5 ml centrifuge tube first, then put the gel in and weigh it again. Subtract the two weights to get the weight of the gel.

[0180] (3) Add 3 times the volume of sol solution DD;

[0181] If the gel weight is 100 mg, its volume can be regarded as 100 μl, and 300 μl of sol solution should be added. If the gel concentration is greater than 2%, 6 times the volume of sol solution should be added;

[0182] (4) Place in a 56°C water bath for 10 minutes (or until the gel is completely dissolved), vortexing every 2 to 3 minutes to help accelerate dissolution;

[0183] (5) Optional, usually not necessary, add 150 μl of isopropanol per 100 mg of initial gel weight and shake to mix;

[0184] Sometimes adding isopropanol can improve the recovery rate. Do not centrifuge after adding it. When recovering fragments larger than 4 Kb, do not add isopropanol. Adding it may sometimes reduce the recovery efficiency.

[0185] Equilibration liquid pretreatment adsorption column:

[0186] Using equilibration solution to pretreat the silica membrane adsorption column is a necessary step. The specific method is: take a new silica membrane adsorption column and put it in a collection tube, pipette 100 μl of equilibration solution into the column, centrifuge at 13000 rpm for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. At this time, the pretreatment of the column with equilibration solution is completed.

[0187] (6) Add the solution obtained in the previous step to the EC adsorption column (the adsorption column is placed in the collection tube), leave it at room temperature for 1 minute, centrifuge it at 12,000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube;

[0188] If the total volume exceeds 750 μl, the solution can be added to the same EC column twice.

[0189] After the filtered sol liquid is mixed with the strong alkaline equilibrium solution remaining in the collection tube, the sol liquid may change from yellow to orange or even purple. This is the normal color change of the phenol red pH indicator under alkaline conditions.

[0190] (7) Add 600 μl of washing buffer (WB) (please check whether anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid;

[0191] (8) Add 600 μl of wash buffer (WB), centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid;

[0192] (9) Place the EC adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 2 min to remove as much rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions.

[0193] (10) Take out the adsorption column EC and put it into a clean centrifuge tube. Add 50 μl of elution buffer EB to the middle of the adsorption membrane (it is better to heat the elution buffer in a 65-70℃ water bath beforehand). Leave it at room temperature for 2 minutes and centrifuge it at 12,000 rpm for 1 minute. If more DNA is needed, add the resulting solution back to the adsorption column and centrifuge it for 1 minute.

[0194] The larger the elution volume, the higher the elution efficiency. If a higher DNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume should not be less than 25 μl. Too small a volume will reduce DNA elution efficiency and yield.

[0195] 2. Acquisition of target gene fragments

[0196] The primers and plasmid templates used in this process were synthesized by Shanghai GeneCare Gene Medical Technology Co., Ltd. The cloning site was Bm I / EcoR The primer sequences of the target fragments corresponding to I are shown in the following table.

[0197] Primer description: Contains exchange pairing bases, restriction sites, and partial sequence at the 5' end of the target gene for PCR to fish for the target gene.

[0198]

[0199] The cloning site is Bm I / Bm The primer sequences of the target fragments corresponding to I are shown in the following table.

[0200]

[0201] The acquisition steps include: PCR amplification of the target gene fragment, configuration of the reaction system, as shown in Table 3, adding various reagents in sequence according to the order in Table 3, gently pipetting and mixing with a pipette, briefly centrifuging and placing in a PCR instrument for reaction, and the reaction conditions are shown in Table 4. The cloning site is Bm I / EcoR The result of the target fragment corresponding to I is as follows Figure 4 As shown, the size of the obtained PCR product was 2129 bp. Figure 4 The markers from top to bottom are: 5 kb, 3 kb, 2 kb, 1.5 kb, 1 Kb, 750 bp, 500bp, 250 bp, and 100 bp.

[0202] The cloning site is Bm I / Bm The result of the target fragment corresponding to I is as follows Figure 5 As shown, the size of the obtained PCR product was 974 bp. 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.

[0203]

[0204] 3. Exchange of PCR products with vectors

[0205] Prepare the reaction system shown in Table 5 in an ice water bath, mix thoroughly (avoid bubbles), react at 37°C for 30 min after rapid centrifugation, cool in an ice water bath for 5 min and then immediately transform to obtain the exchange reaction product. PCR identification primers are shown in the following table. Bm I / EcoR The identification result of the target fragment corresponding to I exchanged into the linear expression vector is as follows Figure 6 As shown, 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 load self-ligation control group), lane 3 is the positive control (GAPDH), lane 4 is the Marker, and from top to bottom they are 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, and 100 bp, and lanes 5 to 12 are transformants 1 to 8, respectively.

[0206]

[0207] The PCR identification primers are shown in the following table. Bm I / Bm The identification result of the target fragment corresponding to I exchanged into the linear expression vector is as follows Figure 7As shown, 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 load self-ligation control group), lane 3 is the positive control (GAPDH), lane 4 is the Marker, and from top to bottom they are 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, and 100 bp, and lanes 5 to 12 are transformants 1 to 8, respectively.

[0208]

[0209] 4. Conversion

[0210] Add 10 μl of the exchange reaction product to a centrifuge tube containing 100 μl of competent cells, flick to mix, and place on ice for 30 min. Heat shock in a 42°C water bath for 90 s, and immediately incubate in an ice water bath for 2 min. Add 500 μl of LB liquid culture medium to the mixture and place it on a shaker at 37°C for 1 h. Take an appropriate amount of bacterial liquid and evenly spread it on a plate containing ampicillin (Amp), and invert and culture it in a constant temperature incubator for 12-16 h.

[0211] 5. Colony PCR Identification

[0212] The primers used in this process were synthesized by Shanghai GeneCare Gene Medicine Technology Co., Ltd.

[0213] The reaction system was prepared according to Table 6. After preparation, the reaction system was evenly shaken and centrifuged quickly. A single colony was picked up in the clean bench and added to 20 μl of the identification system. After mixing, it was placed in a PCR instrument for reaction. The PCR reaction conditions are shown in Table 7.

[0214]

[0215] 6. Sequencing

[0216] The positive clone transformants after identification were inoculated into an appropriate amount of LB liquid medium containing Amp, cultured at 37°C for 12-16 h, and an appropriate amount of bacterial liquid was taken for sequencing, and the sequencing results were compared and analyzed with the target gene sequence. The comparison results of the positive clone transformants of GV400 are shown in SEQ ID NO: 11, which 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 transformants of GV401 are shown in SEQ ID NO: 12, which 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.

[0217] 7. Plasmid Transformation

[0218] The specific steps include: open 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, tap the tube wall several times to mix, and place on ice for 30 min; heat shock at 42°C for 90 s, incubate in an ice water bath for 2 min, then add 500 μl of LB liquid culture medium, and place on a shaker at 37°C for 1 h; after culturing, take an appropriate amount of bacterial liquid and evenly spread it on a plate containing Amp, and culture it in a constant temperature incubator for 12 to 16 h.

[0219] 8. Pick a single clone

[0220] The specific steps include: preparing LB liquid culture medium containing Amp, 200 ml of LB liquid culture medium + 200 μl of Amp, mixing, and boiling in a microwave oven; taking culture bottles after high-pressure sterilization, adding 50 ml of LB liquid culture medium to each culture bottle for later use; taking out the plate in the 37°C constant temperature incubator, looking for a single colony, picking up the colony and putting it into the culture bottle; placing the culture bottle in a constant temperature shaker, setting the speed to 200 rpm, the temperature to 37°C, and shaking the bacteria for 16-20 hours.

[0221] 9. Plasmid Extraction

[0222] The specific steps include: transferring the culture medium in the culture bottle to a 50 ml centrifuge tube, centrifuging at 3000 g for 10 min, and discarding the liquid; adding 4 ml of Buffer P1 reagent to each centrifuge tube and mixing thoroughly; adding 4 ml of Buffer P2 reagent to each centrifuge tube, mixing by inverting until the liquid turns blue and viscous, and letting it stand for 3 min; adding 4 ml of Buffer S3 reagent to each centrifuge tube, mixing by inverting until transparent, filtering the liquid in the centrifuge tube through a syringe, and transferring the filtered liquid to a new 50 ml centrifuge tube; adding 2 ml of Buffer BB to each centrifuge tube; filtering the liquid in the centrifuge tube through a filter plug into a centrifuge column, removing the centrifuge column, centrifuging at 18000 g for 1 min; adding 0.7 ml of Buffer ETR to the centrifuge column, centrifuging at 18000 g for 1 min, and discarding the liquid; adding 0.7 ml of Buffer PE to the centrifuge column, centrifuging at 18000 g for 1 min, and discarding the liquid, and repeating once; 18000 g, empty the centrifuge column for 1 min, discard the liquid, add 200 μl of Elution Buffer, let it stand for 1 min, centrifuge at 18000 g for 1 min, transfer the collected liquid after centrifugation to a new EP tube; take 2 μl of plasmid to detect the plasmid concentration.

[0223] 10. Lentiviral packaging

[0224] The specific steps include: 293T cells were revived in advance, 24 h before transfection, 293T cells in the logarithmic growth phase were digested with trypsin, and the cell density was adjusted to about 5×10 in a medium containing 10% serum. 6 cells / 15 ml, re-inoculated into a six-well plate, cultured in a 37°C, 5% CO2 incubator for 24 h, and used for transfection when the cell density reaches 70%-80%; calculate the amount of plasmid according to the ratio of target plasmid: psPAX2: pMD2G of 3:2:1 and a total mass of 4 μg; add the required volume of plasmid to 400 μl of serum-free DMEM medium, add 6 μl of Turbo and mix well, and leave at room temperature for 15-20 min; slowly add the mixture to the culture dish, shake well, and culture in a 37°C incubator; change the medium after 24 h of culture, collect the virus once at 48 h and once at 72 h, mix the two collected viruses and prepare to concentrate.

[0225] 11. Lentivirus Concentration

[0226] The specific steps include: harvesting the virus, transferring the cell supernatant to a 50 ml centrifuge tube, centrifuging at 3500 g for 10 min; adding the virus concentrate to a new 50 ml centrifuge tube at a volume ratio of cell supernatant: concentrate reagent = 3:1, and precooling in a 4°C refrigerator; transferring the cell supernatant after centrifugation to the precooled virus concentrate reagent, mixing thoroughly, and shaking at a low speed overnight in a 4°C refrigerator; taking the centrifuge tube after overnight, centrifuging at 3500 g, 4°C for 1 h; discarding the supernatant, centrifuging at 3500 g, 4°C for 1 min; preparing the virus resuspension protection solution, wherein the volume ratio of virus resuspension solution: virus protection reagent is 9:1, taking 9 ml of virus resuspension solution and 1 ml of virus protection reagent into a 15 ml centrifuge tube for use; taking out the virus after centrifugation, discarding the supernatant, adding 1%~10% of the volume of the virus stock solution of virus resuspension protection solution, slowly pipetting 20~30 times, and letting it stand for 10 min; 12000 g, 4℃, centrifuge for 3-5min, aspirate the supernatant as the concentrated virus; transfer the concentrated virus to a 1.5 ml centrifuge tube and freeze it in a -80℃ refrigerator.

[0227] 12. Determination of lentiviral titer by fluorescence method

[0228] like Figure 8 As shown, Figure 8 A is a schematic diagram of virus gradient dilution; Figure 8 B is the observation of plasmid transfection under fluorescence microscope; Figure 8 C is the count under fluorescence microscope. The specific measurement steps include: one day before the measurement, 293T adherent cells were plated in a 96-well plate, 4×10 4cells, 100 μl of culture medium; according to the expected titer of the virus, prepare 7 to 10 sterile EP tubes, add 90 μl of serum-free culture medium to each tube; take 10 μl of the virus stock solution to be measured and add it to the first tube, mix it well, and record it as 1E+1 μl. Perform a ten-fold dilution in the second EP tube, and the resulting virus stock solution is 1 / 10 of that in the first EP tube, recorded as 1E+0 μl, and so on. Perform the seventh ten-fold dilution in the eighth EP tube, recorded as 1E-6 μl; select the required cell wells, discard 90 μl of culture medium, add 90 μL of diluted virus solution, and culture in a 37°C, 5% CO2 incubator; after 24 hours, add 100 ml of complete culture medium to the cell wells, and avoid blowing up the cells during this process; after 4 days, observe the fluorescence expression, and the value of fluorescent cells decreases with the increase of dilution times; calculate the virus titer according to the observation results of the fluorescence microscope, where virus titer = number of fluorescent cells / amount of virus stock solution, for example: 1E-6 If one fluorescent cell is observed in the infection well of 1 μl of virus stock solution, it means that at least one virus particle in the well has infected the cell, that is, 1 / (1E-6)=1E+6 (TU / μl), which means that the virus titer is 1×10 9 TU / ml. Example 9

[0229] This embodiment provides a method for preparing CAR-T cells. The preparation process includes: confirming that the T cells are in good condition, setting the speed to 1200 rpm, centrifuging for 5 min, resuspending with 1640 culture medium, and counting the cells; after counting, diluting the cells to 1×10 6 / ml; take a six-well plate, add 500 μl of cell suspension, 500 μl of concentrated virus and 40 μl of transduction reagent to each well, and mix well; after mixing, culture in a 37°C incubator, add fresh culture medium after 8-12 hours and observe the cell status; 3-4 days after infection, observe the fluorescence expression. For cells with slow growth or slow metabolism, the infection time can be appropriately extended before observation, and the medium can be changed in the middle to maintain the growth activity of the cells. Example 10

[0230] This embodiment is a method and test result of detecting the transfection efficiency of CAR-T by flow cytometry. The method of detecting the transfection efficiency of CAR-T includes: taking a flow cytometry tube, labeling the tube, placing untransfected T cells ( Fig. 91 ml of culture medium (denoted as NT in the figure), DR5 CAR-T cells, and DR5 / ASGR1 CAR-T cells were taken out from each well of the six-well plate, the speed was set to 1200 rpm, the cells were centrifuged for 5 min, and the liquid was discarded; 2 ml of PBS was added to resuspend the cells, and the cells were oscillated evenly. The speed was set to 1200 rpm, the cells were centrifuged for 5 min, and the liquid was discarded; 100 μl of PBS was added to resuspend the cells, and the cells were flicked and loaded on the machine; the flow cytometer adjusted the voltage, selected the FITC channel to observe ASGR1, and the PE channel to observe DR5, and observed the transduction efficiency of the two CAR-T cells.

[0231] The results are as follows Fig. 9 As shown, Fig. 9 A The results showed that CD8 + Detection of CD3 by flow cytometry after magnetic bead sorting + The proportion of T cells was 88.7%, of which CD8 + The proportion of T cells was 96.6%; CD3 was detected by flow cytometry after CD3 / CD28 magnetic beads activation + The proportion of T cells was 96.6%, CD8 + The proportion of T cells was 56.6%. Fig. 9 B Results showed that the transduction efficiencies of different CAR-T cells detected by flow cytometry after lentiviral transduction were: DR5 single-target CAR-T: 60%, ASGR1 single-target CAR-T: 63.5%, DR5 / ASGR1 dual-target CAR-T: 35%. Embodiment 11

[0232] This example is a comparison of the killing function of CAR-T cells with different effector-target ratios on liver cancer cells (LDH release test). LDH (lactate dehydrogenase) is an enzyme that is stably present in the cell cytoplasm. Under normal conditions, it only exists inside the cell. When the cell is stimulated to die, the plasma membrane ruptures and LDH is rapidly released outside the cell (in this experiment, LDH is rapidly released into the cell culture medium). Therefore, the number of dead cells can be determined by the amount of LDH released from the damaged cell membrane detected in the LDH release test. The test results of the LDH release test directly reflect the cell mortality rate, that is, the higher the measured absorbance, the stronger the cytotoxicity of the test substance.

[0233] The specific operation steps include: target cell plating, target cells (HepG2 cells) digested and resuspended at 2×10 5 / ml, 50μl / well, the target cells were inoculated into 96-well plates, and 3 parallel wells were set up for each group; effector cells were plated, and the effector cells (dual-target CAR-T, single-target CAR-T or untransfected T cells) were resuspended at 3.2×10 6 / ml, 1.6×10 6 / ml, 8×105 / ml, 4×10 5 / ml, 50 μl / well was mixed with target cells and incubated at 37°C, 5% CO2. The killing time was 16-18 h.

[0234] Specifically, this embodiment quantitatively detects the toxic killing effect on cells by detecting the release of LDH in the cell culture supernatant. The experimental grouping provided in this embodiment is as follows: ① Experimental group: LDH released by target cells + effector cells; ② Spontaneous control group: LDH released spontaneously by target cells; ③ Maximum release group: LDH released after complete lysis of target cells; ④ Effector cell spontaneous group: LDH released spontaneously by effector T cells; ⑤ Volume control group: background value generated by blank culture medium, in which effector cells are untransfected T cells ( Fig.10 NT in the above refers to NT cells, which are equivalent to the untransfected T cells in this example), DR5 single-target CAR-T cells, and DR5 / ASGR1 dual-target CAR-T cells. The detection method was carried out according to the instructions.

[0235] After the killing test, add 10 μl of Lysis Buffer to the high control well and culture in a CO2 incubator at 37°C for 30 min. Add 100 μl of Working Solution to each well, wrap the entire culture plate with aluminum foil, and culture in a light-proof, room temperature condition. Add 50 μL of Stop Solution to each well, immediately place it in a microplate reader, and measure the absorbance at 490 nm.

[0236] This example provides a cytotoxicity calculation formula, specifically: cytotoxicity (%) = (experimental group-spontaneous control group-effect cell spontaneous group) / (maximum release group-effect cell spontaneous group-volume control group)×100%.

[0237] Comparison of the killing effects of CAR-T cells with different effector-target ratios on liver cancer cells Fig.10 Specifically, the LDH release test compared the damage to tumor cells after co-culture of two CAR-T cells with tumor cells Huh7 at effector-target ratios of 2:1, 4:1, 8:1, and 16:1. Fig.10 The results from A to E showed that the damaging effect of DR5 single-target CAR-T cells on tumor cells Huh7 was higher than that of untransfected T cells at all effector-target ratios; the damaging effect of DR5 / ASGR1 dual-target CAR-T cells on tumor cells Huh7 was higher than that of DR5 single-target CAR-T cells at all effector-target ratios. Example 12

[0238] This example is a comparison of the killing function of CAR-T cells on liver cancer cells (liver cancer cell apoptosis detection).

[0239] Fig.11 This is the apoptosis of liver cancer cells detected by flow cytometry when the effector-target ratio is 2:1.

[0240] Flow cytometry was used to detect the apoptosis of tumor cells after co-culture of two types of CAR-T cells with single-target DR5 and dual-target DR5 / ASGR1 with tumor cells Huh7 and HepG2. Fig.11 As shown in A and B, DR5 single-target CAR-T cells promoted Huh7 cell apoptosis better than untransfected T cells (P < 0.05), and DR5 single-target CAR-T cells promoted HepG2 cell apoptosis better than untransfected T cells (P < 0.0001). DR5 / ASGR1 dual-target CAR-T cells promoted Huh7 cell apoptosis better than DR5 single-target CAR-T cells, and the results were statistically significant (P < 0.001). DR5 / ASGR1 dual-target CAR-T cells promoted HepG2 cell apoptosis better than DR5 single-target CAR-T cells, and the results were statistically significant (P < 0.01). Example 13

[0241] This example provides an operating method for a cytotoxicity test (Granzym B release test), and the specific operating steps include: taking out Huh7 / HepG2 cells grown to 90% from an incubator, discarding the supernatant, and washing once with 5 ml of PBS; adding 2 ml of trypsin, digesting in an incubator at 37°C for 3 min, and adding 3 ml of MEM / DMEM complete medium to neutralize; transferring the cells to a 15 ml centrifuge tube, setting the speed to 1200 rpm, centrifuging for 5 min, and 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; take 24-well plates, add 500 μl of cell suspension to each well, and culture; take untransfected T cells and CAR-T cells to a 15 ml centrifuge tube, set the speed to 1200 rpm, and centrifuge for 5 min; add 3 ml of 1640 culture medium to resuspend the cells, take 10 μl of cell suspension for counting, and adjust the cell concentration to 1×10 6 / ml; add 500 μl of untransfected T cell suspension or CAR-T cell suspension to each well of the 24-well plate and culture in an incubator for 18 h; take 1 ml of CAR-T cells from each well of the 24-well plate into a flow tube, set the speed to 1200 rpm, centrifuge for 5 min, discard the liquid, add 1 μl of CD3 antibody, mix well, and incubate in the dark for 30 min; wash once with PBS, add the corresponding reagents in the live / dead kit (calcein AM (live cell indicator), SYTOX™ Deep Red nucleic acid stain (dead cell indicator)), mix well, incubate in the dark for 30 min, wash once with PBS; add Fix / perm buffer, mix well, incubate in the dark for 50 min; add Perm wash buffer and wash once, set the speed to 1200 rpm, centrifuge for 5 min; add an appropriate amount of granzyme B antibody (Granzym B) according to the number of cells, incubate in the dark for 40 min; wash once, discard the supernatant, and put on the machine. Embodiment 14

[0242] This embodiment provides the operation method and test results of the cytotoxicity test (CD107a expression test). CD107a is a highly glycosylated transmembrane protein present in lysosomes. It is one of the most abundant proteins in cytolytic granules and can be used to evaluate the activity of NK cells or T cells. Specifically, the particles reach the serosal surface of NK / T cells, the particle 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 (CTL), which is directly related to the cytotoxic activity of NK cells or CTLs, and can be detected by flow cytometry after specific antibody labeling.

[0243] The specific operation method includes: opening the biosafety cabinet 30 min in advance and irradiating with ultraviolet light for 30 min; adjusting the cell density of HepG2 / Huh7 cells to 1×10 6 / ml, 1 ml was taken from each well and inoculated on a 24-well plate and cultured in a 37°C incubator; the concentrations of untransfected T cells and three types of CAR-T cells were adjusted to 1×10 6 / ml; the cells were divided into three groups, namely, untransfected T cell group, DR5 CAR-T cell group, and DR5 / ASGR1 CAR-T cell group, and 1 ml of the corresponding CAR-T cell suspension was added to the corresponding cell group, and Golgi inhibitors monensin and brefeldin a were added to the culture medium, and co-cultured in a 37°C incubator for 18 h; the cells were taken out from the incubator, transferred to a flow tube, centrifuged at 1200 rpm for 5 min, and the liquid was discarded; 1 ml of PBS was added to resuspend the cells, the speed was set to 1200 rpm, centrifuged for 5 min, and the liquid was discarded; CD107a antibody was added, and incubated in the dark for 30 min; 1 ml of PBS was added to resuspend the cells, the speed was set to 1200 rpm, centrifuged for 5 min, and the liquid was discarded; 100 μl of PBS was added dropwise, and the test was performed on the flow cytometer.

[0244] Fig.12 A is the expression of CD107a detected by flow cytometry after three types of T cells (untransfected T cells, DR5 single-target T cells, DR5 / ASGR1 dual-target T cells) were co-incubated with liver cancer cells Huh-7 and HepG2; Fig.12 B is the expression level of Granzym B detected by flow cytometry after co-incubation of three types of T cells with liver cancer cells Huh-7 and HepG2. .

[0245] Fig.12 The results showed that after co-culture of DR5 single-target CAR-T with Huh7 cells, the amount of CD107a released was higher than that of untransfected T cells (P < 0.01); after co-culture of DR5 / ASGR1 dual-target CAR-T with Huh7 cells, the amount of CD107a released was higher than that of DR5 single-target CAR-T (P < 0.001). After co-culture of DR5 single-target CAR-T with HepG2 cells, the amount of CD107a released was higher than that of untransfected T cells (P < 0.001); after co-culture of DR5 / ASGR1 dual-target CAR-T with HepG2 cells, the amount of CD107a released was higher than that of DR5 single-target CAR-T (P < 0.01). Fig.12A). After DR5 single-target CAR-T cells were co-cultured with Huh7 cells, the release of Granzym B was higher than that of untransfected T cells (P<0.001); after DR5 / ASGR1 dual-target CAR-T cells were co-cultured with Huh7 cells, the release of Granzym B was higher than that of DR5 single-target CAR-T cells (P<0.001). After DR5 single-target CAR-T cells were co-cultured with HepG2 cells, the release of Granzym B was higher than that of untransfected T cells (P<0.01); after DR5 / ASGR1 dual-target CAR-T cells were co-cultured with HepG2 cells, the release of Granzym B was higher than that of DR5 single-target CAR-T cells (P<0.01) ( Fig.12 B). Embodiment 15

[0246] This example provides the experimental results of FZJD-containing serum enhancing the inhibitory effect of DR5 / ASGR1 dual-target CAR-T cells on liver cancer cells.

[0247] In this example, the CCK8 method was used to detect the inhibitory effect of FZJD drug-containing serum combined with DR5 / ASGR1 dual-target CAR-T cells on tumor cell proliferation. Fig.13 As shown in the figure, when the serum concentration was 30%, FZJD-containing serum could effectively increase the inhibitory effect of DR5 / ASGR1 dual-target CAR-T cells on the growth of Huh7 cells, and the results were statistically significant (P < 0.05) ( Fig.13 A). When the serum concentration was 20% and 30%, FZJD-containing serum could effectively increase the inhibitory effect of CAR-T cells on the growth of HepG2 cells ( Fig.13 B), the results were statistically significant. Example 16

[0248] This example provides the experimental results of FZJD-containing serum enhancing the apoptosis effect of DR5 / ASGR1 dual-target CAR-T cells on liver cancer cells. Fig.14 shown.

[0249] In this example, flow cytometry was used to detect the effect of FZJD-containing serum on the apoptosis of DR5 / ASGR1 dual-target CAR-T cells on tumor cells Huh7 and HepG2. Fig.14As shown in A and B, DR5 / ASGR1 dual-target CAR-T cells promoted Huh7 cell apoptosis better than untransfected T cells (P < 0.0001). DR5 / ASGR1 dual-target CAR-T cells promoted HepG2 cell apoptosis better than untransfected T cells (P < 0.001). FZJD-containing serum can effectively increase the effect of DR5 / ASGR1 dual-target CAR-T cells on Huh7 cell apoptosis, and the results were statistically significant (P < 0.01). FZJD-containing serum can effectively increase the effect of DR5 / ASGR1 dual-target CAR-T cells on HepG2 cell apoptosis, and the results were statistically significant (P < 0.05). Embodiment 17

[0250] This example provides the release of Granzym B after the combined action of FZJD drug-containing serum and DR5 / ASGR1 dual-target CAR-T cells.

[0251] The specific operation method for detecting the release of Granzym B after the combined action of FZJD drug-containing serum and DR5 / ASGR1 dual-target CAR-T cells includes: after Huh7 / HepG2 cells grown to 90% are digested, the tumor cell density is adjusted to 1×10 6 / ml; take a 24-well plate, add 500 μl of cell suspension to each well, and culture in a 37°C incubator; take untransfected T cells and dual-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, untransfected T cell group, DR5 / ASGR1 dual-target CAR-T cell group and Fuzheng Jiedu Xiaoji Recipe combined with DR5 / ASGR1 dual-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 each well of the DR5 / ASGR1 dual-target CAR-T cell group and FZJD combined with DR5 / ASGR1 dual-target CAR-T cell group, and 500 μl of culture medium containing normal rat serum was added to the untransfected T cell group and DR5 / ASGR1 dual-target CAR-T cell group, and Fuzheng Jiedu Xiaoji Recipe drug-containing serum was added to the Fuzheng Jiedu Xiaoji Recipe combined with DR5 / ASGR1 dual-target CAR-T cell group, so that the serum concentration of each group was 30%, and cultured in an incubator at 37°C for 18 h; 1 cell was taken from each well of the 24-well plate ml of CAR-T cell suspension was transferred to the flow tube, the speed was set to 1200 rpm, centrifuged for 5 min, the liquid was discarded, 1 μl of CD3 antibody was added, mixed, and incubated in the dark for 30 min; washed once with PBS, live / dead was added, mixed, incubated in the dark for 30 min, and washed once with PBS; Fix permbuffer was added, mixed, and incubated in the dark for 50 min; Perm wash buffer was added, the speed was set to 1200 rpm, and centrifuged for 5 min; according to the number of cells, an appropriate amount of granzyme B antibody was added, and the cells were incubated in the dark for 40 min; washed once, the supernatant was discarded, and the cells were put into the flow tube. Embodiment 18

[0252] This example provides the detection of CD107a expression and test results after the combined action of FZJD drug-containing serum and DR5 / ASGR1 dual-target CAR-T cells. Fig.15 shown.

[0253] The specific operation method for detecting the expression of CD107a after the combined action of FZJD drug-containing serum and DR5 / ASGR1 dual-target CAR-T cells includes: taking out 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 adding 3 ml of MEM / DMEM complete medium to neutralize; transferring the cells to a 15 ml centrifuge tube, setting the speed to 1200 rpm, centrifuging for 5 min, and discarding the supernatant; adding 3 ml of MEM / DMEM complete medium to resuspend the cells, taking 10 μl of the cell suspension for counting, and adjusting the tumor cell density to 1×10 6 / ml; take a 24-well plate, add 500 μl of cell suspension to each well, and culture in a 37°C incubator; take untransfected T cells and DR5 / ASGR1 dual-target CAR-T cells into 15 ml centrifuge tubes, set the speed to 1200 rpm, and centrifuge for 5 min; add 3 ml of 1640 culture medium to resuspend the cells, take 10 μl of cell suspension for counting, and adjust the cell concentration to 1×10 6 / ml; the cells were divided into three groups, namely, untransfected T cell group, DR5 / ASGR1 dual-target CAR-T cell group and Fuzheng Jiedu Xiaoji Recipe combined with DR5 / ASGR1 dual-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 each well of the DR5 / ASGR1 dual-target CAR-T cell group and Fuzheng Jiedu Xiaoji Recipe combined with DR5 / ASGR1 dual-target CAR-T cell group, and 500 μl of culture medium containing normal rat serum was added to the untransfected T cell group and DR5 / ASGR1 dual-target CAR-T cell group, and Fuzheng Jiedu Xiaoji Recipe drug-containing serum was added to the Fuzheng Jiedu Xiaoji Recipe combined with DR5 / ASGR1 dual-target CAR-T cell group to make the serum concentration of each group 30%, and Golgi inhibitors monensin and brefeldin a were added to the culture medium, and the cells were cultured in an incubator at 37°C for 18 h; remove the cells from the incubator, transfer the cells to the flow tube, set the speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add 1 ml of PBS to resuspend the cells, set the speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add CD107a antibody and incubate in the dark for 30 min; add 1 ml of PBS to resuspend the cells, set the speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add 100 μl of PBS and detect on the machine.

[0254] Fig.15 A is the expression of CD107a on T cells detected by flow cytometry; Fig.15 B is the expression of Granzym B in T cells detected by flow cytometry. . After DR5 / ASGR1 dual-target CAR-T cells were co-cultured with Huh7 cells, the amount of CD107a released was higher than that of untransfected T cells (P < 0.001), and FZJD-containing serum could significantly increase the amount of CD107a released after DR5 / ASGR1 dual-target CAR-T cells were co-cultured with Huh7 cells (P < 0.01). After DR5 / ASGR1 dual-target CAR-T cells were co-cultured with HepG2 cells, the amount of CD107a released was higher than that of untransfected T cells (P < 0.0001), and FZJD-containing serum could significantly increase the amount of CD107a released after DR5 / ASGR1 dual-target CAR-T cells were co-cultured with HepG2 cells (P < 0.05) ( Fig.15 A). After DR5 / ASGR1 dual-target CAR-T cells were co-cultured with Huh7 cells, the release of Granzym B was higher than that of untransfected T cells (P<0.0001), and FZJD-containing serum could significantly increase the release of Granzym B after DR5 / ASGR1 dual-target CAR-T cells were co-cultured with Huh7 cells (P<0.001). After DR5 / ASGR1 dual-target CAR-T cells were co-cultured with HepG2 cells, the release of Granzym B was higher than that of untransfected T cells (P<0.001), and FZJD-containing serum could significantly increase the release of Granzym B after DR5 / ASGR1 dual-target CAR-T cells were co-cultured with HepG2 cells, and the release of Granzym B was higher than that of DR5 / ASGR1 dual-target CAR-T cells (P<0.05) ( Fig.15 B). Embodiment 19

[0255] This example provides a method for detecting phenotypic changes of FZJD combined with DR5 / ASGR1 dual-target CAR-T cells by flow cytometry and test results. Fig.16 shown.

[0256] Studies have shown that STAT3 signal activation can increase the expression of PD-L1 in tumor cells, leading to the exhaustion of T cells in the tumor, and the coordinated expression of IL-6 and PD-L1 can significantly inhibit CD8 + T cell infiltration capacity in tumors.

[0257] The specific operation method for detecting the phenotypic changes of FZJD combined with DR5 / ASGR1 dual-target CAR-T cells by flow cytometry includes: preparing CD3 / CD4 / CD8 / CD45RA / CCR7 / CD38 / HLA4 / PD1 / TIGIT / TIM3 / CTLA4 mixed antibodies; taking CAR-T cells from the T25 cell culture flask into a 15 ml centrifuge tube, setting the speed to 1200 rpm for 5 min, resuspending the cells in 1640 culture medium, counting, and adjusting the cell concentration to 1×10 6 / ml; transfer the cell suspension to a 24-well plate and divide it into a FZJD drug-containing serum group and a normal rat serum group; add FZJD drug-containing serum and normal rat serum to the corresponding wells and culture in a 37°C incubator for 24 h; remove the cells, set the speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add 1 ml of PBS to resuspend the cells, set the speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add mixed antibodies and incubate in the dark for 30 min; add 1 ml of PBS to resuspend the cells, set the speed to 1200 rpm, centrifuge for 5 min, and discard the liquid; add 100 μl of PBS and detect on the machine.

[0258] The results are as follows Fig.16 As shown, Fig.16 A and B are the results of T cell phenotype detection by flow cytometry; Fig.16 C is flow cytometry to detect CD4 + T cell ratio; Fig.16 D is flow cytometry to detect tumor cell phenotype. . FZJD drug-containing serum can increase CD4 + The ratio of naïve T cells (naïve cells) and central memory T cells (TCM) in CAR-T cells. Among them, FZJD-containing serum had no statistically significant effect on the increase of naïve T cells (P>0.05), but FZJD-containing serum had a statistically significant effect on the increase of central memory T cells (P<0.01). FZJD-containing serum can increase CD8 + The proportion of naïve T cells (Naïve cells) and central memory T cells (TCM) in CAR-T cells, and the effect of FZJD-containing serum on the increase of naïve T cells and central memory T cells was statistically significant (P < 0.05) ( Fig.16A). In this example, flow cytometry was used to detect the effect of FZJD-containing serum on the expression of immunostimulatory receptors and immunoinhibitory receptors on the surface of CAR-T cells. The results showed that FZJD-containing serum can significantly increase the expression of immunostimulatory receptors on the surface of CAR-T cells (P < 0.01), and significantly reduce the expression of immunoinhibitory receptor TIGIT on the surface of CAR-T cells (P < 0.05). In addition, FZJD-containing serum has a tendency to reduce the expression of immunoinhibitory receptors PD-1, TIM3 and CTLA-4 on the surface of CAR-T cells ( Fig.16 B). Flow cytometry was used to detect whether FZJD-containing serum could change the expression of CAR-T cell CD8 + There was no significant difference in the proportion of T cells. Fig.16 C). Flow cytometry was used to detect whether FZJD-containing serum could reduce the expression of PD-L1 on the surface of tumor cells. The results showed that FZJD-containing serum had a tendency to reduce the expression of PD-L1 on the surface of tumor cells ( Fig.16 D). Embodiment 20

[0259] This example provides the experimental results of FZJD-containing serum reducing the level of inflammatory cytokine release during the killing of target cells by liver cancer cells and DR5 / ASGR1 dual-target CAR-T.

[0260] Cytokine storm (CRS) is a common adverse event related to CAR-T immunotherapy, and its incidence is high. Interleukin 6 (IL-6) is a proinflammatory cytokine that is widely involved in various inflammatory responses of the body and plays an important role in the "cytokine storm" that occurs in CAR-T immunotherapy. IL-6 is both an activating factor of STAT3 and a downstream product of the STAT3 pathway. STAT3 activation can promote the secretion of IL-6 by liver cancer cells, forming a positive feedback effect, aggravating the cytokine storm, causing a persistent chronic inflammatory environment in the local tumor, and promoting tumor progression through multiple pathways. In the Phase I study of GPC3 single-target CAR-T conducted in CRS, 70% of patients developed CRS. Four patients received high-dose hormone pulse therapy, and two of them also received tocilizumab, a monoclonal antibody targeting the IL-6 receptor. Therefore, inhibiting IL-6 can effectively inhibit the cytokine storm and reduce adverse reactions during treatment. In addition, the activation of STAT3 can reduce CD8 + The expression of chemokine receptor CXCR3 on the surface of T cells and the reduction of NK and CD4 + T cells secrete IFN-γ and CD8 + T chemokine CXCL10 levels, leading to CD8 + It is difficult for cells to chemotaxis toward tumor tissue.

[0261] In this example, ELISA was used to detect the expression of IL-6 in the cell supernatant after co-culture of control serum, FZJD-containing serum, DR5 / ASGR1 dual-target CAR-T cells, and FZJD combined with DR5 / ASGR1 dual-target CAR-T cells and target cells HepG2, and the change of IL-6 release by FZJD-containing serum was observed. Fig.17 As shown in the results, co-culture of DR5 / ASGR1 dual-target CAR-T cells with target cells significantly increased the expression of proinflammatory factor IL-6 (P < 0.01), and FZJD-containing serum could effectively reduce the release of proinflammatory factor IL-6 after co-culture of DR5 / ASGR1 dual-target CAR-T cells with target cells, thereby effectively inhibiting the formation of cytokine storm in CAR-T therapy (P < 0.01). Embodiment 21

[0262] This example provides the experimental results that FZJD-containing serum increases the level of interferon release when DR5 / ASGR1 dual-target CAR-T cells kill liver cancer cells.

[0263] In this example, ELISA was used to detect the expression of interferon gamma (IFN-γ) in the cell supernatant after co-culture of FZJD combined with DR5 / ASGR1 dual-target CAR-T cells and target cells HepG2, reflecting the promotion effect of FZJD medicated serum on the killing of target cells by DR5 / ASGR1 dual-target CAR-T cells. The results are as follows Fig.18 shown.

[0264] The results are as follows Fig.18 As shown in A (target cells are HepG2) and B (target cells are HepG3B), FZJD-containing serum can effectively enhance the ability of DR5 / ASGR1 dual-target CAR-T cells to secrete IFN-γ after co-culture and improve the killing function (P < 0.05). Embodiment 22

[0265] This example provides the test results of FZJD-containing serum increasing the level of T cell chemokine release in liver cancer cells. Fig.19 shown.

[0266] The CXCR3 / CXCL10 chemotactic axis is an important signaling pathway for T cell migration, and CXCL9 / 10 / 11 can recruit CD8 +Various immune cells of T cells can directly kill tumor cells or play an anti-tumor role, thereby improving the overall survival rate of patients. The defective expression of CXCL9 / 10 / 11 in the tumor site may be one of the mechanisms of tumor immune escape. In this example, ELISA was used to detect the level of T cell chemokine release of liver cancer cells by FZJD drug-containing serum. The results are as follows Fig.19 As shown in A~C, The results showed that the levels of CXCL9 / 10 / 11 were significantly increased in the FZJD group and the STAT3 inhibitor (stattic) group (p<0.05). Embodiment 23

[0267] 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.

[0268] A and B can be TNFRSF10B and ASGR1, and their positions can be interchanged; the positions of VH and VL of the same protein can be interchanged.

[0269] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-linker-ASGR1 VHH.

[0270] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VH.

[0271] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VL-ASGR1 VH.

[0272] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VHH-Linker-ASGR1 VH-ASGR1 VL.

[0273] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can be TNFRSF10B VH-linker-TNFRSF10B VL-Linker-ASGR1 VHH.

[0274] According to a preferred embodiment, the structure of the tandem chimeric antigen receptors can also be TNFRSF10B VL-linker-TNFRSF10B VH-Linker-ASGR1 VHH.

[0275] According to a preferred embodiment, the structure of the tandem chimeric antigen receptors can also be ASGR1 VHH -linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0276] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

[0277] According to a preferred embodiment, the structure of the tandem chimeric antigen receptors can also be ASGR1 VL-linker-ASGR1 VH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0278] According to a preferred embodiment, the structure of the tandem chimeric antigen receptors can also be ASGR1 VL-linker-ASGR1 VH -linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

[0279] According to a preferred embodiment, the structure of the tandem chimeric antigen receptors can also be ASGR1 VH-linker-ASGR1 VL-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0280] According to a preferred embodiment, the structure of the tandem chimeric antigen receptors can also be ASGR1 VH-linker-ASGR1 VL-linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

[0281] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VH-linker-TNFRSF10B VH-Linker-TNFRSF10B VL.

[0282] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VH-linker-TNFRSF10B VL-Linker-TNFRSF10B VH.

[0283] According to a preferred embodiment, the structure of the tandem chimeric antigen receptor can also be ASGR1 VHH-linker-TNFRSF10B VHH.

[0284] The present invention reserves the right to transform the heavy chain variable domain VH of scFv or humanized heavy chain antibody into the heavy chain variable domain VHH of camelid antibody.

[0285] It should be noted that the above-mentioned specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention specification and its drawings are illustrative and do not constitute a limitation of the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present invention specification contains multiple inventive concepts, such as "preferably" and "according to a preferred embodiment", both of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as a must-have setting. Therefore, the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A pharmaceutical composition, characterized in that The method comprises TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells and a Fuzheng Jiedu Xiaoji recipe, wherein the dual-target chimeric antigen receptor T cells express TNFRSF10B / ASGR1 dual-target chimeric antigen receptor on their membrane surface. The dual-target chimeric antigen receptor T cell of TNFRSF10B / ASGR1 comprises a first CAR and a second CAR, The first CAR comprises a first antigen binding region targeting TNFRSF10B, and the second CAR comprises a second antigen binding region targeting ASGR1. 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 as shown in SEQ ID NO: 1, and the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is as 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 as shown in SEQ ID NO: 3, The prescription for strengthening the body, detoxifying and eliminating accumulation is: Codonopsis pilosula 15 g, raw Astragalus 15 g, Atractylodes macrocephala 15 g, Poria 15 g, Adenophora 15 g, Ophiopogon japonicus 15 g, Angelica 15 g, Rehmannia glutinosa 15 g, Polygonum multiflorum 15 g, Curcuma 9 g, and Pinellia 9 g.

2. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for treating liver cancer.

3. A T cell expressing a dual-target chimeric antigen receptor, characterized in that: The dual-target chimeric antigen receptor includes a first CAR and a second CAR. The first CAR comprises: a first antigen binding region targeting TNFRSF10B, which is sequentially connected from the carboxyl terminus to the amino terminus; a first hinge region; a first transmembrane binding region and CD3ζ, 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 as shown in SEQ ID NO: 1, and the amino acid sequence of the anti-TNFRSF10B antibody heavy chain variable region is as shown in SEQ ID NO: 2; The second CAR comprises: a signal peptide connected sequentially from the carboxyl terminus to the amino terminus; 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 in SEQ ID NO:

3.

4. Use of the T cell expressing the dual-target chimeric antigen receptor as claimed in claim 3 in the preparation of a drug for treating liver cancer.

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