Pharmaceutical composition and use thereof

Abstract

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

Description

A pharmaceutical composition and its application Technical Field

[0001] The present invention relates to the field of biomedicine technology and belongs to IPC classification number A61K. It specifically relates to a pharmaceutical composition and its application, and more particularly 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 its application in improving the efficacy of treating primary liver cancer after being used in combination with the Fuzheng Jiedu Xiaoji Recipe (FZJD). Background Art

[0002] Liver cancer, also known as malignant tumors of the liver, is the sixth most common cancer worldwide and the fourth leading cause of cancer-related death, with a five-year survival rate of 18%. Hepatocellular carcinoma (HCC) accounts for 90% of cases and is a primary liver cancer, a malignant tumor that originates in liver cells. my country has among the highest incidence and mortality rates of liver cancer globally, placing a heavy medical burden on the country. Traditional treatments for liver cancer include surgery, transcatheter arterial chemoembolization, local ablation, and targeted therapy, but these approaches have limited efficacy, leading to the increasing importance of combined tumor immunotherapy.

[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 step 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. However, 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 activity and gradually become exhausted. At the same time, approximately 30% of liver cancer tissues lack infiltration of T cells and inflammatory cells, so the expression of antigen targets of ICIs is low, and there is innate resistance to them. In short, the current efficacy of ICIs in liver cancer is not satisfactory.

[0004] Chimeric Antigen Receptor T-Cell Immunotherapy (abbreviated as "CAR-T cell therapy") is one of the emerging means of tumor immunotherapy and is the focus of current tumor treatment research. Chimeric Antigen Receptor (also known as "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, a large number of CAR-expressing T cells (abbreviated as "CAR-T cells") that can express CAR are prepared. 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 capable of self-proliferation and activation and have powerful tumor-killing ability. Therefore, they are regarded as one of the hopes for completely curing tumors in the future.

[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, a variety of CAR therapies targeting tumor cell surface antigens have entered Phase 1 and 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) and carcinoembryonic antigen (CEA) expressed by colorectal cancer and breast cancer, and targeting prostate-specific membrane antigen (PSMA) for prostate cancer. WO2020017479A1 discloses a CAR containing a single-chain antibody that can specifically bind to GPC3 on the cell membrane. By introducing the gene encoding CAR into immune-competent cells, immune-competent cells expressing CAR with excellent cancer cell toxicity and IFN-γ production ability are constructed. However, the ability of single-target CAR to recognize and kill tumor cells is limited, which may cause some tumor cells to escape the attack of CAR-T cells, increasing the risk of tumor recurrence. US2024043490A1 discloses the construction of targeted cytokines for engineered cell therapy, wherein the targeted cytokine construct selectively activates engineered cells with a potency of 10 times or greater, thereby achieving the purpose of treating cancer. The main mechanism of recurrence after CAR-T cell therapy is the limited persistence of CAR-T cells, inhibition of CAR-T cell function and antigen escape. In order to minimize the risk of recurrence due to target escape, clinical trials are actively exploring the strategy of dual-target CAR-T to recognize more than one tumor-associated antigen. In preclinical models and clinical trials for hematological malignancies, dual-target CAR-T cell therapy uses at least three antigen combinations: CD19 / CD20, CD19 / CD22, and BCMA / CD38. In liver cancer, CAR-Ts targeting Asialoglycoprotein Receptor 1 (ASGR1) and Glypican-3 (GPC3) are currently under development. "Off-target effects" are a widespread challenge in CAR-T applications. In solid tumors, CAR-T cells' recognition of non-tumor tissues expressing target antigens often leads to clinically severe off-target toxicities. Existing GPC3-only CAR-Ts have low sensitivity and specificity for liver cancer recognition. High-grade cytokine release syndrome (Grade 5) has been reported in clinical applications of GPC3-only CAR-Ts, and their safety needs to be further improved.There is currently a GPC3 dual-target CAR-T therapy, but its killing effect has not been significantly improved compared to the GPC3 single-target CAR-T. Summary of the Invention

[0006] While targets for hematologic malignancies and gliomas have good specificity, the number of antigens on the surface of liver cancer cells that can serve as targets for CAR-T cells is relatively limited. For example, the GPC3 positivity rate in liver cancer tissue is approximately 60%, meaning that approximately 40% of patients' liver cancer cells are not recognized by GPC3-targeted CAR-T cells. Therefore, the development of other CAR-Ts with greater targeting, sensitivity, and specificity is urgently needed. In liver cancer treatment, CAR-Ts are hampered by poor tumor-specific chemotaxis, limited infiltration into solid tumors, and T cell exhaustion induced by the persistent inflammatory environment within tumors and high PD-L1 expression in liver cancer cells. Furthermore, most tumor-associated antigens expressed on liver cancer cells are non-tumor-specific and can be expressed on other normal tissues. This makes these tissues susceptible to CAR-T cell attack, leading to a systemic "cytokine storm" characterized by elevated interleukin-6 (IL-6), which can lead to multiple organ failure and death.

[0007] GPC3 is a member of the heparan sulfate proteoglycan family and is highly expressed in HCC, but not expressed or 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 GPC3 positive, 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 explorations 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. 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 parental MHCC-97L or MHCC-97L -GPC3+ , MHCC-97L -ASGR1+ and MHCC-97L -GPC3+ASGR1+In overexpression cell lines, including Huh-7 and HepG2 cells naturally expressing GPC3 and ASGR1, dual-targeted αGPC3-Z + αASGR1-28BB-modified T cells did not exhibit enhanced cytotoxicity compared to single-targeted first-generation CAR-T cells, αGPC3-Z-modified T cells. At effector-target ratios of 3:1 and 1:1, the cytotoxicity percentages were approximately 30% and 60%, respectively, with no significant difference between the two. Furthermore, a comparison of the therapeutic efficacy of third-generation CAR-T cells (GPC3-28BBZ) and dual-targeted CAR-T cells against Huh-7 subcutaneous xenografts revealed that the dual-targeted CAR-T cells exhibited significantly inferior in vivo tumor killing compared to the third-generation CAR-T. In summary, it is possible that the ASGR1 single-chain antibody (scFv) aggregates with certain specific domains of the adjacent GPC3 chimeric antigen antibody, leading to persistent phosphorylation and ultimately resulting in suboptimal anticancer efficacy. Therefore, the combination of GPC3 and ASGR1 as dual-targeting CAR-Ts still presents difficult technical challenges. In terms of clinical research safety, cytokine storm (CRS) is a common adverse event related to CAR-T immunotherapy, with a high incidence rate of 80% to 90%. However, severe CRS, that is, CRS of degree three or above, has a low incidence rate of about 10% to 30%. The current Phase I study of GPC3 single-target CAR-T has included 13 patients, 9 of whom developed cytokine release syndrome (CRS), 8 of whom were grade 1 to 2 and 1 was grade 5. Four patients received high-dose hormone pulse therapy, two of whom also received tocilizumab, a monoclonal antibody targeting the interleukin-6 receptor. One patient received 20.0×10 8 Grade 5 CRS occurred even with only 10 cells (low-dose group), and this is the first reported case of grade 5 CRS in an HCC patient after CAR-T cell therapy. In summary, the safety of single-target GPC3 CAR-T cell therapy in clinical application still needs to be further improved, and cytokine release needs to be further effectively controlled.

[0008] In response to the deficiencies of the prior art, the present invention provides, on one hand, a pharmaceutical composition comprising 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.

[0009] 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 depletion effectively enhances 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, suppressing the "inflammatory cytokine storm" caused by excessive release of IL-6, thereby reducing inflammation-related damage during treatment.

[0010] According to a preferred embodiment, the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor 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.

[0011] According to a preferred embodiment, the intracellular region includes co-stimulatory signal factors, CD3ζ and 4-1BB (CD137), and CD28.

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

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

[0014] 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.

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

[0016] 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 specifically binds 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 specifically binds to ASGR1 on the surface of liver cancer cells.

[0017] 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.

[0018] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription includes: Codonopsis pilosula, raw Astragalus membranaceus, Atractylodes macrocephala, Poria cocos, Adenophora scutellariae, Ophiopogon japonicus, Chinese Angelica sinensis, Rehmannia glutinosa, Paridis septififlora, Curcuma zedoaria, and Pinellia ternata. Specifically, the Fuzheng Jiedu Xiaoji prescription comprises: Codonopsis pilosula 15g, raw Astragalus membranaceus 15g, Atractylodes macrocephala 15g, Poria cocos 15g, Adenophora scutellariae 15g, Ophiopogon japonicus 15g, Chinese Angelica sinensis 15g, Rehmannia glutinosa 15g, Paridis septififlora 15g, Curcuma zedoaria 9g, and Pinellia ternata 9g.

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

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

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

[0022] 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.

[0023] 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.

[0024] 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.

[0025] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription increases CD4 + In CAR-T cells, the initial T cells ( cells) and central memory T cells (TCM), so that the aforementioned pharmaceutical composition can improve the therapeutic effect of liver cancer.

[0026] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription increases CD8 + In CAR-T cells, the initial T cells ( cells) and central memory T cells (TCM), so that the aforementioned pharmaceutical composition can improve the therapeutic effect of liver cancer.

[0027] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by increasing the expression of TNFRSF10B / ASGR1 dual-target CAR-T cell surface immunostimulatory receptors.

[0028] 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 immunoinhibitory receptor TIGIT on the surface of 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 reducing the expression of PD-L1 on the surface of tumor 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 pro-inflammatory factor IL-6.

[0031] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription enhances the killing function of TNFRSF10B / ASGR1 dual-target CAR-T cells by increasing their ability to secrete IFN-γ, thereby enabling the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer.

[0032] According to a preferred embodiment, the Fuzheng Jiedu Xiaoji prescription enables the aforementioned pharmaceutical composition to improve the therapeutic effect of liver cancer by inhibiting the STAT3 signaling pathway.

[0033] 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.

[0034] 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.

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

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

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

[0038] 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.

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

[0040] 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 specifically binds 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 specifically binds to ASGR1 on the surface of liver cancer cells.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] According to a preferred embodiment, the first CAR can be expressed as TNFRSF10BScFv-Hinge-TM-CD3ζ.

[0045] 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 through a cleavable domain or another promoter.

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

[0047] According to a preferred embodiment, the signaling domain of the first CAR includes CD8α (signal peptide).

[0048] According to a preferred embodiment, the first hinge region of the first CAR includes CD8αH (can be expressed as Hinge).

[0049] According to a preferred embodiment, the first transmembrane binding region is capable of anchoring and binding to the cell membrane.

[0050] According to a preferred embodiment, the second transmembrane binding region is capable of anchoring and binding to the cell membrane.

[0051] According to a preferred embodiment, the first transmembrane binding region comprises CD8αTM.

[0052] 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.

[0053] According to a preferred embodiment, CD3ζ is an intracellular signaling domain.

[0054] According to a preferred embodiment, the first CAR can be expressed as CD8αsignal peptide-TNFRSF10B VH-linker-TNFRSF10B VL-CD8αH-CD8αTM-CD3ζ.

[0055] According to a preferred embodiment, the second CAR can be expressed as CD8αsignal peptide-ASGR1VH-CD8αH-CD8αTM-CD137-CD28.

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

[0057] According to a preferred embodiment, the signaling domain of the second CAR includes CD8α (signal peptide).

[0058] According to a preferred embodiment, the second hinge region of the second CAR includes CD8αH (can be expressed as Hinge).

[0059] According to a preferred embodiment, the second transmembrane binding region comprises CD8αTM.

[0060] According to a preferred embodiment, the costimulatory signal binding region includes a 4-1BB (CD137) costimulatory signal domain and a CD28 costimulatory signal domain.

[0061] According to a preferred embodiment, the intracellular co-stimulatory signaling domain can comprise a CD3ζ intracellular signaling domain that can activate immune response cells.

[0062] According to a preferred embodiment, the second CAR can be expressed as CD8αsignal peptide-ASGR1 VH-CD8αH-CD8αTM-CD137-CD28.

[0063] According to a preferred embodiment, the immune response cells include T lymphocytes, NK cells, and NKT cells.

[0064] 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, or 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.

[0065] According to a preferred embodiment, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO:5.

[0066] According to a preferred embodiment, the amino acid sequence of the CD8αTM transmembrane binding region is shown in SEQ ID NO: 6.

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

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

[0069] According to a preferred embodiment, CD3ζ is an intracellular signaling domain, and its amino acid sequence is shown in SEQ ID NO:9.

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

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

[0072] Another aspect of the present invention provides a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0073] Another aspect of the present invention provides a vector comprising a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0074] According to a preferred embodiment, the vector can be one or more of adenovirus, lentivirus, adeno-associated virus or retrovirus.

[0075] The present invention provides another dual-target chimeric antigen receptor, wherein the dual-target chimeric antigens are linked in series. According to a preferred embodiment, the series-linked chimeric antigen receptor can be TNFRSF10B VHH-linker-ASGR1 VHH.

[0076] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VH-linker-TNFRSF10B VL-Linker-ASGR1 VH.

[0077] According to a preferred embodiment, the tandem chimeric antigen receptor can be TNFRSF10B VL-linker-TNFRSF10B VH-Linker-ASGR1 VH.

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

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

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

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

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

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

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

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

[0086] 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.

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

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

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

[0090] According to a preferred embodiment, the dual-target chimeric antigen receptor comprises: CD8a signal peptide, ASGR1scFv, G4S Linker, DR5scFv, CD8a hinge region, CD8α transmembrane region, 4-1BB, CD28 co-stimulatory molecule, and CD3ζ signal transduction molecule.

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

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

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

[0094] 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.

[0095] Another aspect of the present invention provides a host cell, which contains the aforementioned vector or has the aforementioned exogenous nucleic acid molecule integrated into its chromosome.

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

[0097] 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.

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

[0099] 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.

[0100] 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:

[0101] Providing immune cells to be modified; transducing a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor or a vector containing a nucleic acid molecule encoding a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor into the immune cells, thereby obtaining modified immune cells.

[0102] 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 target antigens at the same time. In addition to TNFRSF10B, the target molecule that can mediate the transmission of the first signal (CAR) after being recognized, ASGR1, the 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 dual-target CAR-T cells of TNFRSF10B / ASGR1 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 of TNFRSF10B in pan-cancer and liver cancer is shown in Figure 20: Figure 20A shows the expression of DR5 protein in human liver cancer tissues detected by immunohistochemistry and immunofluorescence; Figure 20B shows the correlation between TNFRSF10B expression in liver cancer and prognosis, as queried from the TCGA database; and Figure 20C shows the expression of TNFRSF10B in pan-cancer pathological tissues, as queried from the Human Protein Atlas database. TNFRSF10B expression in liver cancer tissues is greater than 75%, higher than the average expression of GPC3; TNFRSF10B expression is extremely low in normal tissues. The correlation between TNFRSF10B and prognosis in liver cancer patients suggests that this target is more widely distributed in liver cancer tissues, and CAR-T targeting it may have higher sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] FIG1 is a schematic diagram of the structure of the lentiviral backbone vector GV400 and GV401 plasmid provided by the present invention;

[0104] FIG2 is the result of the enzyme digestion with the restriction enzyme sites of BamH I / EcoR I provided by the present invention;

[0105] FIG3 is the result of the enzyme digestion with the restriction site BamH I / BamH I provided by the present invention;

[0106] FIG4 is a PCR result of a target fragment with a cloning site of BamH I / EcoR I provided by the present invention;

[0107] FIG5 is a PCR result of a target fragment with a cloning site of BamH I / BamH I provided by the present invention;

[0108] FIG6 is an identification result of BamH I / EcoR I corresponding target fragment exchanged into a linear expression vector provided by the present invention;

[0109] FIG7 is an identification result of BamH I / BamH I corresponding target fragment exchanged into a linear expression vector provided by the present invention;

[0110] FIG8 is a schematic diagram of the dilution of the virus titer by fluorescence detection provided by the present invention, and the observation of plasmid transfection and counting by fluorescence microscopy;

[0111] FIG9 is the result of flow cytometry detection of CAR-T transfection efficiency provided by the present invention;

[0112] FIG10 is a comparison of the killing effects of CAR-T cells with different effector-target ratios provided by the present invention on liver cancer cells;

[0113] FIG11 is a graph showing apoptosis of liver cancer cells detected by flow cytometry when the effector-target ratio provided by the present invention is 2:1;

[0114] FIG12 is a flow cytometry analysis of 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, as provided by the present invention;

[0115] FIG13 is a graph showing the inhibition of tumor cell proliferation detected by the combined treatment of FZJD drug-containing serum and DR5 / ASGR1 dual-target CAR-T cells using the CCK8 assay provided by the present invention;

[0116] FIG14 is an experimental result showing that the FZJD-containing serum provided by the present invention enhances the apoptosis of liver cancer cells by DR5 / ASGR1 dual-target CAR-T cells;

[0117] FIG15 shows 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;

[0118] FIG16 is the experimental results of flow cytometry detection of 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) provided by the present invention;

[0119] FIG17 is an ELISA result provided by the present invention for detecting the expression of cytokine IL-6 after co-culture of dual-target CAR-T and HepG2;

[0120] FIG18 is an ELISA result provided by the present invention for detecting the effect of FZJD drug-containing serum on the level of interferon release when DR5 / ASGR1 dual-target CAR-T cells kill liver cancer cells;

[0121] FIG19 is an experimental result showing that a FZJD-containing serum provided by the present invention increases the level of T cell chemokine release in liver cancer cells (Stattic is a small molecule inhibitor of STAT3);

[0122] FIG20 shows the expression of TNFRSF10B protein in human liver cancer tissue provided by the present invention and the related expression of TNFRSF10B found in the TCGA database and The Human Protein Atlas database.

[0123] FIG21 is the flow cytometry detection result of the present invention for detecting residual tumor cells in the killing of lung cancer calu-3 cells by TNFRSF10B / ASGR1 dual-target tandem CAR-T;

[0124] FIG22 is a flow cytometry analysis of the statistical results of residual tumor cells detected by TNFRSF10B / ASGR1 dual-target tandem CAR-T cells killing lung cancer calu-3 cells provided by the present invention;

[0125] FIG23 is a graph showing the fluorescence expression intensity (%) of tumor cells killed by different groups of cells on lung cancer Calu-3 cells by luciferase fluorescence detection provided by the present invention;

[0126] FIG24 is a statistical result of LDH release of different groups of cells after killing lung cancer Calu-3 cells detected by the LDH release test provided by the present invention;

[0127] FIG25 is a statistical result of the interferon release of different groups of cells killing lung cancer Calu-3 cells by ELISA provided by the present invention;

[0128] FIG26 is the flow cytometry detection result of residual tumor cells detected by TNFRSF10B / ASGR1 dual-target tandem CAR-T cells killing hepG2 liver cancer cells provided by the present invention;

[0129] FIG27 is a flow cytometry analysis of the statistical results of residual tumor cells detected by TNFRSF10B / ASGR1 dual-target tandem CAR-T cells killing hepG2 liver cancer cells provided by the present invention;

[0130] FIG28 is a statistical result of the ELISA detection of the amount of interferon (IFN-γ) released by different groups of cells to kill liver cancer HepG2 cells provided by the present invention;

[0131] FIG29 is a statistical result of LDH release of different groups of cells after killing HepG2 liver cancer cells detected by the LDH release test provided by the present invention;

[0132] Figure 30.1 shows the comparative results of the killing effects of TNFRSF10B-28z single-target CAR-T, TNFRSF10B / ASGR1 dual-target tandem CAR-T, and TNFRSF10B / ASGR1 dual-target parallel CAR-T on HepG2 liver cancer cells at different effector-target ratios provided by the present invention;

[0133] Figure 30.2 shows the statistical results of the killing effects of the TNFRSF10B-28z single-target CAR-T, TNFRSF10B / ASGR1 dual-target tandem CAR-T, and TNFRSF10B / ASGR1 dual-target parallel CAR-T on HepG2 liver cancer cells provided by the present invention;

[0134] FIG31 is a schematic diagram of the TNFRSF10B / ASGR1 dual-target tandem CAR-T provided by the present invention;

[0135] FIG32 is a schematic diagram of the structure of a chimeric antigen receptor with TNFRSF10B / ASGR1 dual targets in series provided by the present invention;

[0136] FIG33 is a TNFRSF10B / ASGR1 dual-target tandem CAR lentiviral vector provided by the present invention. DETAILED DESCRIPTION

[0137] The following is a detailed description with reference to the accompanying drawings.

[0138] TNFRSF10B is also referred to as DR5 in this application. TNFRSF10B / ASGR1 dual-targeting CAR-T cells are equivalent to DR5 / ASGR1 dual-targeting CAR-T cells.

[0139] The Fuzheng Jiedu Xiaoji recipe is also represented as FZJD in this application.

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

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

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

[0143] 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, or to an antibody molecule fragment that retains the ability to bind to an immunogen, and 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 are interconnected by disulfide bonds, or an antigen-binding portion thereof, which may include two heavy (H) chains and two light (L) chains. Each H chain includes a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each L chain includes a light chain variable region (abbreviated herein as VL) and a light chain constant region. scFv is a single-chain antibody fragment composed of VH and VL combined by a connecting peptide. 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 nanobodies, which can be modified from traditional VH.

[0144] The TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and the immune response cells expressing it, which are 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 an immune response, or their progenitor cells, or their progeny cells. The TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and the 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 the 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 the immune response cells expressing it are administered to the circulatory system and indirectly provided to the target organ. Before, during, or after administration of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor and immune response cells expressing it, an effective dose of FZJD can be administered to enhance the therapeutic effect of liver cancer. Immune response cells expressing the TNFRSF10B / ASGR1-specific chimeric antigen receptor and compositions containing them can be obtained from a single subject and administered to the same subject or a different compatible subject. The peripheral blood-derived T cells and their progeny of the disclosed subject matter can be administered by systemic injection, local injection, intravenous injection, or parenteral administration. For example, administering at least 1×10 5 cells, eventually reaching 1×10 12 or more.

[0145] When administering the pharmaceutical composition disclosed herein, it can be formulated 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 additives, carriers or other diluents are used, they must be compatible with the immune response cells expressing the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor disclosed herein. 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 to about 1×10 12 , about 1×10 6 to about 1×10 11 , about 1×10 7 to about 1×10 10 , about 1×10 8 to about 1×10 9 In some embodiments, a smaller or larger number of immune response cells of the present disclosure 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 Individual immune response cells of the presently disclosed subject matter are administered to a subject.

[0146] Those skilled in the art can determine the cells and optional additives or vehicles in the pharmaceutical composition and determine the specific mode of administration. For example, the additives (except the active cells and / or active substances) are present in an amount of about 0.001 wt% to about 50 wt% of a solution in phosphate buffered saline, and the active ingredient is present in an amount 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%.

[0147] 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 exacerbation, improving the therapeutic effect, 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 given 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, "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 obtained or achieved.

[0148] Example 1

[0149] This embodiment provides a pharmaceutical composition comprising TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells and FZJD, wherein the dual-target chimeric antigen receptor T cells express the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor on their membrane surface. Specifically, FZJD comprises: 15g of Codonopsis pilosula, 15g of raw Astragalus membranaceus, 15g of Atractylodes macrocephala, 15g of Poria cocos, 15g of Adenophora adenophora, 15g of Ophiopogon japonicus, 15g of Angelica sinensis, 15g of Rehmannia glutinosa, 15g of Paridis chinensis, 9g of Curcuma zedoaria, and 9g of Pinellia ternata.

[0150] Example 2

[0151] This embodiment provides a chimeric antigen receptor comprising a signal peptide, a first antigen-binding region targeting TNFRSF10B, a second antigen-binding region targeting ASGR1, a hinge region, a transmembrane region, and an intracellular region. According to a preferred embodiment, the structure of the first antigen-binding region targeting TNFRSF10B is a specific antigen-binding domain designed for 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 comprises an anti-TNFRSF10B antibody light chain variable region and an anti-TNFRSF10B antibody heavy chain variable region. Preferably, the anti-TNFRSF10B antibody light chain variable region and the anti-TNFRSF10B antibody heavy chain variable region are connected by a linker or a connecting peptide. Preferably, the 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 thereof can be VL-linker-VH or VH-linker-VL. The chimeric antigen receptor is a parallel chimeric antigen receptor.

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

[0153] 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.

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

[0155] 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 specifically binds 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 specifically binds to ASGR1 on the surface of liver cancer cells.

[0156] 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.

[0157] 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.

[0158] According to a preferred embodiment, the first CAR can be expressed as TNFRSF10B ScFv-Hinge-TM-CD3ζ.

[0159] 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 through a cleavable domain or another promoter.

[0160] 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).

[0161] 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 CD8αTM.

[0162] 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.

[0163] According to a preferred embodiment, CD3ζ is an intracellular signaling domain.

[0164] Specifically, the first CAR can be expressed as CD8αsignal peptide-TNFRSF10B VH-linker-TNFRSF10B VL-CD8αH-CD8αTM-CD3ζ.

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

[0166] According to a preferred embodiment, the second CAR can be expressed as CD8αsignal peptide-ASGR1 VH-CD8αH-CD8αTM-CD137(4-1BB)-CD28.

[0167] 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).

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

[0169] According to a preferred embodiment, the costimulatory signal binding region includes a 4-1BB (CD137) costimulatory signal domain and a CD28 costimulatory signal domain.

[0170] According to a preferred embodiment, the intracellular co-stimulatory signaling domain can comprise a CD3ζ intracellular signaling domain that can activate immune response cells.

[0171] According to a preferred embodiment, the immune response cells include T lymphocytes, NK cells, and NKT cells.

[0172] 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, or 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.

[0173] According to a preferred embodiment, the amino acid sequence of the CD8α hinge region is shown in SEQ ID NO:5.

[0174] According to a preferred embodiment, the amino acid sequence of the CD8αTM transmembrane binding region is shown in SEQ ID NO: 6.

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

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

[0177] According to a preferred embodiment, CD3ζ is an intracellular signaling domain, and the amino acid sequence is shown in SEQ ID NO:9.

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

[0179] 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.

[0180] Example 3

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

[0182] Example 4

[0183] This embodiment provides a vector comprising 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 an adenovirus, a lentivirus, an adeno-associated virus, or a retrovirus.

[0184] Example 5

[0185] This embodiment provides a host cell containing the aforementioned vector or the aforementioned exogenous nucleic acid molecule integrated into its chromosome. According to a preferred embodiment, the host cell expresses a TNFRSF10B / ASGR1 dual-target chimeric antigen receptor.

[0186] Example 6

[0187] 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.

[0188] Example 7

[0189] This embodiment 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:

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

[0191] Example 8

[0192] This example provides the specific steps for constructing a CAR plasmid. The construction of the CAR plasmid includes: vector digestion, extraction of the target gene fragment, exchange of the PCR product with the vector, transformation, colony PCR identification, sequencing, plasmid transformation, single clone selection, plasmid midi extraction, lentiviral packaging, lentiviral concentration, and determination of lentiviral titer by fluorescence.

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

[0194] 1. Vector digestion

[0195] Double-digest vector GV400 with BamH I and EcoR I, and vector GV401 with BamH I. Prepare 50 μl of the enzyme digestion system as shown in Tables 1 and 2. Add the reagents in the order listed in Table 1, gently pipette to mix, and incubate at 37°C for 3 hours or overnight.

[0196] Table 1

[0197] Table 2

[0198] After the reaction was completed, the vector digestion products were subjected to agarose gel electrophoresis. The results of the digestion at the BamH I / EcoR I site are shown in Figure 2. In Figure 2, a 10 kb marker was used for electrophoresis. 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, and 250 bp. Lane 2 is the vector digestion product. Lane 3 is the undigested vector. The results of the digestion at the BamH I / EcoR I site are shown in Figure 3. In Figure 3, a 10 kb marker was used for electrophoresis. 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, and 250 bp. Lane 2 is the vector digestion product. Lane 3 is the undigested vector.

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

[0200] Recover the target band. The recovery process includes:

[0201] (1) Under long-wave ultraviolet light, use a clean blade to cut the DNA band to be recovered, and try to remove the gel without DNA, so that the gel volume is as small as possible;

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

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

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

[0205] 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;

[0206] (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 accelerate dissolution;

[0207] (5) Optional, usually not necessary, add 150 μl of isopropanol per 100 mg of initial gel weight and shake to mix;

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

[0209] Equilibration liquid pretreatment adsorption column:

[0210] Using equilibration liquid to pretreat the silica membrane adsorption column is a necessary step. The specific method is: take a new silica membrane adsorption column and place it in a collection tube, draw 100μl of equilibration liquid into the column, centrifuge at 13000rpm 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 equilibration liquid pretreatment of the column is completed.

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

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

[0213] 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.

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

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

[0216] (9) Place the EC adsorption column back into the empty collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much of the rinse solution as possible to prevent residual ethanol in the rinse solution from inhibiting downstream reactions;

[0217] (10) Remove the adsorption column EC and place it in a clean centrifuge tube. Add 50 μl of elution buffer EB to the middle of the adsorption membrane (preheat the elution buffer in a 65-70°C water bath for better results). Incubate at room temperature for 2 minutes and centrifuge at 12,000 rpm for 1 minute. If more DNA is needed, add the resulting solution back to the adsorption column and centrifuge for 1 minute.

[0218] 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 25μl. Too small a volume will reduce DNA elution efficiency and yield.

[0219] 2. Acquisition of target gene fragments

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

[0221] Primer description: Contains exchange pairing bases, restriction enzyme cutting sites, and partial sequence of the 5' end of the target gene for PCR fishing of the target gene.

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

[0223] The acquisition steps include PCR amplification of the target gene fragment, preparing the reaction system as shown in Table 3, adding the reagents in the order listed in Table 3, gently pipetting to mix, briefly centrifuging, and then performing the reaction in a PCR instrument. The reaction conditions are shown in Table 4. The results for the target fragment corresponding to the BamH I / EcoR I cloning site are shown in Figure 4. The resulting PCR product is 2129 bp in size. The markers in Figure 4 are, from top to bottom, 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, and 100 bp.

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

[0225] Table 3

[0226] Table 4

[0227] 3. Exchange of PCR products with vectors

[0228] The reaction system shown in Table 5 was prepared in an ice-water bath, mixed thoroughly (to avoid bubbles), and reacted at 37°C for 30 minutes after rapid centrifugation. After cooling in an ice-water bath for 5 minutes, the reaction was immediately transformed to obtain the exchange reaction product. PCR identification primers are shown in the table below. The identification results of the target fragment corresponding to BamH I / EcoR I exchanged into the linear expression vector are shown in Figure 6. Among them, the PCR product size of the positive transformant is 997 bp. Lane 1 is a negative control (ddH2O), lane 2 is a negative control (empty vector self-ligation control group), lane 3 is a positive control (GAPDH), and lane 4 is a marker. From top to bottom, the following are 5 kb, 3 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp, and 100 bp. Lanes 5 to 12 represent transformants 1 to 8, respectively.

[0229] PCR identification primers are shown in the table below. The identification results of the target fragment corresponding to BamH I / BamH I exchanged into the linear expression vector are shown in Figure 7, wherein the size of the PCR product of the positive transformant is 1120 bp, lane 1 is a negative control (ddH2O), lane 2 is a negative control (empty vector self-ligation control group), lane 3 is a positive control (GAPDH), lane 4 is a marker, and 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. Lanes 5 to 12 represent transformants 1 to 8, respectively.

[0230] Table 5

[0231] 4. Conversion

[0232] 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 minutes. Heat shock the mixture in a 42°C water bath for 90 seconds, then immediately incubate in an ice bath for 2 minutes. Add 500 μl of LB liquid medium to the mixture and incubate on a shaker at 37°C for 1 hour. Spread an appropriate amount of the bacterial solution evenly on a plate containing ampicillin (Amp) and incubate in an incubator upside down for 12-16 hours.

[0233] 5. Colony PCR Identification

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

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

[0236] Table 6

[0237] Table 7

[0238] 6. Sequencing

[0239] The identified positive clone transformants were inoculated into an appropriate amount of LB liquid medium containing Amp and cultured at 37°C for 12-16 hours. An appropriate amount of the bacterial culture was then collected for sequencing, and the sequencing results were compared with the target gene sequence. The alignment results of the positive clone transformants of GV400 are shown in SEQ ID NO: 11. SEQ ID NO: 11 is the complete nucleic acid sequence of the first antigen-binding region targeting TNFRSF10B, including the nucleic acid sequence of the light chain variable region of the anti-TNFRSF10B antibody and the nucleic acid sequence of the heavy chain variable region of the anti-TNFRSF10B antibody. The alignment results of the positive clone transformants of GV401 are shown in SEQ ID NO: 12. SEQ ID NO: 12 is the nucleic acid sequence of the second antigen-binding region targeting ASGR1, including the nucleic acid sequence of the heavy chain variable region of the anti-ASGR1 antibody.

[0240] 7. Plasmid Transformation

[0241] The specific steps include: opening the water bath in advance and heating it to 42°C; adding 10μl of plasmid to a centrifuge tube containing 100μl of competent cells, gently tapping the tube wall several times to mix, and placing it on ice for 30 minutes; heat shock at 42°C for 90 seconds, incubate in an ice water bath for 2 minutes, then add 500μl of LB liquid culture medium, and place it on a shaker at 37°C for 1 hour; after incubation, take an appropriate amount of bacterial liquid and evenly spread it on a plate containing Amp, and incubate it in a constant temperature incubator for 12 to 16 hours.

[0242] 8. Pick single clones

[0243] The specific steps include: preparing LB liquid culture medium containing Amp, 200ml of LB liquid culture medium + 200μl of Amp, mixing, and boiling in a microwave oven; taking culture bottles that have been sterilized by high pressure, adding 50ml of LB liquid culture medium to each culture bottle for later use; taking out the plates in the 37°C constant temperature incubator, looking for single colonies, picking up the colonies and placing them in the culture bottles; placing the culture bottles in a constant temperature shaker, setting the speed to 200rpm, the temperature to 37°C, and shaking the bacteria for 16-20 hours.

[0244] 9. Plasmid Extraction

[0245] The specific steps include: transferring the culture medium in the culture flask to a 50ml centrifuge tube, centrifuging at 3000g for 10 minutes, and discarding the liquid; adding 4ml of Buffer P1 reagent to each centrifuge tube and mixing thoroughly; adding 4ml of Buffer P2 reagent to each centrifuge tube, mixing by inverting until the liquid turns blue and viscous, and letting it stand for 3 minutes; adding 4ml 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 50ml centrifuge tube; adding 2ml 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 18000g for 1 minute; adding 0.7ml of Buffer ETR to the centrifuge column, centrifuging at 18000g for 1 minute, and discarding the liquid; adding 0.7ml of Buffer PE, 18000g, centrifuge for 1 min, discard the liquid, repeat once; 18000g, empty the centrifuge column for 1 min, discard the liquid, add 200μl of Elution Buffer, let it stand for 1 min, 18000g, centrifuge for 1 min, transfer the collected liquid to a new EP tube; take 2μl of plasmid to test the plasmid concentration.

[0246] 10. Lentiviral Packaging

[0247] The specific steps include: pre-recovery of 293T cells, digestion of 293T cells in logarithmic growth phase with trypsin 24 h before transfection, and adjusting the cell density to approximately 5 × 10 cells using a medium containing 10% serum. 6cells / 15ml, re-inoculated into a six-well plate, and cultured in a 37℃, 5% CO2 incubator for 24 hours. When the cell density reaches 70%-80%, it can be used for transfection; calculate the plasmid dosage according to the target plasmid: psPAX2:pMD2G ratio of 3:2:1 and a total mass of 4μg; add the required volume of plasmid to 400μl of serum-free DMEM medium, add 6μl of Turbo and mix well, and let it stand at room temperature for 15-20 minutes; slowly add the mixture to the culture dish, shake well, and culture in a 37℃ incubator; change the medium after 24 hours of culture, collect the virus once at 48 hours and once at 72 hours, and mix the two collected viruses for concentration.

[0248] 11. Lentivirus Concentration

[0249] The specific steps include: harvesting the virus, transferring the cell supernatant to a 50ml centrifuge tube, centrifuging at 3500g for 10 minutes; adding the virus concentrate to a new 50ml centrifuge tube at a volume ratio of cell supernatant: concentrate reagent = 3:1, and pre-cooling in a 4℃ refrigerator; transferring the centrifuged cell supernatant to the pre-cooled virus concentrate reagent, mixing thoroughly, and shaking at low speed in a 4℃ refrigerator overnight; taking the centrifuge tube after overnight, centrifuging at 3500g at 4℃ for 1 hour; discarding the supernatant, and centrifuging at 3500g at 4℃ for 1 minute; preparing the virus concentrate. Virus resuspension protection solution, wherein the volume ratio of virus resuspension solution to virus protection reagent is 9:1. Take 9 ml of virus resuspension solution and 1 ml of virus protection reagent and put them into a 15 ml centrifuge tube for later use; remove the virus after centrifugation, discard the supernatant, add virus resuspension protection solution with a volume of 1% to 10% of the virus stock solution, slowly pipette 20 to 30 times, and let it stand for 10 minutes; centrifuge at 12000g, 4℃, for 3 to 5 minutes, and aspirate the supernatant to obtain the concentrated virus; transfer the concentrated virus to a 1.5 ml centrifuge tube and freeze at -80℃.

[0250] 12. Determination of Lentivirus Titer by Fluorescence Method

[0251] As shown in Figure 8, Figure 8A is a schematic diagram of virus gradient dilution; Figure 8B is the observation of plasmid transfection under a fluorescence microscope; and Figure 8C is the counting under a fluorescence microscope. The specific measurement steps include: one day before the measurement, 293T adherent cells were plated in a 96-well plate, with 4×10 cells per well. 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, 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. Similarly, 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 incubate at 37°C, 5% The cells were cultured in a CO2 incubator. After 24 hours, 100 ml of complete culture medium was added to the cell wells. During this process, the cells were not blown away. After 4 days, the fluorescence expression was observed. The number of fluorescent cells decreased with the increase of the dilution factor. The virus titer was calculated based on the observation results of the fluorescence microscope. The virus titer = number of fluorescent cells / volume of virus stock solution. For example, if one fluorescent cell was observed in a well infected with 1E-6 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.

[0252] Example 9

[0253] This example 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 minutes, 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 500μl of cell suspension, 500μl of concentrated virus and 40μl of transduction reagent to each well, and mix well; after mixing, culture in a 37℃ incubator, add fresh culture medium after 8-12 hours and observe the cell status; observe the fluorescence expression 3-4 days after infection. 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 cell growth activity.

[0254] Example 10

[0255] This example describes a method and results for detecting CAR-T transfection efficiency by flow cytometry. The method for detecting CAR-T transfection efficiency includes: taking a flow cytometer, labeling the tube, and removing 1 ml of ASGR1CAR-T cells, DR5CAR-T cells, and DR5 / ASGR1CAR-T cells from each well of a six-well plate, setting the speed to 1200 rpm, centrifuging for 5 minutes, and discarding the liquid; adding 2 ml of PBS to resuspend the cells, vortexing evenly, setting the speed to 1200 rpm, centrifuging for 5 minutes, and discarding the liquid; adding 100 μl of PBS to resuspend the cells, flicking them evenly, and loading them onto the flow cytometer; adjusting the voltage of the flow cytometer, selecting the FITC channel to observe ASGR1, and the PE channel to observe DR5, to observe the transfection efficiency of the two CAR-T cells.

[0256] The results are shown in Figure 9. The CAR positivity rate of DR5CAR-T was 50.22%; the CAR positivity rate of ASGR1CAR-T was 54.33%; the single DR5z CAR positivity rate of the dual-target DR5+ASGR1CAR-T was 26.75%; the single ASGR1CAR positivity rate was 53.80%; and the DR5+ASGR1 dual-target CAR positivity rate was 20.59%.

[0257] Example 11

[0258] 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 fluid). Therefore, the amount of LDH released from the damaged cell membrane detected in the LDH release test can be used to determine the number of dead cells. The test results of the LDH release test directly reflect the cell mortality rate, that is, the higher the absorbance measured, the stronger the cytotoxicity of the test substance.

[0259] 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, 8×10 5 / ml, 4×10 5 / ml, 50μl / well was mixed with target cells and incubated at 37℃, 5% CO2. The killing time was 16-18h.

[0260] 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, wherein the effector cells are untransfected T cells (NT in Figure 10 refers to NT cells, which is equivalent to the untransfected T cells in this embodiment), DR5 single-target CAR-T cells, and DR5 / ASGR1 dual-target CAR-T cells. The detection method is carried out in accordance with the instructions.

[0261] After the killing test, add 10μl of Lysis Buffer to the high control wells and incubate in a CO2 incubator at 37°C for 30 minutes. After adding 100μl of Working Solution to each well, wrap the entire culture plate with aluminum foil and incubate in a dark place at room temperature. After adding 50μL of Stop Solution to each well, immediately place it in a microplate reader and measure the absorbance at 490nm.

[0262] This example provides a cytotoxicity calculation formula, specifically: Cytotoxicity (%) = (experimental group - spontaneous control group - effector cell spontaneous group) / (maximum release group - effector cell spontaneous group - volume control group) × 100%.

[0263] Figure 10 shows a comparison of the cytotoxic effects of CAR-T cells at different effector-target ratios on liver cancer cells. Specifically, an LDH release assay compared the damage to Huh7 tumor cells after co-culture of the two CAR-T cells with the tumor cells at effector-target ratios of 2:1, 4:1, 8:1, and 16:1. Figures 10A-E show that the cytotoxic effects of DR5-targeted CAR-T cells on Huh7 tumor cells were greater than those of untransfected T cells at all effector-target ratios. Furthermore, the cytotoxic effects of DR5 / ASGR1-targeted CAR-T cells on Huh7 tumor cells were greater than those of DR5-targeted CAR-T cells at all effector-target ratios.

[0264] Example 12

[0265] This example shows the comparative results of the killing function of CAR-T cells on liver cancer cells (liver cancer cell apoptosis detection).

[0266] Figure 11 shows the apoptosis of liver cancer cells detected by flow cytometry when the effector-target ratio was 2:1.

[0267] Flow cytometry was used to assess tumor cell apoptosis after co-culture of DR5 single-target and DR5 / ASGR1 dual-target CAR-T cells with Huh7 and HepG2 tumor cells. As shown in Figures 11A and 11B, DR5 single-target CAR-T cells promoted apoptosis in Huh7 cells more effectively than untransfected T cells (P < 0.05), and DR5 single-target CAR-T cells promoted apoptosis in HepG2 cells more effectively than untransfected T cells (P < 0.0001). DR5 / ASGR1 dual-target CAR-T cells promoted apoptosis in Huh7 cells more effectively than DR5 single-target CAR-T cells, with statistically significant results (P < 0.001). DR5 / ASGR1 dual-target CAR-T cells promoted apoptosis in HepG2 cells more effectively than DR5 single-target CAR-T cells, with statistically significant results (P < 0.01).

[0268] Example 13

[0269] This example provides a method for a cytotoxicity test (Granzym B release test). The specific steps include: removing 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, incubating in a 37°C incubator for 3 minutes, and neutralizing by adding 3 ml of MEM / DMEM complete medium; transferring the cells to a 15 ml centrifuge tube, setting the speed to 1200 rpm, centrifuging for 5 minutes, 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 a 24-well plate, add 500μl of cell suspension to each well and culture; take untransfected T cells and CAR-T cells to a 15ml centrifuge tube, set the speed to 1200rpm, and centrifuge for 5min; add 3ml of 1640 culture medium to resuspend the cells, take 10μl of cell suspension for counting, and adjust the cell concentration to 1×10 6 / ml; add 500μl of untransfected T cell suspension or CAR-T cell suspension to each well of a 24-well plate and culture in an incubator for 18h; take 1ml of CAR-T cells from each well of the 24-well plate into a flow tube, set the speed to 1200rpm, centrifuge for 5min, discard the liquid, add 1μl of CD3 antibody, mix well, and incubate in the dark for 30min; wash once with PBS, add the corresponding reagents in the live / dead kit (calcein AM (live cell indicator), SYTOX TMDeep Red nucleic acid stain (dead cell indicator), mix well, incubate in the dark for 30 minutes, wash once with PBS; add Fix / perm buffer, mix well, incubate in the dark for 50 minutes; add Perm wash buffer and wash once, set the speed to 1200 rpm, and centrifuge for 5 minutes; add an appropriate amount of granzyme B antibody (Granzym B) according to the cell number, incubate in the dark for 40 minutes; wash once, discard the supernatant, and load onto the machine.

[0270] Example 14

[0271] This embodiment provides the operating method and test results of the cytotoxicity test (CD107a expression experiment). 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 NK cell or T cell activity. Specifically, the particles reach the serosal surface of NK / T cells, the granule membrane fuses with the cell membrane, and CD107a is exposed on the cell membrane surface. The membrane expression of CD107a molecules can directly reflect the degranulation process of NK cells or cytotoxic T cells (CTLs), and is directly related to the cytotoxic activity of NK cells or CTLs. It can be detected by flow cytometry after specific antibody labeling.

[0272] The specific operation method includes: opening the biosafety cabinet 30 minutes in advance, irradiating with ultraviolet light for 30 minutes; adjusting the cell density of HepG2 / Huh7 cells to 1×10 6 / ml, 1 ml was taken from each well and inoculated into 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, DR5CAR-T cell group, and DR5 / ASGR1CAR-T cell group, and 1ml 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 the cells were co-cultured in a 37°C incubator for 18 hours; the cells were removed from the incubator, transferred to a flow tube, the speed was set to 1200 rpm, centrifuged for 5 minutes, and the liquid was discarded; 1ml of PBS was added to resuspend the cells, the speed was set to 1200 rpm, centrifuged for 5 minutes, and the liquid was discarded; CD107a antibody was added, and the cells were incubated in the dark for 30 minutes; 1ml of PBS was added to resuspend the cells, the speed was set to 1200 rpm, centrifuged for 5 minutes, and the liquid was discarded; 100μl of PBS was added dropwise, and the cells were tested on the machine.

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

[0274] The results in Figure 12 show 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) (Figure 12A). When DR5 single-target CAR-T cells were co-cultured with Huh7 cells, the amount of Granzym B released was higher than that of untransfected T cells (P < 0.001); when DR5 / ASGR1 dual-target CAR-T cells were co-cultured with Huh7 cells, the amount of Granzym B released was higher than that of DR5 single-target CAR-T cells (P < 0.001). When DR5 single-target CAR-T cells were co-cultured with HepG2 cells, the amount of Granzym B released was higher than that of untransfected T cells (P < 0.01); when DR5 / ASGR1 dual-target CAR-T cells were co-cultured with HepG2 cells, the amount of Granzym B released was higher than that of DR5 single-target CAR-T cells (P < 0.01) (Figure 12B).

[0275] Example 15

[0276] This example provides experimental results showing that FZJD-containing serum enhances the inhibitory effect of DR5 / ASGR1 dual-target CAR-T cells on liver cancer cells.

[0277] In this example, the inhibitory effect of FZJD-containing serum combined with DR5 / ASGR1 dual-target CAR-T cells on tumor cell proliferation was detected by CCK8 method. The results are shown in Figure 13. When the serum concentration was 30%, FZJD-containing serum was able to effectively increase the effect of DR5 / ASGR1 dual-target CAR-T cells in inhibiting the growth of Huh7 cells, and the results were statistically significant (P < 0.05) (Figure 13A). When the serum concentration was 20% and 30%, FZJD-containing serum was able to effectively increase the effect of CAR-T cells in inhibiting the growth of HepG2 cells (Figure 13B), and the results were statistically significant.

[0278] Example 16

[0279] This example provides experimental results of FZJD-containing serum enhancing the apoptotic effect of DR5 / ASGR1 dual-target CAR-T cells on liver cancer cells, as shown in FIG14 .

[0280] In this example, flow cytometry was used to detect whether FZJD-containing serum increased the apoptosis effect of DR5 / ASGR1 dual-target CAR-T cells on tumor cells Huh7 and HepG2. The results are shown in Figures 14A and B. The effect of DR5 / ASGR1 dual-target CAR-T cells in promoting Huh7 cell apoptosis was better than that of untransfected T cells (P < 0.0001). The effect of DR5 / ASGR1 dual-target CAR-T cells in promoting HepG2 cell apoptosis was better than that of untransfected T cells (P < 0.001). FZJD-containing serum can effectively increase the apoptosis effect of DR5 / ASGR1 dual-target CAR-T cells in promoting Huh7 cells, and the results are statistically significant (P < 0.01). FZJD-containing serum can effectively increase the apoptosis effect of DR5 / ASGR1 dual-target CAR-T cells in promoting HepG2 cells, and the results are statistically significant (P < 0.05).

[0281] Example 17

[0282] 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.

[0283] The specific operation method for detecting the release of Granzym B after the combination of FZJD drug-containing serum and DR5 / ASGR1 dual-target CAR-T cells includes: digesting Huh7 / HepG2 cells grown to 90%, adjusting the tumor cell density to 1×10 6 / ml; take a 24-well plate, add 500μl of cell suspension to each well, and culture in a 37℃ incubator; take untransfected T cells and dual-target CAR-T cells into 15ml 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. 500μl of untransfected T cell suspension was added to the untransfected T cell group, 500μl of CAR-T cell suspension was added to each well of DR5 / ASGR1 dual-target CAR-T cell group and FZJD combined with DR5 / ASGR1 dual-target CAR-T cell group, and 500μl of CAR-T cell suspension was added to each well of the untransfected T cell group and DR5 / ASGR1 dual-target CAR-T cell group. Add 500 μl of culture medium containing normal rat serum, and add Fuzheng Jiedu Xiaoji Recipe combined with DR5 / ASGR1 dual-target CAR-T cell group with drug-containing serum of Fuzheng Jiedu Xiaoji Recipe to make the serum concentration of each group 30%, and culture in a 37°C incubator for 18 h; take 1 ml of CAR-T cell suspension from each well of the 24-well plate into the 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 live / dead and 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 and centrifuge for 5 min; add an appropriate amount of granzyme B antibody according to the cell number, incubate in the dark for 40 min; wash once, discard the supernatant, and load the machine.

[0284] Example 18

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

[0286] The specific operation method for detecting the expression of CD107a after the combination 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, incubating at 37°C for 3 minutes, 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 minutes, 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 500μl of cell suspension to each well, and culture in a 37℃ incubator; take untransfected T cells and DR5 / ASGR1 dual-target CAR-T cells into 15ml centrifuge tubes respectively, set the speed to 1200rpm, and centrifuge for 5min; add 3ml of 1640 culture medium to resuspend the cells, take 10μl 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, 500μl of untransfected T cell suspension was added to the untransfected T cell group, 500μl of CAR-T cell suspension was added to each well of DR5 / ASGR1 dual-target CAR-T cell group and Fuzheng Jiedu Xiaoji recipe combined with DR5 / ASGR1 dual-target CAR-T cell group, and 500μl 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 500μl of culture medium containing normal rat serum was added to the untransfected T cell group and DR5 / ASGR1 dual-target CAR-T cell group. The / ASGR1 dual-target CAR-T cell group was supplemented with Fuzheng Jiedu Xiaoji recipe-containing serum to make the serum concentration of each group 30%, and Golgi inhibitors monensin and brefeldin a were added to the culture medium and cultured in a 37°C incubator for 18 hours; the cells were removed from the incubator and transferred to a flow cytometer, the speed was set to 1200 rpm, the cells were centrifuged for 5 minutes, and the liquid was discarded; 1 ml of PBS was added to resuspend the cells, the speed was set to 1200 rpm, the cells were centrifuged for 5 minutes, and the liquid was discarded; CD107a antibody was added and incubated in the dark for 30 minutes; 1 ml of PBS was added to resuspend the cells, the speed was set to 1200 rpm, the cells were centrifuged for 5 minutes, and the liquid was discarded; 100 μl of PBS was added dropwise, and the cells were tested on the flow cytometer.

[0287] Figure 15A shows the expression of CD107a on T cells as assessed by flow cytometry; Figure 15B shows the expression of Granzym B on T cells as assessed by flow cytometry. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3. Co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with Huh7 cells resulted in a higher release of CD107a than that of untransfected T cells (P<0.001). FZJD-containing serum significantly increased the release of CD107a after co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with Huh7 cells (P<0.01). After co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with HepG2 cells, the amount of CD107a released was higher than that of untransfected T cells (P < 0.0001). FZJD-containing serum significantly increased the amount of CD107a released after co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with HepG2 cells (P < 0.05) (Figure 15A). After co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with Huh7 cells, the amount of Granzym B released was higher than that of untransfected T cells (P < 0.0001). FZJD-containing serum significantly increased the amount of Granzym B released after co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with Huh7 cells (P < 0.001). After co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with HepG2 cells, the amount of Granzym B released was higher than that of untransfected T cells (P < 0.001). FZJD-containing serum could significantly increase the amount of Granzym B released after co-culture of DR5 / ASGR1 dual-targeting CAR-T cells with HepG2 cells, and the amount of Granzym B released was higher than that of DR5 / ASGR1 dual-targeting CAR-T cells (P < 0.05) (Figure 15B).

[0288] Example 19

[0289] This example provides a method for detecting phenotypic changes of FZJD combined with DR5 / ASGR1 dual-target CAR-T cells by flow cytometry and the experimental results, the results of which are shown in FIG16 .

[0290] 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 synergistic expression of IL-6 and PD-L1 can significantly inhibit CD8 + T cell infiltration capacity in tumors.

[0291] The specific operation method for flow cytometry detection of phenotypic changes of FZJD combined with DR5 / ASGR1 dual-target CAR-T cells 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 15ml centrifuge tube, setting the speed to 1200rpm for 5min, resuspending the cells in 1640 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 hours; remove the cells, set the speed to 1200 rpm, centrifuge for 5 minutes, and discard the liquid; add 1 ml of PBS to resuspend the cells, set the speed to 1200 rpm, centrifuge for 5 minutes, and discard the liquid; add mixed antibodies and incubate in the dark for 30 minutes; add 1 ml of PBS to resuspend the cells, set the speed to 1200 rpm, centrifuge for 5 minutes, and discard the liquid; add 100 μl of PBS and detect on the machine.

[0292] The results are shown in FIG16 . FIG16A and B show the results of flow cytometry detection of T cell phenotypes; FIG16C shows the results of flow cytometry detection of CD4 + T cell ratio; Figure 16D is the flow cytometry analysis of tumor cell phenotype, *P<0.05, **P<0.01, vs Control, n=3. FZJD-containing serum can increase CD4 + In CAR-T cells, the initial T cells ( FZJD-containing serum had no statistically significant effect on the increase of initial T cells (P>0.05), but had a statistically significant effect on the increase of central memory T cells (P<0.01). + In CAR-T cells, the initial T cells ( The ratio of T cells) and central memory T cells (TCM) was significantly increased by FZJD drug-containing serum, and the increase of initial T cells and central memory T cells by FZJD drug-containing serum was statistically significant (P < 0.05) (Figure 16A). In this example, the effect of FZJD drug-containing serum on the expression of immunostimulatory receptors and immunoinhibitory receptors on the surface of CAR-T cells was detected by flow cytometry. The results showed that FZJD drug-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 drug-containing serum has a trend to reduce the immunoinhibitory receptors PD-1, TIM3 and CTLA-4 on the surface of CAR-T cells (Figure 16B). Flow cytometry was used to detect whether FZJD drug-containing serum can change the expression of CAR-T cell CD8 + There was no significant difference in the proportion of T cells (Figure 16C). Flow cytometry was used to detect whether FZJD-containing serum reduced the expression of PD-L1 on the surface of tumor cells. The results showed that FZJD-containing serum had a trend of reducing the expression of PD-L1 on the surface of tumor cells (Figure 16D).

[0293] Example 20

[0294] This example provides experimental results showing that FZJD-containing serum reduces the levels of inflammatory cytokine release during the killing of target cells by liver cancer cells and DR5 / ASGR1 dual-target CAR-T cells.

[0295] Cytokine storm (CRS) is a common adverse event related to CAR-T immunotherapy, and its incidence is high. Interleukin 6 (IL-6) is a 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 a 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 (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, 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.

[0296] 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 with target cells HepG2, and the change in IL-6 release by FZJD-containing serum was observed. As shown in Figure 17, DR5 / ASGR1 dual-target CAR-T cells can significantly increase the expression of the proinflammatory factor IL-6 after co-culture with target cells (P < 0.01), and FZJD-containing serum can effectively reduce the release of the 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).

[0297] Example 21

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

[0299] In this example, ELISA was used to detect the expression of interferon γ (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 promoting effect of FZJD medicated serum on the killing of target cells by DR5 / ASGR1 dual-target CAR-T cells. The results are shown in Figure 18.

[0300] The results are shown in Figure 18A (target cells are HepG2) and B (target cells are HepG3B). FZJD drug-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).

[0301] Example 22

[0302] This example provides the experimental results of FZJD-containing serum increasing the level of T cell chemokine release in liver cancer cells, as shown in FIG19 .

[0303] The CXCR3 / CXCL10 chemotactic axis is an important signaling pathway for T cell migration, and CXCL9 / 10 / 11 can recruit CD8 +A variety of immune cells of T cells can directly kill tumor cells or play an anti-tumor effect, thereby improving the overall survival rate of patients. The expression defect of CXCL9 / 10 / 11 in the tumor site may be one of the mechanisms of action that causes tumor immune escape. In this example, FZJD drug-containing serum was detected by ELISA to increase the level of T cell chemokine release in liver cancer cells. The results are shown in Figures 19A to C, *P<0.05, **P<0.01, ***P<0.001, n=3. The results showed that the content of CXCL9 / 10 / 11 was significantly increased in the FZJD group and the STAT3 inhibitor (stattic) group (p<0.05).

[0304] Example 23

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

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

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

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

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

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

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

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

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

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

[0315] 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.

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

[0317] 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.

[0318] 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.

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

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

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

[0322] Example 24

[0323] This embodiment provides a TNFRSF10B / ASGR1 dual-target tandem CAR-T. Figure 31 is a schematic diagram of the TNFRSF10B / ASGR1 dual-target tandem CAR-T. The structure of the TNFRSF10B / ASGR1 dual-target tandem chimeric antigen receptor is shown in Figure 32. Specifically, the TNFRSF10B / ASGR1 dual-target tandem CAR comprises: CD8a signal peptide, ASGR1 scFv, G4S Linker, DR5 scFv, CD8a hinge region, CD8α transmembrane region, 4-1BB, CD28 co-stimulatory molecule, and CD3ζ signal transduction molecule. At the same time, the TNFRSF10B / ASGR1 dual-target tandem CAR is connected to the mCherry gene sequence via T2A. In this embodiment, after synthesizing the ASGR1 scFv molecular sequence gene, it is connected to the DR5 scFv CD28 Costi CD3ζCAR lentiviral vector, as shown in Figure 33. According to this embodiment, the nucleotide sequence of the TNFRSF10B / ASGR1 dual-target tandem CAR is shown in SEQ ID NO: 13. The amino acid sequence of the TNFRSF10B / ASGR1 dual-target tandem CAR is shown in SEQ ID NO: 14.

[0324] This example provides flow cytometry detection results of residual tumor cells killed by TNFRSF10B / ASGR1 dual-target tandem CAR-T against lung cancer Calu-3 cells, as shown in Figures 21 to 25. In Figure 21, the dual-target tandem CAR-T kills lung cancer Calu-3 cells at effector-target ratios of 1:1, 2:1, and 5:1, respectively, with a killing time of 24 hours. The control group is T cells transfected with a plasmid without scFv (represented by Mock T in Figure 21). In Figure 21, the abscissa (FITC-H) represents the intensity of the fluorescence signal, which reflects the intensity of binding to FITC-labeled antibodies (such as tumor-specific antigen antibodies) on the cell surface or inside, and is used to distinguish tumor cells (positive signal) from non-tumor cells (negative or low signal). The ordinate (SSC-H) represents the intensity of side scattered light, which is used to distinguish different cell types. In P5 / E7, P5 refers to the tumor cell gate defined by SSC-H and FITC-H, and E7 represents the number of events (cell number) within the gate, indicating that specific residual tumor cells have been detected. When the effector-target ratio was 1:1, the residual calu-3 cells in the Mock T control group were 40.01%, while the residual calu-3 cells in the Tan DR5z-ASGR1 CART group were 25.29%; when the effector-target ratio was 2:1, the residual calu-3 cells in the Mock T control group were 32.79%, while the residual calu-3 cells in the Tan DR5z-ASGR1 CART group were 11.57%; when the effector-target ratio was 5:1, the residual calu-3 cells in the Mock T control group were 16.08%, while the residual calu-3 cells in the Tan DR5z-ASGR1 CART group were 4.79%.

[0325] Figure 22 is the statistical result of the residual tumor cells killed by TNFRSF10B / ASGR1 dual-target tandem CAR-T on lung cancer calu-3 cells by flow cytometry detection in Figure 21, wherein the horizontal axis is the effect-target ratio of the control group (represented by Mock T+calu-3 in Figure 22) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (represented by CART+calu-3 in Figure 22), the vertical axis is the residual tumor cell ratio (%), and the killing time is 24 hours. When the effect-target ratio (E:T) is 1:1, 2:1 and 5:1, the proportion of residual tumor cells in the control group is significantly greater than that in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0326] Figure 23 shows the fluorescence expression intensity of tumor cells killed by different groups of cells on lung cancer calu-3 cells by luciferase fluorescence detection, wherein the horizontal axis is the effect-target ratio of the control group (represented by Mock T+calu-3 in Figure 23) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (represented by CART+calu-3 in Figure 23), the vertical axis is luciferase activity (%), and the killing time is 24 hours. When the effect-target ratio (E:T) is 1:1, 2:1 and 5:1, the tumor cell mortality rate in the control group is significantly lower than that in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0327] Figure 24 is the statistical results of the LDH release test of different groups of cells killing lung cancer calu-3 cells after the tumor cells were killed, wherein the horizontal axis is the effect-target ratio of the control group (represented by Mock T+calu-3 in Figure 24) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (represented by CAR-T+calu-3 in Figure 24), the vertical axis is the LDH release rate (%), and the killing time is 24 hours. When the effect-target ratio (E:T) is 1:1, 2:1 and 5:1, the tumor cell death rate in the control group is significantly lower than that in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0328] Figure 25 is the statistical result of the release of interferon (IFN-γ) by ELISA detection of different groups of cells killing lung cancer calu-3 cells, wherein the horizontal axis is the effect-target ratio of the control group (represented by Mock T+calu-3 in Figure 25) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (represented by CART+calu-3 in Figure 25), and the vertical axis is the release of IFN-γ (pg / ml), and the killing time is 24 hours. When the effect-target ratio (E:T) is 1:1, 2:1 and 5:1, the release of IFN-γ in the control group is significantly lower than that in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0329] The results in Figures 21 to 25 show that compared with the Mock T control group, the TNFRSF10B / ASGR1 dual-target tandem CAR-T group had a better killing effect on lung cancer cells. Specifically, the TNFRSF10B / ASGR1 dual-target tandem CAR-T group had a significantly reduced proportion of residual tumor cells, a significantly increased tumor cell mortality rate, a significantly increased LDH release, and a significantly increased IFN-γ release. In other words, the TNFRSF10B / ASGR1 dual-target tandem CAR-T cells can significantly enhance the killing effect of tumor cells compared to T cells.

[0330] Example 25

[0331] This example provides flow cytometry detection results of residual tumor cells in hepG2 liver cancer cells killed by TNFRSF10B / ASGR1 dual-target tandem CAR-T, as shown in Figures 26 to 29. In Figure 26, the dual-target tandem CAR-T kills hepG2 liver cancer cells at effector-target ratios of 1:1, 2:1, and 5:1, respectively, with a killing time of 24 hours. The control group is T cells transfected with a plasmid without scFv (represented by Mock T in Figure 26). When the effector-target ratio was 1:1, the residual hepG2 cells in the mock T control group were 47.36%, while the residual hepG2 cells in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group were 28.89%. When the effector-target ratio was 2:1, the residual hepG2 cells in the mock T control group were 26.08%, while the residual hepG2 cells in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group were 22.33%. When the effector-target ratio was 5:1, the residual hepG2 cells in the mock T control group were 17.92%, while the residual hepG2 cells in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group were 8.23%. The statistical results of residual tumor cells detected by flow cytometry corresponding to Figure 26 are shown in Figure 27. In Figure 27, the horizontal axis is the effect-target ratio of the control group (represented by Mock T+HepG2 in Figure 27) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (represented by CART+HepG2 in Figure 27), and the vertical axis is the proportion of residual tumor cells (%), and the killing time is 24 hours. When the effect-target ratio is 1:1, 2:1, and 5:1, the proportion of residual tumor cells in the control group is significantly greater than that in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group, among which the effect-target ratios of 1:1 and 5:1 are statistically significant.

[0332] Figure 28 is the statistical result of the interferon (IFN-γ) release of different groups of cells to kill liver cancer HepG2 cells by ELISA, wherein the horizontal axis is the effect-target ratio of the control group (represented by Mock T+HepG2 in Figure 28) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (represented by CART+HepG2 in Figure 28), and the vertical axis is the release of IFN-γ (pg / ml), and the killing time is 24 hours. When the effect-target ratio (E:T) is 1:1, 2:1 and 5:1, the release of IFN-γ in the control group is significantly lower than that in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (all statistically significant).

[0333] Figure 29 is the statistical result of LDH release after the tumor cells of different groups of cells killed liver cancer HepG2 cells detected by LDH release test, wherein the horizontal axis is the effect-target ratio of the control group (represented by Mock T+HepG2 in Figure 29) and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group (represented by CART+HepG2 in Figure 29), the vertical axis is the LDH release rate (%), and the killing time is 24 hours. When the effect-target ratio (E:T) is 1:1, 2:1 and 5:1, the tumor cell death rate in the control group is significantly lower than that in the TNFRSF10B / ASGR1 dual-target tandem CAR-T group, among which the effect-target ratios of 2:1 and 5:1 are statistically significant.

[0334] The results in Figures 26 to 29 show that compared with the Mock T control group, the TNFRSF10B / ASGR1 dual-target tandem CAR-T group had a better killing effect on liver cancer cells. Specifically, the TNFRSF10B / ASGR1 dual-target tandem CAR-T group had a significantly reduced proportion of residual tumor cells, a significantly increased tumor cell mortality rate, a significantly increased IFN-γ release, and a significantly increased LDH release. In other words, the TNFRSF10B / ASGR1 dual-target tandem CAR-T cells can significantly enhance the killing effect of tumor cells compared to T cells.

[0335] Example 26

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

[0337] Figure 30.1 shows the flow cytometry results of residual tumor cells detected by CAR-T cells in each group killing hepG2 liver cancer cells at different effector-target ratios.

[0338] Figure 30.2 shows the percentage of dead tumor cells in each group at different effector-target ratios, as well as the statistical results of IFN-γ release by cells from different groups in killing HepG2 liver cancer cells. In Figure 30.2B, the horizontal axis represents the effector-target ratio, and the vertical axis represents the percentage of dead tumor cells (%). At effector-target ratios of 1:1 and 2:1, the killing efficacy of TNFRSF10B / ASGR1 dual-target parallel CAR-T cells and TNFRSF10B / ASGR1 dual-target tandem CAR-T cells against HepG2 liver cancer cells was superior to that of single-target CAR-T cells, and the killing efficacy of TNFRSF10B / ASGR1 dual-target parallel CAR-T cells and TNFRSF10B / ASGR1 dual-target tandem CAR-T cells was similar.

[0339] Figure 30.2C shows the statistical results of IFN-γ release in response to HepG2 liver cancer cell lines treated with different cell groups. The horizontal axis represents the effector-target ratio, and the vertical axis represents IFN-γ release (pg / ml). At effector-target ratios of 1:1 and 2:1, IFN-γ release was significantly higher in the TNFRSF10B / ASGR1 dual-target parallel CAR-T group and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group than in the TNFRSF10B-28z single-target CAR-T group. When the effector-target ratio was 1:1, the IFN-γ release amounts of the TNFRSF10B / ASGR1 dual-target parallel CAR-T group and the TNFRSF10B / ASGR1 dual-target tandem CAR-T group were similar, while when the effector-target ratio was 2:1, the IFN-γ release amount of the TNFRSF10B / ASGR1 dual-target tandem CAR-T group was significantly higher than that of the TNFRSF10B / ASGR1 dual-target parallel CAR-T group, that is, the ability of TNFRSF10B / ASGR1 dual-target tandem CAR-T cells to release IFN-γ was better than that of TNFRSF10B / ASGR1 dual-target parallel CAR-T cells.

[0340] 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.

Claims

1. A pharmaceutical composition, characterized in that, Comprising TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells and Fuzheng Jiedu Xiaoji Recipe, The dual-target chimeric antigen receptor of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells comprises a signal peptide, a first antigen-binding region targeting TNFRSF10B, a second antigen-binding region targeting ASGR1, a hinge region, a transmembrane region, and an intracellular region.

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

3.

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

4. The pharmaceutical composition according to claim 1 or 2, wherein The TNFRSF10B / ASGR1 dual-target chimeric antigen receptor comprises a first CAR and a second CAR. Among them, the first CAR contains: a first antigen-binding region targeting TNFRSF10B connected in sequence from the carboxyl terminus to the amino terminus; a first hinge region; a first transmembrane-binding region and CD3ζ; the second CAR contains: 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.

5. The pharmaceutical composition according to claim 1, characterized in that, The dual-target chimeric antigen receptor of the TNFRSF10B / ASGR1 dual-target chimeric antigen receptor T cells is of the tandem type.

6. The pharmaceutical composition according to claim 5, characterized in that, The dual-target chimeric antigen receptor contains: CD8a signal peptide, ASGR1 scFv, G4S Linker, DR5 scFv, CD8a hinge region, CD8α transmembrane region, 4-1BB, CD28 co-stimulatory molecule, CD3ζ signal transduction molecule.

7. The pharmaceutical composition according to claim 1, wherein The Fuzheng Jiedu Xiaoji Recipe is: 15g of Codonopsis pilosula, 15g of Astragalus membranaceus, 15g of Atractylodes macrocephala, 15g of Poria cocos, 15g of Adenophora stricta, 15g of Ophiopogon japonicus, 15g of Angelica sinensis, 15g of Rehmannia glutinosa, 15g of Paris polyphylla, 9g of Rhizoma zedoariae, 9g of Pinellia ternata.

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

9. The cell according to claim 8, wherein The signal peptide can be a human CD8α signal peptide, a human GM-CSF signal peptide, a human insulin signal peptide, a human IL-2 signal peptide, or a human trypsinogen signal peptide.

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

2.

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

3.

12. The cell according to claim 8, wherein The TNFRSF10B / ASGR1 bispecific chimeric antigen receptor is of the parallel type. The TNFRSF10B / ASGR1 bispecific chimeric antigen receptor comprises a first CAR and a second CAR. The first CAR comprises: the first antigen-binding region targeting TNFRSF10B connected in sequence 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 the variable region of the light chain of an anti-TNFRSF10B antibody and the variable region of the heavy chain of an anti-TNFRSF10B antibody. Among them, the amino acid sequence of the variable region of the light chain of the anti-TNFRSF10B antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the heavy chain of the anti-TNFRSF10B antibody is as shown in SEQ ID NO:

2. The second CAR comprises: a signal peptide connected in sequence from the carboxyl terminus to the amino terminus; the 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 the variable region of the heavy chain of an anti-ASGR1 antibody. Among them, the amino acid sequence of the variable region of the heavy chain of the anti-ASGR1 antibody is as shown in SEQ ID NO:

3.

13. The cell according to claim 8, wherein The bispecific chimeric antigen receptor of the cell expressing the TNFRSF10B / ASGR1 bispecific chimeric antigen receptor is of the tandem type.

14. The cell according to claim 13, characterized in that, The bispecific chimeric antigen receptor comprises: a CD8a signal peptide, an ASGR1 scFv, a G4S Linker, a DR5 scFv, a CD8a hinge region, a CD8α transmembrane region, 4-1BB, a CD28 co-stimulatory molecule, and a CD3ζ signal transduction molecule.

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

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