Application of HMGB1 overexpression agent in the preparation of drugs for improving the anti-tumor effect of CD8 T cells

Abstract

The present invention discloses the use of an HMGB1 overexpression agent in the preparation of a drug for improving the anti-tumor effect of CD8T cells. The present invention finds through research that the HMGB1 target can promote the anti-tumor effect of CD8T cells by promoting the expression of IFN-γ in CD8T cells, and that targeting CD8T cell HMGB1 has anti-tumor potential. The present invention finds an important target HMGB1 that promotes the anti-tumor effect of CD8T cells in immunotherapy, and by promoting the overexpression of the HMGB1 target, the tumor effect of CD8T cells can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug application, and more specifically, relates to the application of an HMGB1 overexpression agent in the preparation of a drug for improving the anti-tumor effect of CD8T cells. Background Art

[0002] Tumor immunotherapy is a treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. It includes monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, and small molecule inhibitors.

[0003] Anti-programmed death 1 (PD-1) antibodies are currently the most studied and fastest-growing immunotherapy. PD-1 acts in the effector phase of the immune response. It is expressed in activated T cells, B cells and myeloid cells. It has two ligands, namely programmed death ligand 1 (PD-L1) and PD-L2. PD-L1 / L2 are expressed in antigen-presenting cells, and PD-L1 is also expressed in a variety of tissues. The binding of PD-1 and PD-L1 mediates the co-inhibitory signal of T cell activation, inhibits the killing function of T cells, and plays a negative regulatory role in the human immune response. Chen Lieping, a Chinese-American scientist, first discovered that PD-L1 is highly expressed in tumor tissues and regulates the function of tumor-infiltrating CD8 T cells.

[0004] Tumor immunotherapy mainly utilizes the anti-tumor effect of immune cells for anti-tumor treatment. Immune cells can secrete a variety of immune factors to kill tumors. For example, CD8 T cells can kill tumor cells by secreting IFN-g.

[0005] CD8 T cells are the main immune cells that exert anti-tumor effects in the human body. This cell participates in a variety of tumor immunotherapy therapies. Improving the anti-tumor ability of this cell can significantly improve the efficacy of tumor immunotherapy. For example, Chinese patent CN116270990A discloses the use of ADAM17 in the preparation of drugs for the treatment of effector CD8+T cell differentiation-related diseases. In a mouse tumor model, CD8T cells modified by adoptive ADAM17 can significantly inhibit tumor growth compared to wild-type CD8T cells, and the proportion of exhausted CD8T cells in the tumor is reduced. Therefore, ADAM17 can be used as a potential therapeutic target for effector CD8T cell-related diseases. However, the existing important targets for promoting the anti-tumor effect of CD8 T cells in immunotherapy are still limited. Discovering more targets that can improve the anti-tumor effect of CD8 T cells is expected to further promote the efficacy of tumor immunotherapy. Summary of the invention

[0006] In view of the above existing technical problems, the primary purpose of the present invention is to provide the use of an HMGB1 overexpression agent in the preparation of an anti-tumor drug. The present invention has found through research that the HMGB1 target can promote the anti-tumor effect of CD8 T cells by promoting the expression of IFN-γ in CD8 T cells, and that targeting the CD8 T cell HMGB1 target has anti-tumor potential. The present invention has found an important target HMGB1 for promoting the anti-tumor effect of CD8 T cells in immunotherapy, and by promoting the overexpression of the HMGB1 target, the tumor effect of CD8 T cells can be improved.

[0007] The second object of the present invention is to provide the use of an HMGB1 overexpression agent in the preparation of a drug for improving the anti-tumor effect of CD8 T cells.

[0008] The third object of the present invention is to provide an application of a pharmaceutical composition in the preparation of anti-tumor drugs.

[0009] The fourth object of the present invention is to provide a use of a pharmaceutical composition in the preparation of a drug for improving the anti-tumor effect of CD8 T cells.

[0010] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0011] The present invention seeks to protect the use of an HMGB1 overexpression agent in the preparation of an anti-tumor drug.

[0012] HMGB1 in the cell nucleus plays an important regulatory role. For example, HMGB1 in the nucleus of keratinocytes can reduce allergic dermatitis by inhibiting IL-24 gene expression. For example, HMGB1 in the nucleus of hepatocytes can inhibit non-alcoholic fatty liver disease by negatively regulating liver X receptors.

[0013] The present invention uses the cre-loxp system to knock out the HMGB1 gene of mouse CD8 T cells and CD4 T cells, so that the mouse CD8 T cells and CD4 T cells do not express HMGB1, and then the inventors analyze the expression level of IFN-γ of CD8 T cells and CD4 T cells. Next, the inventors overexpressed HMGB1 in CD8 T cells by using HMGB1 overexpression lentivirus, and then co-cultured the CD8 T cells overexpressing HMGB1 with B16 tumor cells to perform an in vitro simulated tumor killing experiment. Through the above experiments, the inventors were surprised to find that HMGB1 can promote the expression of IFN-γ, a key tumor killing factor of CD8 T cells, and after knocking out the HMGB1 gene of CD4 T cells, the expression of IFN-γ of CD4 T cells was not affected. To further confirm the above conclusion, the inventors infected CD8 T cells with HMGB1-overexpressing lentivirus. The experiment found that the number of tumor cells in CD8 T cells infected with HMGB1-overexpressing lentivirus was significantly lower than that in the normal group, which shows that the overexpression of target HMGB1 can significantly promote the anti-tumor effect of CD8 T cells. These results show that the HMGB1 target targeting CD8 T cells has anti-tumor potential and can promote the anti-tumor effect of CD8 T cells in immunotherapy.

[0014] Compared with PD-1 immunotherapy, it requires that the patient's tumor cells have a higher PD-L1 expression level, so the therapy has poor efficacy for clinical patients with lower PD-L1 expression levels. However, the present invention has lower requirements for PD-L1 expression levels, and all CD8 T cells express the HMGB1 gene. Based on the present invention, the expression of HMGB1 in CD8 T cells is promoted, which can promote the anti-tumor ability of CD8 T cells, improve the efficacy of immunotherapy, and has a wider range of application scenarios.

[0015] Preferably, the HMGB1 overexpressing agent is a HMGB1 overexpressing lentivirus.

[0016] Preferably, the accession number of the HMGB1 overexpressing lentivirus sequence in the NCBI database is NM_001313894.1.

[0017] Preferably, the tumor is one or more of melanoma, colorectal cancer, and lymphoma.

[0018] Furthermore, the present invention seeks to protect the use of an HMGB1 overexpressing agent in the preparation of a drug for improving the anti-tumor effect of CD8 T cells.

[0019] Preferably, the HMGB1 overexpressing agent is a HMGB1 overexpressing lentivirus.

[0020] Preferably, the accession number of the HMGB1 overexpressing lentivirus sequence in the NCBI database is NM_001313894.1.

[0021] Preferably, the tumor is one or more of melanoma, colorectal cancer, lymphoma and the like.

[0022] Specifically, improving the anti-tumor effect of CD8 T cells refers to promoting the expression of IFN-γ in CD8 T cells.

[0023] Furthermore, the present invention seeks to protect the use of a pharmaceutical composition in the preparation of an anti-tumor drug or in the preparation of a drug for improving the anti-tumor effect of CD8 T cells, wherein the pharmaceutical composition comprises an HMGB1 overexpression agent.

[0024] Preferably, the HMGB1 overexpression agent is a HMGB1 overexpression lentivirus. Further preferably, the sequence of the HMGB1 overexpression lentivirus is NCBI Reference Sequence: NM_001313894.1.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention has discovered a new potential anti-tumor target HMGB1 on CD8 T cells through experiments. By promoting the overexpression of HMGB1 in CD8 T cells, the anti-tumor effect of CD8 T cells can be promoted. The present invention has a low requirement for the expression level of PD-L1. All CD8 T cells express the HMGB1 gene. On the basis of the present invention, promoting the expression of HMGB1 in CD8 T cells can promote the anti-tumor ability of CD8 T cells and improve the efficacy of immunotherapy. Therefore, it has a wider application scenario for PD-L1 immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The expression of HMGB1 and IFN-γ in CD8 T cells and CD4 T cells after knocking out HMGB1 in CD8 T cells and CD4 T cells. Figure 1 A shows the expression of IFN-γ in CD8 T cells after knocking out HMGB1 in CD8 T cells; Figure 1 B shows the expression of IFN-γ in CD4 T cells after knocking out HMGB1 in CD4 T cells.

[0028] Figure 2 This is a schematic diagram showing that the HMGB1 overexpression lentivirus promotes the upregulation of CD8 T cell tumor killing ability. Figure 2A shows the expression of HMGB1 in CD8 T cells after transfection with HMGB1 overexpression lentivirus. Figure 2 B shows the expression of IFN-γ in CD8 T cells after transfection with HMGB1 overexpression lentivirus. Figure 2 C is the proportion of B16 cells remaining after 12 h of co-culture of CD8 T cells and B16 cells in mice overexpressing HMGB1. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] Example 1 HMGB1 knockout test in CD8 T cells and CD4 T cells

[0031] (1) Experimental operation

[0032] Since Cre recombinase is selective for LoxP sequences, it can cut out the DNA between two LoxP sequences. In order to obtain CD8T cells and CD4T cells with HMGB1 knockout mice, we chose CD2-Cre mice here, and obtained heterozygous mice (HMGB1-f / +) (heterozygous mice (HMGB1-f / +) are vectors that sandwich the mouse HMGB1 gene between two LoxP sequences. This vector is inserted into the genome of mouse germ cells through gene targeting to obtain heterozygous mice. Its source is: Enterprise Saiye Bio, product name Hmgb1-flox, product number By mating S-CKO-02901) with CD2-Cre mice, we first obtained heterozygous mice with CD2-cre (HMGB1-f / +; CD2-cre), and then mated heterozygous mice with CD2-cre (HMGB1-f / +; CD2-cre) with each other, and finally we obtained homozygous mice with CD2-cre (HMGB1-f / f; CD2-cre), in which HMGB1 of CD8 T cells and CD4 T cells was knocked out.

[0033] The spleens of homozygous mice with CD2-cre (HMGB1-f / f; CD2-cre) and control mice (HMGB1-f / f) were obtained, and then the spleens were made into cell suspensions, and CD8, CD4, and IFN-γ were stained by flow cytometry, and the expression of CD8 T cells and CD4 T cells IFN-γ in the spleen was detected by flow cytometry using Cytek Northernlight.

[0034] (2) Experimental results

[0035] Figure 1 The expression of HMGB1 and IFN-γ in CD8 T cells and CD4 T cells after knocking out HMGB1 in CD8 T cells and CD4 T cells. Figure 1 A shows the expression of IFN-γ in CD8 T cells after knocking out HMGB1 in CD8 T cells; Figure 1 B shows the expression of IFN-γ in CD4 T cells after knocking out HMGB1 in CD4 T cells.

[0036] Depend on Figure 1 It can be seen that HMGB1 of CD8 T cells can promote the expression of IFN-γ in CD8 T cells. After knocking out HMGB1 of CD8 T cells, the expression of IFN-γ in CD8 T cells was significantly downregulated (such as Figure 1 A), CD4 T cell IFN-γ expression was not affected after knocking out CD4 T cell HMGB1 (as shown in Figure 1 B).

[0037] Example 2 Experiment on the anti-tumor effect of lentivirus overexpressing HMGB1 on CD8 T cells

[0038] (1) Experimental operation

[0039] Mouse spleens were removed and made into single cell suspensions. CD8 T cells were sorted and plated in 48-well plates (5*10 4 / well). On the second day, HMGB1 overexpression lentivirus (accession number NM_001313894.1) was used for transfection. Two days after transfection, some cells were taken for flow cytometry analysis of CD8 T cell HMGB1 (A) and IFN-γ expression levels (n=3) (B), and the remaining cells were taken as effector cells. B16 cells (mouse melanoma cells) were labeled with CFSE high (5 mM) as target cells, and the same number of WT spleen cells (1x10 4 / well) labeled with CFSE low (0.5 mM) was used as an internal cell number control. Different numbers of effector cells were mixed with 1x10 4 The cells were mixed (CD8:B16=1:1, 5:1, 10:1) and co-cultured for 24 hours, and the percentage of remaining target cells was analyzed by FACS (H, n=4).

[0040] (2) Experimental results

[0041] Figure 2This is a schematic diagram showing that the HMGB1 overexpression lentivirus promotes the upregulation of CD8 T cell tumor killing ability. Figure 2 A shows the expression of HMGB1 in CD8 T cells after transfection with HMGB1 overexpression lentivirus. Figure 2 B shows the expression of IFN-γ in CD8 T cells after transfection with HMGB1 overexpression lentivirus. Figure 2 C is the proportion of B16 cells remaining after 12 h of co-culture of CD8 T cells and B16 cells in mice overexpressing HMGB1.

[0042] like Figure 2 A and Figure 2 As shown in B, compared with the control group, the expression of HMGB1 and IFN-γ in CD8 T cells transfected with HMGB1 overexpression lentivirus was significantly increased. Figure 2 As shown in C, compared with the control group, in mice overexpressing HMGB1, the proportion of remaining B16 cells was significantly reduced after 12 hours of co-culture of CD8 T cells and B16 cells, and the B16 cells decreased more significantly with the increase in the amount of CD8 T cells. This indicates that lentivirus overexpressing HMGB1 can promote the anti-tumor effect of CD8 T cells.

[0043] The foregoing embodiments are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the inventor's actual test results. Therefore, the inventor's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. Use of an HMGB1 overexpression agent in the preparation of a drug for improving the anti-melanoma effect of CD8 T cells, characterized in that: The HMGB1 overexpression agent is a HMGB1 overexpression lentivirus, and the accession number of the HMGB1 overexpression lentivirus sequence in the NCBI database is NM_001313894.

1.

2. Use of a pharmaceutical composition in the preparation of a drug for improving the anti-melanoma effect of CD8 T cells, characterized in that: The pharmaceutical composition comprises an HMGB1 overexpression agent, which is a HMGB1 overexpression lentivirus, and the accession number of the sequence of the HMGB1 overexpression lentivirus in the NCBI database is NM_001313894.1.

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