Application of macrophages in improving immunotherapy stomach cancer

Abstract

The invention relates to the technical field of biology, and particularly discloses application of macrophages in improving immunotherapy stomach cancer. The synergistic effect of CLEC2D + macrophages and a CD161 receptor in gastric cancer immune escape is focused for the first time, the single attention on macrophage M1 / M2 polarization and T cell checkpoint molecules in traditional research is broken through, a new T cell depletion mechanism driven by direct interaction of myeloid cells and lymphocytes is disclosed, and supplement is provided for the tumor immune escape theory; a novel blocking agent (such as a CD161 targeting antibody and a small molecule inhibitor) is developed on the basis of a mechanism of activating and mediating T cell depletion by a CD161 receptor, a basic mechanism research is directly converted into an innovative treatment scheme, and a differentiated strategy is provided for gastric cancer immunotherapy in combination with an existing PD-1 / PD-L1 inhibitor.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a macrophage in improving immunotherapy for gastric cancer. BACKGROUND

[0002] Gastric cancer is a serious situation in the field of global malignant tumors, which seriously endangers people's life and health. Because of the occult early symptoms of gastric cancer, most patients are diagnosed at an advanced stage. Traditional surgical, radiotherapy and chemotherapy have poor efficacy for advanced gastric cancer, and the prognosis is not optimistic.

[0003] Immunotherapy targeting PD-1 / PD-L1 immune checkpoints is a new cancer treatment strategy, which can activate or strengthen the body's immune system to accurately identify and attack tumor cells. Immunotherapy has the advantages of strong specificity, low recurrence rate and mild side effects, and is widely used in clinical practice. However, in the field of gastric cancer immunotherapy, tumor cell immune escape is common, which seriously hinders its therapeutic effect.

[0004] In the tumor microenvironment, a variety of complex factors work together to enable tumor cells to cleverly evade the monitoring and attack of the body's immune system, leading to immune escape. Among them, CD8 ＋ T cells play a very key effector cell role in anti-tumor immune response and are the core force to eliminate tumor cells. However, studies have shown that CD8 ＋ T cells are easily induced to differentiate into exhausted state under continuous antigen stimulation or specific immunosuppressive microenvironment.

[0005] CD8 ＋ T cell exhaustion mainly shows many characteristics, such as significantly reduced proliferation capacity, difficulty in expanding the size of the immune "troop", abnormal secretion of key cytokines, leading to the inability to regulate and activate other immune cells to kill tumor cells, and reduced secretion of perforin, granzyme and other toxic granules, leading to a significant decrease in the direct killing ability of tumor target cells. Therefore, CD8 ＋ T cell exhaustion greatly reduces the efficiency of the immune system in eliminating tumor cells, and becomes an important internal factor for tumor immune escape. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art and provide a macrophage for improving immunotherapy for gastric cancer.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is as follows: The present application provides a macrophage for improving immunotherapy for gastric cancer, wherein the macrophage comprises CLEC2D ＋ macrophage.

[0008] In the gastric cancer microenvironment, the composition and functional status of macrophage subsets affect CD8. ＋ T cells play a crucial role. In-depth analysis of specific macrophage subsets, especially those playing a key role in immune escape from gastric cancer, and their influence on CD8... ＋ The regulatory pathways of T cell function can not only further improve the theoretical system of tumor immune escape and reveal the deep-seated mechanism of immune escape in gastric cancer, but more importantly, it can provide new targets and ideas for the development of innovative immunotherapy strategies targeting macrophages.

[0009] This invention collects CD8 ＋ Gastric cancer patient samples with varying degrees of T cell depletion showed an enrichment of immune cells (CD45). ＋ Single-cell sequencing was performed to screen for drivers of CD8. ＋ Key immune cells and signaling pathways involved in T cell depletion. Research results show that CD8+ is present in gastric cancer tissue. ＋ T cell exhaustion was positively correlated with a significant increase in the proportion of macrophages, indicating that macrophages are key drivers of CD8 cell depletion in the gastric cancer immune microenvironment. ＋ T cell depletion is a key immune cell that mediates immune escape.

[0010] This invention provides CLEC2D ＋ Application of macrophages and / or CD161 receptors in products that improve immune escape in gastric cancer.

[0011] As a preferred embodiment of the application described in this invention, the CLEC2D ＋ Macrophages promote CD8 ＋ T cell exhaustion mediates immune escape from gastric cancer; it promotes CD8 cell activation by activating CD161 receptors. ＋ T cell depletion.

[0012] This invention's analysis revealed that the expression of macrophage membrane protein CLEC2D is upregulated, and it is associated with CD8. ＋ The significantly increased binding of T cell receptor CD161 suggests that the CLEC2D-CD161 axis may be involved in and affect CD8. ＋ T cell exhaustion. Macrophage membrane protein CLEC2D expression and CD8 ＋ T cell depletion is associated with this, and thus plays a role in the immunotherapy of gastric cancer.

[0013] This invention provides CLEC2D ＋ Macrophages promote the production of CD8 ＋ Applications of T cell depletion reagents.

[0014] As a preferred embodiment of the application described in this invention, CD8 is present in gastric cancer tissue.+ T cell exhaustion was positively correlated with a significant increase in the proportion of macrophages.

[0015] This invention provides the application of macrophage membrane protein CLEC2D in combination with PD-1 monoclonal antibody or PD-L1 monoclonal antibody in the preparation of drugs for treating gastric cancer.

[0016] This invention proposes a novel strategy for treating gastric cancer by targeting the CLEC2D-CD161 signaling axis in combination with PD-1 monoclonal antibody therapy, providing a new treatment option for improving the efficacy of immunotherapy for gastric cancer.

[0017] In a preferred embodiment of the application described in this invention, the expression of macrophage membrane protein CLEC2D is upregulated, and it is associated with CD8. + The binding of T cell receptor CD161 was significantly increased.

[0018] This invention provides the application of inhibitors of macrophage membrane protein CLEC2D or CD161 receptors in the preparation of immunotherapeutic drugs for gastric cancer.

[0019] In a preferred embodiment of the application described in this invention, the inhibitor comprises a specific monoclonal antibody.

[0020] The specific monoclonal antibody includes the CLEC2D inhibitor-ZM008 or the CD161 monoclonal antibody-IMT-009.

[0021] Experiments show that CLEC2D ＋ Macrophage enrichment can promote CD8 ＋ T cell exhaustion; activation of CD161 receptor can promote CD8... ＋ T cell depletion. Inhibition of CD8. ＋ T cell depletion can significantly reduce the efficiency of the immune system in clearing tumor cells and reduce tumor immune escape.

[0022] This invention utilizes flow cytometry analysis of CLEC2D in gastric cancer tissue samples and ascites samples. ＋ CD68 ＋ CD11b ＋ Macrophages and CD161 ＋ CD8 ＋ The proportion of T cells was found to be significantly enriched. Finally, using organoid and mouse models, this invention demonstrated that in a gastric cancer immune model, antibody blocking of CLEC2D / CD161 resulted in CD8... + T cell infiltration and function were significantly enhanced, and the exhaustion phenotype was significantly reduced.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention focuses for the first time on CLEC2D ＋The synergistic effect of macrophages and CD161 receptors in gastric cancer immune escape breaks through the traditional focus on macrophage M1 / M2 polarization and T cell checkpoint molecules, revealing a new mechanism of T cell exhaustion driven by direct interaction between myeloid cells and lymphocytes, thus supplementing the theory of tumor immune escape. Based on the mechanism of T cell exhaustion mediated by CD161 receptor activation, novel blocking agents (such as CD161-targeting antibodies and small molecule inhibitors) are being developed, directly translating basic mechanism research into innovative treatment strategies. Combined with existing PD-1 / PD-L1 inhibitors, this provides a differentiated strategy for gastric cancer immunotherapy. Attached Figure Description

[0024] Figure 1 Macrophages enriched with CD8 ＋ T cell exhaustion is positively correlated with (where, Figure 1 In the middle, A represents the clustering of the captured cells; Figure 1 In section B, the proportions of immune cells in the two groups are compared. **** p <0.001, n=7); Figure 2 Expression of macrophage membrane protein CLEC2D and CD8 ＋ T cell exhaustion is correlated (among others) Figure 2 In section A, differential analysis of macrophage-related genes is performed. Figure 2 B is macrophages and CD8 ＋ (Molecular strength analysis of T cell interactions) Figure 3 To block CLEC2D ＋ CD8 can be reversed after macrophages ＋ T cell depletion and enhancing its anti-tumor function (among which, Figure 3 A in the middle represents the flow cytometry analysis CLEC2D. ＋ Macrophages (CD11b) ＋ CLEC2D ＋ )Proportion; Figure 3 B in the diagram represents the experimental procedure flowchart for this part. Figure 3 C represents the flow cytometry blocking CLEC2D. ＋ Apoptosis of gastric cancer organoids following macrophages; Figure 3 D in the middle represents the flow cytometry blocking CLEC2D. ＋ CD8 in the macrophage microenvironment ＋ T cell infiltration status; Figure 3 E represents the depletion rate of each group in the flow cytometry analysis (CD8). ＋ T cells (CD8) ＋ PD1 ＋ TIM3 ＋ Proportional situation; Figure 3The IFN-γ in each group was analyzed by flow cytometry. ＋ CD8 + T cells and GranB ＋ CD8 ＋ T cell ratio. *** p <0.001, n=3.); Figure 4 CD8 in Example 3 ＋ Figure showing the interaction between T cell exhaustion and macrophage CLEC2D expression; Figure 5 In Example 3, activation of the CD161 receptor promotes CD8... ＋ Image showing the results of T cell depletion. Detailed Implementation

[0025] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0026] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0027] In the research of this project, CD8 was first collected. ＋ Gastric cancer patient samples with varying degrees of T cell depletion showed an enrichment of immune cells (CD45). ＋ Single-cell sequencing was performed to screen for drivers of CD8. ＋ Key immune cells and signaling pathways of T cell depletion.

[0028] The study results showed that CD8 was present in gastric cancer tissue. ＋ T cell exhaustion was positively correlated with a significant increase in the proportion of macrophages; further analysis revealed that the expression of macrophage membrane protein CLEC2D was upregulated and correlated with CD8. ＋ The significantly increased binding of T cell receptor CD161 suggests that the CLEC2D-CD161 axis may be involved in and affect CD8. ＋ T cell depletion.

[0029] Example 1 To screen for CD8-mediated immune microenvironment in gastric cancer ＋ This invention addresses key immune cell and molecular mechanisms of T cell exhaustion, and utilizes flow cytometry pre-analysis of CD8+ in gastric cancer patient tissues. ＋ The condition of T cell exhaustion was analyzed and divided into a non-exhausted group (TIM3). + PD1 + CD8 +(low percentage of T cells, n=4) and exhaustion group (Exhaust, TIM3) + PD1 + CD8 + T cells accounted for a large proportion (n=4), and immune cells (CD45) were sorted using magnetic beads. ＋ Single-cell sequencing was performed.

[0030] By comparing immune cells in the non-exhaust and exhaustion groups: CD8 ＋ T cells, tumor-associated macrophages, CD4 ＋ The proportions of T cells, DC cells, NK cells, B cells, and neutrophils were analyzed, and it was found that compared with the non-exhausted group, the exhausted group had a higher proportion of macrophages (CD11b). + CD86 + The increase in the proportion was most significant.

[0031] Furthermore, flow cytometry analysis showed that macrophages were enriched in gastric cancer tissue along with CD8+. ＋ There is a positive correlation between T cell exhaustion and T cell depletion. This suggests that macrophages may be a driving force behind CD8 cell depletion in the gastric cancer immune microenvironment. ＋ T cell exhaustion is a key immune cell mediated by immune escape (such as...) Figure 1 (As shown).

[0032] Example 2 In order to screen for macrophage-mediated CD8 activation ＋ This invention compares the expression of macrophage-related cytokines and membrane protein genes in non-exhaust and exhaustion groups, focusing on key subpopulations and analytical mechanisms of T cell exhaustion.

[0033] like Figure 2 As shown, compared with the unexhausted group, the expression of the macrophage membrane protein CLEC2D gene was most significantly upregulated; further, this invention analyzed the interaction between macrophages and CD8+. ＋ The strength of T cell interaction molecules was analyzed, and the results showed that, compared with the non-exhausted group, the signaling pathway composed of CLEC2D and CD161 (CD161 is encoded by the KLRB1 gene) was significantly enhanced in the exhausted group. This part of the study indicates that the expression of the macrophage membrane protein CLEC2D is related to CD8. ＋ T cell exhaustion may be related (e.g.) Figure 2 (As shown).

[0034] Example 3 To verify CD8 ＋This invention investigates the interaction between T cell exhaustion and macrophage CLEC2D expression, firstly by flow cytometry screening of CLEC2D in patient-derived gastric cancer tissue. ＋ Macrophages (CD11b) ＋ CLEC2D ＋ The sample with a higher proportion ( Figure 3 (A), and gastric cancer organoids and immune cells were cultured separately, then co-cultured, and the monoclonal antibody ZM-008 targeting the CLEC2D protein was added for intervention (experimental procedure as follows). Figure 3 (Chinese B) Data shows that intervention with the monoclonal antibody ZM008, which specifically blocks CLEC2D, significantly increased the apoptosis rate of gastric cancer organoids. Figure 3 (C), CD8 ＋ The proportion of T cell infiltration increased significantly ( Figure 3 (D), PD1 ＋ TIM3 ＋ CD8 ＋ The proportion of exhausted T cells was significantly reduced. Figure 3 (E), and IFN-γ ＋ CD8 ＋ Cells, GranB ＋ CD8 ＋ The proportion of T cells increased significantly ( Figure 4 (FG). The above evidence indicates that CLEC2D ＋ Macrophage enrichment can promote CD8 ＋ T cell depletion.

[0035] Similarly, this invention also detected a certain proportion of CD161 in ascites immune cells. ＋ CD8 ＋ T cells ( Figure 4 (A). To confirm that the CD161 receptor mediates CD8. ＋ T cell exhaustion was addressed using the same method in this invention, with the addition of the CD161-blocking specific monoclonal antibody IMT-009 (experimental procedure as follows). Figure 4 (B) Data shows that blocking the CD161 receptor significantly increases the apoptosis rate in gastric cancer organoids. Figure 4 (C), LAG3 ＋ CD8 ＋ The proportion of exhausted T cells was significantly reduced. Figure 4 (D).

[0036] Meanwhile, this study extracted CD8 from peripheral blood lymphocytes of healthy individuals. ＋ T cells, and through a plasmid that stably transfects CD161 overexpression ( Figure 5 (A) Research found that CD8 ＋After T cells overexpress CD161, their depletion of CD8 ＋ T cells (CD8) + PD1 + TIM3 + The proportion has increased significantly. Figure 5 (B), and its toxicity IFN-γ + CD8 + Cells, GranB + CD8 + The proportion of T cells decreased significantly ( Figure 5 Medium CD).

[0037] This part of the research shows that activation of the CD161 receptor can promote CD8... ＋ T cell depletion.

[0038] In summary, this invention has preliminarily discovered CLEC2D in gastric cancer. ＋ Macrophage enrichment and CD8 ＋ There is a positive correlation between T cell depletion and T cell exhaustion.

[0039] This invention focuses for the first time on CLEC2D ＋ The synergistic effect of macrophages and CD161 receptors in gastric cancer immune escape breaks through the traditional focus on macrophage M1 / M2 polarization and T cell checkpoint molecules, revealing a new mechanism of T cell exhaustion driven by direct interaction between myeloid cells and lymphocytes, and providing a supplement to the theory of tumor immune escape.

[0040] Based on the mechanism by which CD161 receptor activation mediates T cell exhaustion, we will develop novel blocking agents (such as CD161-targeting antibodies and small molecule inhibitors) to directly translate basic mechanism research into innovative treatment plans. Combined with existing PD-1 / PD-L1 inhibitors, this will provide a differentiated strategy for the immunotherapy of gastric cancer.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An application of macrophages in improving immunotherapy for gastric cancer, characterized in that, The macrophages include CLEC2D ＋ Macrophages. 2.CLEC2D ＋ Application of macrophages and / or CD161 receptors in products that improve immune escape in gastric cancer.

3. The application as described in claim 2, characterized in that, The CLEC2D ＋ Macrophages promote CD8 + T cell exhaustion mediates gastric cancer immune escape; Activation of CD161 receptor promotes CD8 ＋ T cell depletion. 4.CLEC2D ＋ Macrophages promote the production of CD8 ＋ Applications of T cell depletion reagents.

5. The application as described in claim 4, characterized in that, CD8 in gastric cancer tissue ＋ T cell exhaustion was positively correlated with a significant increase in the proportion of macrophages.

6. Application of macrophage membrane protein CLEC2D in combination with PD-1 monoclonal antibody or PD-L1 monoclonal antibody in the preparation of drugs for treating gastric cancer.

7. The application as described in claim 6, characterized in that, Macrophage membrane protein CLEC2D expression is upregulated and correlates with CD8. ＋ The binding of T cell receptor CD161 was significantly increased.

8. Application of inhibitors of macrophage membrane protein CLEC2D or CD161 receptors in the preparation of immunotherapy drugs for gastric cancer.

9. The application as described in claim 8, characterized in that, The inhibitors include specific monoclonal antibodies.

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