Application of TREM2 + MARCO + TAMs subgroup as drug target in screening or preparing drug for improving cancer immunotherapy response

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a TREM2 + MARCO + TAMs subgroup as a drug target in screening or preparing a drug for improving cancer immunotherapy response. According to the application, CD8 < + > T cell terminal depletion and bladder cancer ICB non-response are taken as entry points, a brand-new TREM2 < + > MARCO < + > TAMs subgroup is identified for the first time by combining single cell RNA sequencing with space transcriptome sequencing, it is confirmed that the subgroup is related to high enrichment of terminal depletion CD8 < + > T cells and ICB non-response of bladder cancer, and further in-vivo functional experiments confirm that the subgroup is a new subgroup. Conditional knockout of the TREM2 + MARCO + TAMs subgroup reverses CD8 + T cell terminal depletion to greatly improve the response of bladder cancer ICB treatment, a new target is provided for developing bladder cancer immunotherapy drugs, and a new treatment strategy is expected to be provided for patients who do not respond to solid malignant tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to TREM2 + MARCO + Application of TAMs subpopulations as drug targets in screening or preparing drugs that improve cancer immunotherapy responses. Background Art

[0002] In recent years, immunotherapy based on immune checkpoint blockade (ICB) has greatly changed the treatment landscape of malignant tumors. Despite the success of this therapy, a considerable proportion of patients still fail to respond. The current mainstream view is that ICB non-response is usually due to tumor-infiltrating tumor-specific CD8 + T cells enter a terminally exhausted state under continuous tumor antigen encounter, which limits the effect of ICB on CD8 + The reactivation of T cells and their anti-tumor efficacy are weakened. A key challenge is to reveal the role of CD8 + Complex regulatory mechanisms of T cell terminal exhaustion.

[0003] There is growing evidence that blockade of PD-1 in combination with other checkpoints (such as CTLA4, TIGIT, and TIM3) can inhibit CD8 + T cell terminal exhaustion can improve efficacy when traditional single PD-1 inhibitors are ineffective. However, patients vary greatly in their response to this treatment strategy, suggesting that there is an unknown critical checkpoint that exacerbates T cell terminal exhaustion. Recent clinical trial results show that combined inhibition of LAG-3 and PD-1 synergistically improves survival and reduces CD8 + T cell exhaustion. The LAG-3 antibody relatlimab was approved by the FDA in 2022, establishing LAG-3 as a third-generation immune checkpoint and blocking CD8 + T cells are terminally exhausted. However, the key ligand of LAG3 is still controversial. Its known ligands MHC-II and FGL1 bind to LAG3 to inhibit T cell activation, while LAG3 can induce CD8 + T cells enter a terminally exhausted state. MHC-II mainly affects CD4 + T cells, while LAG3 mutants lacking FGL1 binding ability can still effectively inhibit CD8 + Therefore, CD8 + The key ligand of LAG3 for terminal T cell exhaustion is still unclear.

[0004] Recent studies have shown that the ligands that T cell checkpoints bind to are mainly expressed on myeloid antigen presenting cells (APCs) and tumor cells. Recent studies have shown that macrophages, especially tumor-associated macrophages (TAMs), as APCs, bind to CD8 + Long-term interaction with T cells drives their exhaustive differentiation. This finding suggests that there may be an unidentified ligand on macrophages that interacts with LAG3. Due to the heterogeneity of macrophages, further analysis of the specific macrophage subsets that provide LAG3 ligands and transmit terminal exhaustion signals in the tumor microenvironment is crucial for developing next-generation therapeutic modalities to improve the efficacy of ICB and improve the prognosis of cancer patients.

[0005] At present, there is still a lack of clinical treatment strategies and drug targets for immunotherapy resistance of malignant tumors, especially bladder cancer, and the effect on improving the prognosis of patients is poor; the most important factor is that the mechanism of non-response to bladder cancer immunotherapy and anti-tumor CD8+ T cell dysfunction is still unclear, and there is still a lack of more effective diagnostic and therapeutic targets. Therefore, it is of great application value to clarify the microenvironment mechanism of bladder cancer immunotherapy resistance and find new therapeutic targets and strategies that can enhance the efficacy of PD-1 inhibitors in bladder cancer for the treatment of bladder cancer. Summary of the invention

[0006] In order to overcome at least one technical problem existing in the prior art, the present invention provides TREM2 + MARCO + Application of TAMs subpopulations as drug targets in screening or preparing drugs that improve cancer immunotherapy responses.

[0007] The above technical problem to be solved by the present invention is achieved by adopting the following technical solution: The present invention first provides TREM2 + MARCO + Application of TAMs subpopulations as drug targets in screening or preparing drugs that improve cancer immunotherapy responses.

[0008] Preferably, the cancer is bladder cancer.

[0009] Preferably, the immunotherapy refers to immunotherapy using PD1 antibodies.

[0010] Preferably, the PD1 antibody is tislelizumab.

[0011] The present invention also provides TREM2 + MARCO +Application of TAMs subset inhibitors in the preparation of drugs for improving cancer immunotherapy response.

[0012] Preferably, the cancer is bladder cancer.

[0013] Preferably, the immunotherapy refers to immunotherapy using PD1 antibodies.

[0014] Preferably, the PD1 antibody is tislelizumab.

[0015] The present invention also provides a pharmaceutical composition comprising TREM2 + MARCO + TAMs subset inhibitors and anticancer drugs.

[0016] The present invention also provides the use of the above-mentioned pharmaceutical composition in preparing anti-cancer drugs.

[0017] Preferably, the anticancer drug is an anti-bladder cancer drug.

[0018] Beneficial effect: The present invention uses CD8 + Using T cell terminal exhaustion and bladder cancer ICB non-response as the entry point, single-cell RNA sequencing combined with spatial transcriptome sequencing identified a new TREM2 for the first time + MARCO + TAMs subsets and confirmed their relationship with terminally exhausted CD8 + High enrichment of T cells is associated with ICB non-response in bladder cancer. Further in vivo functional experiments confirmed that conditional knockout of TREM2 + MARCO + TAMs subsets reverse CD8 + Terminal depletion of T cells can greatly improve the response to ICB treatment of bladder cancer, which will provide a new target for the development of immunotherapy drugs for bladder cancer and is expected to provide a new treatment strategy for patients who do not respond to immunotherapy for solid malignancies.

[0019] It can be seen that further integration of TREM2 + MARCO + TAMs subpopulations can be used as drug targets to screen and prepare drugs for improving the immunotherapy response of bladder cancer; TREM2 + MARCO + TAMs subset inhibitors can inhibit TREM2 + MARCO + Furthermore, TREM2 can be used to regulate the expression of TAMs subpopulations, thereby improving the response to bladder cancer immunotherapy. + MARCO +The TAMs subgroup inhibitor and anticancer drug combination is used to improve the immunotherapy response of bladder cancer, thereby improving the therapeutic effect of bladder cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Identification of highly enriched TREM2 in ICB-unresponsive bladder cancer using single-cell sequencing combined with spatial transcriptome sequencing + MARCO + TAMs subpopulation experimental results.

[0021] Figure 2 Flow cytometry, multicolor tissue immunofluorescence, and multicenter clinical data analysis confirmed TRE2M + MARCO + TAMs subsets and CD8 + Figure 3. Experimental results showing the correlation between terminal T cell exhaustion and poor prognosis in patients with bladder cancer who do not respond to ICB.

[0022] Figure 3 Trem2-Marco - / - The spontaneous tumorigenesis mouse model of bladder cancer confirmed that conditional knockout of TRE2M + MARCO + TAMs inhibit CD8 + Figure 3. Experimental results showing improvement of ICB treatment response in bladder cancer by terminal depletion of T cells.

[0023] Figure 4 TREM2 + MARCO + The spontaneous tumorigenesis mouse model of bladder cancer using adoptive transfer of TAMs confirmed that adoptive transfer of TRE2M + MARCO + TAMs promote CD8 + Figure 3. Experimental results showing terminal exhaustion of T cells promoting non-response to ICB therapy in bladder cancer. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1 TREM2 + MARCO + TAMs subpopulation is highly enriched in bladder cancer that does not respond to ICB To explore the complex heterogeneity of the tumor immune microenvironment and its relationship with terminally exhausted CD8 + T cells (terminally exhausted CD8 + T cells, CD8 + To investigate the relationship between Tex-Term and bladder cancer, we performed single-cell RNA sequencing (n=10) and spatial transcriptome sequencing (n=8) on pre-treatment samples from patients with bladder cancer who received neoadjuvant ICB. Through unbiased analysis, we identified 63,854 high-quality cells and discovered 11 specific cell types. Notably, CD8 + Tex-Term showed a significant increase in bladder cancer patients who did not respond to ICB, and these cells highly expressed TOX, HAVCR2, and LAYN. In contrast, the number of these cells was lower in patients who responded to bladder cancer treatment. Spatial transcriptome sequencing analysis further revealed the spatial distribution of different cell types identified in single-cell sequencing data in tumor tissues and showed that immunosuppressive macrophages were more prominent in CD8 + The proportion and proximity scores of Tex-Term cells were significantly higher than those of other cell types. Further analysis showed that the immunosuppressive macrophage population macrophage C1 was mainly associated with CD8 + Tex-Term cells are spatially distributed adjacent to cell subsets. Further, in the Imvigor210 clinical trial and the extended analysis of the TCGA database, the proportion of macrophage C1 group was higher in bladder cancer patients who did not respond to ICB, and was closely related to CD8 + The enrichment of Tex-Term cells was significantly correlated. More importantly, the enrichment of macrophage C1 group was closely related to the poor prognosis of bladder cancer patients. In order to explore the membrane markers of macrophage C1 group, we determined through membrane protein analysis that TREM2 and MARCO are markers of macrophage C1 group, and the expression of these markers was significantly increased in bladder cancer patients who did not respond to ICB. The above results show that TREM2 + MARCO + The TAMs subpopulation marks a macrophage subpopulation that does not respond to bladder cancer ICB. Figure 1 .

[0026] Example 2 TREM2 + MARCO + TAMs are associated with non-response to ICB and poor prognosis in bladder cancer patients To further evaluate TREM2 + MARCO +The clinical significance of TAMs in bladder cancer immunotherapy was verified by collecting 163 multicenter clinical tissue samples of bladder cancer. Flow cytometry analysis confirmed that TREM2 was expressed in bladder cancer tissues that did not respond to ICB. + MARCO + The proportion of TAMs was significantly higher than that of responding patients. In addition, automated multiplex immunofluorescence (mIF) analysis showed that TREM2 + MARCO + TAMs are enriched in non-responsive bladder cancer tissues and associate with CD8 + Analysis of mIF images using HALO software further showed that TREM2 + MARCO + TAMs and CD8 + Tex-Term cells had more close contacts (within 20 μm), and these contacts were consistent at different distance thresholds (15 μm and 25 μm). + The degree of terminal exhaustion of T cells is also related to their interaction with TREM2 + MARCO + The survival analysis results showed that TREM2 + MARCO + The enrichment of TAMs was associated with shorter progression-free survival and overall survival in bladder cancer patients. Univariate and multivariate Cox regression analysis validated TREM2 + MARCO + TAMs infiltration has independent prognostic significance for overall survival and progression-free survival in bladder cancer patients. In addition, Imvigor210 and TCGA databases validated TREM2 + MARCO + TAMs infiltration scores are higher in bladder cancer patients who do not respond to immunotherapy and are associated with CD8 + These results suggest that TREM2 is positively correlated with the degree of Tex-Term invasion and is positively correlated with poor prognosis in patients with bladder cancer. + MARCO + TAMs subsets and CD8 + The infiltration of Tex-Term cells is closely related to the non-response of bladder cancer to ICB. Figure 2 .

[0027] Example 3 Construction of conditional knockout of TREM2 + MARCO + Spontaneous tumorigenesis mouse model of bladder cancer by TAMs and TREM2 + MARCO + TAMs drive CD8+ Terminal T cell exhaustion and nonresponse to ICB therapy in bladder cancer We plan to further evaluate TREM2 by constructing a transgenic mouse model + MARCO + TAMs regulate CD8 in vivo + We first purchased immune-competent C57BL / 6 mice (4-5 weeks old, weighing 18-20 g) and knocked in Marco in C57BL / 6 mice using CRISPR / Cas9 gene editing technology. flox / flox Gene. The tail DNA was detected by PCR to screen the offspring carrying the Marco allele and then compared with the constructed Trem2 cre Mice were crossed to further generate Trem2 cre MARCO flox / clox The offspring of the knockout gene expression of Marco in Trem2-expressing macrophages. - / - A 0.75% chemical carcinogen (N-butyl-N-(4-hydroxybutyl)-nitrosamine, BBN) was added to the drinking water of transgenic mice for 6 months to induce spontaneous bladder cancer in mice. TREM2 was isolated from spontaneous bladder cancer tissues. + TAMs were tested by PCR with specific primers to verify the effect of conditional knockout. The bladder tumor was then partially surgically removed and the tissue was stained by IHC. PD-L1-positive mice were treated with αPD-1 twice a week for three weeks. Bladder tumor volume was monitored by magnetic resonance imaging, and tissue was retained for subsequent immune cell detection and sequencing to evaluate the knockout of TREM2. + MARCO + The impact of TAMs on the immune microenvironment of bladder cancer and the efficacy of immunotherapy.

[0028] After constructing a transgenic mouse model of spontaneous bladder cancer, magnetic resonance imaging results showed that conditional knockout of TREM2 + MARCO + TAMs significantly reduced tumor growth and volume, accompanied by a significant decrease in the number and incidence of metastatic lymph nodes and prolonged survival. + MARCO + TAMs significantly reduced TIM3 expression in spontaneously formed bladder cancer tissues in mice + TOX + CD8 + The proportion of T cells confirmed TREM2+ MARCO + TAMs exacerbate CD8 + We then evaluated the expression of TREM2 + MARCO + Whether TAMs knockout affects the response of bladder cancer to ICB. The results showed that compared with the control group, conditional knockout of TREM2 + MARCO + TAMs enhanced the effect of αPD1 treatment in inhibiting tumor growth. In addition, compared with control mice, TREM2 + MARCO + TAMs conditional knockout mice have a lower incidence of lymph node metastasis and longer survival. These results suggest that TREM2 + MARCO + TAMs induce CD8 + Terminal exhaustion of T cells and suppression of anti-tumor CD8 + T cell response to PD-1 inhibitors. The above experimental results are shown in Figure 3 .

[0029] Example 4 TREM2 + MARCO + Construction of TAM adoptive transfer model confirmed TREM2 + MARCO + The full role of TAMs in ICB non-response in bladder cancer To further investigate TREM2 + MARCO + TAMs induce CD8 + Given the full role of T cell terminal exhaustion and resistance to ICB therapy, we designed an ICB-based TREM2 + MARCO + TAM adoptive transfer mouse model, in which selected TAM subsets from donor mice were transferred into bladder cancer of recipient mice to evaluate their effects on CD8 + Effect of T cells on the response to PD-1 inhibitors. Magnetic resonance imaging showed that compared with the control group, the injection of TREM2 + MARCO + TAMs significantly promoted tumor growth. TREM2 was associated with an increased incidence of metastatic lymph nodes and a shortened survival time. + MARCO + Adoptive transfer of TAMs significantly increased the expression of CD8 + Terminal exhaustion of T cells. + MARCO +-d TAMs compared to TREM2 + MARCO + TAMs and CD8 + T cells are also spatially distributed more proximal within the tumor immune microenvironment. Importantly, TREM2 + MARCO + TAMs significantly inhibited the inhibitory effect of PD-1 inhibitor treatment on tumor growth, accompanied by a higher rate of lymph node metastasis and a shorter survival time. These results confirm that TREM2 + MARCO + TAMs exacerbate CD8 + The terminal exhaustion of T cells mediates resistance to ICB therapy in bladder cancer. Figure 4 .

[0030] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0031] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. TREM2 + MARCO + Application of TAMs subpopulations as drug targets in screening or preparing drugs that improve cancer immunotherapy responses.

2. The use according to claim 1, characterized in that: The cancer is bladder cancer.

3. The use according to claim 1, characterized in that: The immunotherapy refers to immunotherapy using PD1 antibodies.

4. The use according to claim 3, characterized in that: The PD1 antibody is tislelizumab.

5. TREM2 + MARCO + Application of TAMs subset inhibitors in the preparation of drugs for improving cancer immunotherapy response.

6. The use according to claim 5, characterized in that: The cancer is bladder cancer.

7. The use according to claim 5, characterized in that: The immunotherapy refers to immunotherapy using PD1 antibodies.

8. The use according to claim 5, characterized in that: The PD1 antibody is tislelizumab.

9. A pharmaceutical composition, characterized in that Contains TREM2 + MARCO + TAMs subset inhibitors and anticancer drugs.

10. Use of the pharmaceutical composition according to claim 9 in the preparation of anti-cancer drugs.

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