IGLL5 + Application of B cells as drug targets in screening or preparing drugs to improve cancer immunotherapy response

Abstract

The present invention relates to the field of biomedicine technology, and in particular to IGLL5 + The application of B cells as drug targets in screening or preparing drugs to improve cancer immunotherapy response. This invention takes the tertiary lymphoid structure and tumor immunotherapy as the entry point, and innovatively identifies a new tumor-related IGLL5 through single-cell sequencing combined with spatial transcriptome sequencing. + B cell subsets, and confirmed that they were negatively correlated with the tertiary lymphoid structure and immunotherapy response in bladder cancer tumors. Further in vivo functional experiments confirmed that IGLL5 + B cell subpopulations inhibit the formation of tertiary lymphoid structures, leading to bladder cancer non-responsiveness to immunotherapy. This will provide a new target for the development of bladder cancer immunotherapy drugs and is expected to provide a new treatment idea for patients who do not respond to immunotherapy for solid malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to IGLL5 + The application of B cells as drug targets in screening or preparing drugs to improve cancer immunotherapy response. Background Art

[0002] In the past decade, effective immunotherapy has revolutionized the treatment of malignant tumors, but only a small number of patients have achieved long-term tumor control. Recent studies have shown that the lack of tertiary lymphoid structure (TLS) leads to the failure of immunotherapy, which has prompted us to interpret the significance of intratumoral TLS in supporting anti-tumor immunity through multiple models: ① Speed: Rapid activation of effector T cells in situ in TLS bypasses lymph node trafficking; ② Efficiency: Local TLS promotes stronger immune response activation by providing more opportunities for cognate antigen presentation; ③ Maintenance: TLS-associated cytokine niches maintain cytotoxic CD8 + T cell homeostasis and function. However, not all tumors contain TLS, especially in patients who do not respond to immunotherapy, which may directly lead to their high metastasis rate and poor prognosis. Therefore, the current major challenge is to elucidate the regulatory mechanism of TLS formation and develop therapeutic strategies to induce TLS formation to assist immunotherapy of immune-inert tumors.

[0003] The current cutting-edge view is that, in addition to the local interaction of dendritic cells and T and B cells, the formation of TLS is highly dependent on the specialized neogenesis of high endothelial venules (HEV) to provide support for the aggregation of lymphocytes, but there is currently a lack of attention. Lymphotoxin β receptor (LTβR) mediates the non-canonical NF-κB signaling pathway and is a key evolutionary node in the functional phenotype of HEV. In vivo experiments reported that LTβR-specific knockout mice exhibited the collapse of HEV and the absence of TLS, resulting in a lack of adaptive immune response. However, it is worth noting that recent studies have shown that several vascular regulation strategies using LTβR agonist transfection cannot completely reverse the destruction of HEV and TLS in malignant tumors. These special evidences suggest that there may be unknown inhibitory ligands for LTβR, which may hinder the formation of HEV-mediated TLS and thus inhibit antitumor immune responses.

[0004] With the booming development of spatial transcriptomics, researchers have gradually found that tumor-infiltrating B cells show greater aggregation and consistency with TLS distribution compared with T cells and other cellular components, indicating that in TLS, persistent interactions between tumor-associated B cells and HEV are more likely to occur. In the tumor microenvironment, the mode of interaction between tumor-associated B cells and HEV is widely involved in the regulation of HEV phenotype and the formation of TLS. However, tumor-associated B cells show multifaceted heterogeneity in tissue distribution, cancer type preference, B cell receptor repertoire, and transcriptome gene profile. Well-differentiated effector B cells secrete high-affinity antibodies to help effector immune cells eliminate tumors, while some regulatory B cells secrete immunosuppressive factors to destroy anti-tumor immunity. Therefore, dissecting the phenotypic diversity and diversified effects of tumor-associated B cells is expected to improve immunotherapy responses.

[0005] There is a lack of molecular targeted drugs for bladder cancer immunotherapy resistance in clinical practice, and the effect on improving patient prognosis is poor; the main reason is that the microenvironmental characteristics and molecular mechanisms of bladder cancer immunotherapy non-response are not fully understood, and there is still a lack of effective diagnostic and therapeutic targets. Therefore, clarifying the microenvironmental characteristics and molecular mechanisms of bladder cancer immunotherapy non-response, and finding new therapeutic targets and strategies that can enhance the efficacy of bladder cancer immunotherapy have important application value 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 IGLL5 + The application of B cells as drug targets in screening or preparing drugs to improve cancer immunotherapy response.

[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 IGLL5 + The application of B cells as drug targets in screening or preparing drugs to improve cancer immunotherapy response.

[0008] Preferably, the cancer is bladder cancer.

[0009] Preferably, the immunotherapy refers to immunotherapy using PD1 antibodies or immunotherapy using a pharmaceutical composition containing PD1 antibodies.

[0010] Further preferably, the pharmaceutical composition containing PD1 antibody specifically comprises PD1 antibody, gemcitabine and cisplatin.

[0011] Most preferably, the PD1 antibody is tislelizumab.

[0012] The present invention also provides an IGLL5+ Use of B cell inhibitors in the preparation of drugs for improving cancer immunotherapy response.

[0013] Preferably, the cancer is bladder cancer.

[0014] More preferably, the immunotherapy refers to immunotherapy using PD1 antibodies or immunotherapy using a pharmaceutical composition containing PD1 antibodies.

[0015] Most preferably, the pharmaceutical composition containing PD1 antibody specifically includes PD1 antibody, gemcitabine and cisplatin.

[0016] The present invention also provides a pharmaceutical composition comprising IGLL5 + B cell inhibitors and anticancer drugs.

[0017] Preferably, the anticancer drug is a PD1 antibody.

[0018] Most preferably, the PD1 antibody is tislelizumab.

[0019] The present invention also provides an application of the above-mentioned pharmaceutical composition in preparing a drug with anti-cancer effect.

[0020] Preferably, the anticancer drug is an anti-bladder cancer drug.

[0021] Beneficial effects: This paper takes the tertiary lymphoid structure and tumor immunotherapy as the entry point, and innovatively identifies a new tumor-related IGLL5 through single-cell sequencing combined with spatial transcriptome sequencing. + B cell subsets, and confirmed that they were negatively correlated with the tertiary lymphoid structure and immunotherapy response in bladder cancer tumors. Further in vivo functional experiments confirmed that IGLL5 + B cell subpopulations inhibit the formation of tertiary lymphoid structures, leading to bladder cancer non-responsiveness to immunotherapy. This will provide a new target for the development of bladder cancer immunotherapy drugs and is expected to provide a new treatment idea for patients who do not respond to immunotherapy for solid malignant tumors.

[0022] It can be seen that further + B cells can be used as drug targets to screen and prepare drugs for improving the immunotherapy response of bladder cancer; at the same time, IGLL5 + B cell inhibitors, which can inhibit IGLL5 + Furthermore, IGLL5 + The B cell 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

[0023] Figure 1 Identification of IGLL5 for single-cell sequencing combined with spatial transcriptome sequencing + B cells and confirmed that they are highly enriched in bladder cancer tumor tissues lacking TLS.

[0024] Figure 2 Gene set-based spatial transcriptome sequencing mapping, immunofluorescence staining, and clinical data analysis confirmed IGLL5 + Experimental results showing that B cells are negatively correlated with TLS and immunotherapy response in bladder cancer tumors.

[0025] Figure 3 To construct humanized IGLL5 + Diagram of the experimental process for constructing a spontaneous tumor-forming mouse model of bladder cancer with overexpression of B cells.

[0026] Figure 4 3D immunofluorescence staining for tissue clearing confirmed the expression of IGLL5 in a spontaneous tumorigenesis mouse model of bladder cancer + Figure 3. Experimental results of B cells inhibiting the formation of TLS.

[0027] Figure 5 In vivo experiments confirmed that IGLL5 + Figure 3. Experimental results showing that B cells hinder immunotherapy response by inhibiting TLS formation. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1 IGLL5 + B cells are highly enriched in TLS-deficient bladder cancer tumor tissues We collected spatial transcriptome sequencing of 8 bladder cancer tumor tissue samples that received immunotherapy, and single-cell RNA sequencing of 14 bladder cancer tumor tissue samples that received immunotherapy. In the joint analysis, we revealed that B cells are the most abundant and spatially localized cell type in the tumor microenvironment TLS. Furthermore, we re-clustered all B cells into 8 different B cell subsets. Among them, representative B cell subsets corresponding to different maturation stages were identified, including CD24 + Naive B cells, EGR1 + Activated B cells, CD27 +Memory B cells, germinal center B cells, and plasma cells. Notably, we discovered a new subpopulation that had been largely uncharacterized before: the B cell subpopulation B_08, which had a significantly increased infiltration amount in TLS-deficient bladder cancer tumor tissues compared with TLS-rich bladder cancer tumor tissues. To further characterize this particular B cell subpopulation, we screened for the top 10 characteristic expressed genes encoding membrane-localized proteins in the B_08 subpopulation compared with other subpopulations by single-cell sequencing analysis. B cells were sorted separately from bladder cancer tumor tissues by flow cytometry and further validated using qRT-PCR analysis. It was found that IGLL5 was the most significantly upregulated gene in B cells derived from TLS-deficient bladder cancer tumor tissues, indicating that IGLL5 + B cells are significantly enriched in TLS-deficient bladder cancer tumor tissues. Figure 1 .

[0030] Example 2 IGLL5 + B cells are negatively correlated with intratumoral TLS and immunotherapy response in bladder cancer We then calculated the IGLL5 containing differentially expressed genes by analyzing single-cell RNA sequencing data. + B cell expression gene set; In addition, based on Cloupe software, combined with the regional characteristics of HE staining, we defined TLS and non-TLS regions, and performed differential gene expression analysis on spatial points marked as TLS and non-TLS through differential expression analysis and AUC verification to identify genes significantly upregulated in the TLS region and define the TLS imprinted gene set. Next, we respectively + The B cell gene set and TLS signature gene set were mapped to the spatial transcriptome sequencing results, and IGLL5 was confirmed + B cell subsets were significantly negatively correlated with TLSs. We also found that IGLL5 + B cells were mainly located outside TLS and were significantly negatively correlated with the presence of TLS. In addition, combined with the analysis of the patient's clinical pathological characteristics, IGLL5 was significantly increased in bladder cancer tumor tissues lacking TLS and not responding to immunotherapy, compared with bladder cancer tumor tissues enriched with TLS and responding to immunotherapy. + Importantly, statistical analysis combined with survival follow-up data from a multicenter clinical sample cohort showed that IGLL5 was significantly increased in bladder cancer patients receiving immunotherapy. + The higher the infiltration amount of B cell subsets, the worse the patient's prognosis. In addition, we also obtained the gene expression matrix and survival follow-up information of bladder cancer patients in the Imvigor210 immunotherapy public database. The correlation and survival analysis also confirmed that IGLL5 +B cell subsets were significantly negatively correlated with patients' overall survival (OS) and progression-free survival (PFS). Figure 2 .

[0031] Example 3 Spontaneous bladder cancer mouse model confirms IGLL5 + B cells inhibit the formation of TLS We plan to further evaluate IGLL5 by constructing a transgenic mouse model + B cells regulate the function of TLS and anti-tumor immunity in bladder cancer tumors. Since the IGLL5 gene is lacking in rodents, we first constructed the human IGLL5 conditional knock-in gene in C57BL / 6J mice using CRISPR / CAS-mediated genome editing technology. Mouse ROSA26 gene gRNA, donor vector containing human IGLL5 coding sequence, and Cas9 mRNA were co-injected into mouse fertilized eggs to produce targeted humanized IGLL5 knock-in offspring. F0 maternal mice were identified by PCR, followed by Sanger sequencing analysis and nucleic acid electrophoresis, and cultured into wild-type mice for germline transmission and F1 animal generation tests. Next, heterozygous mice were screened for hybridization to produce homozygous mice. Then, homozygous humanized IGLL5 knock-in mice were hybridized with homozygous Mb1-Cre mice to produce homozygous B cell conditional humanized IGLL5 conditional knock-in homozygous mice (IGLL5-Hu). Subsequently, IGLL5-Hu mice were exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) to establish a spontaneous bladder cancer mouse model for testing the efficacy of intratumoral TLS and immunotherapy. Figure 3 shown.

[0032] After constructing humanized IGLL5 + After establishing a spontaneous tumor-forming mouse model of bladder cancer with overexpression of B cells, we first removed the mouse bladder cancer tumor tissue and obtained primary tumor cells after dissociation. Flow cytometry confirmed that humanized IGLL5 + A large amount of IGLL5 is present in bladder cancer tumor tissues of mice with overexpression of B cells + IGLL5 was not detected in bladder cancer tumor tissues of wild-type mice + B cells, indicating that humanized IGLL5 +A transgenic mouse model with overexpression of B cells was successfully constructed. We further analyzed the intratumoral TLS by tissue clearing 3D staining. First, the mouse bladder cancer tumor tissue was fixed in PBS containing 4% paraformaldehyde and the tissue was cleared using the CUBIC-L reagent in the CUBIC-HV1 3D staining kit. Subsequently, the tissue was incubated with CD3 and CD79a primary antibodies and then stained with fluorescein-labeled secondary antibodies. All antibodies were stained in a 3D immunostaining buffer containing casein. Subsequently, the entire tissue was rinsed three times with 3D immunofluorescence staining wash buffer and then fixed in 1% formaldehyde solution. Images were captured using a laser scanning confocal microscope and then analyzed using Imaris 9.0 software. The results showed that compared with wild-type mice, humanized IGLL5 + TLS was significantly absent in bladder cancer tumor tissues of mice with B cell overexpression, confirming that IGLL5 + B cells inhibited the formation of TLS in tumors. Figure 4 .

[0033] Example 5 In vivo experiments confirm that IGLL5 + B cells impede immunotherapy response by inhibiting TLS formation We combined PD-1 antibody (Tislelizumab) with gemcitabine and cisplatin by tail vein injection into humanized IGLL5 + The researchers treated C57BL / 6J mice with spontaneous bladder cancer and wild-type mice with B cell overexpression. Dynamic monitoring by magnetic resonance imaging showed that the size of bladder tumors in wild-type mice was significantly reduced after treatment. In contrast, immunotherapy failed to reduce the expression of humanized IGLL5 lacking TLS. + In addition, PET-CT imaging confirmed that humanized IGLL5 + Overexpression of B cells significantly promoted tumor growth and lymph node metastasis, resulting in a significantly shortened survival of mice and a poor response to immunotherapy. These experimental results indicate that IGLL5 + B cells inhibit the formation of TLS in mouse tumors, hindering the response of bladder cancer to immunotherapy. Figure 5 .

[0034] 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.

[0035] 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. IGLL5 + The application of B cells as drug targets in screening or preparing drugs to improve cancer immunotherapy response.

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 or immunotherapy using a pharmaceutical composition containing PD1 antibodies.

4. The use according to claim 3, characterized in that: The pharmaceutical composition containing PD1 antibody specifically includes PD1 antibody, gemcitabine and cisplatin. 5.IGLL5 + Use of B cell inhibitors in the preparation of drugs for improving cancer immunotherapy responses.

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 or immunotherapy using a pharmaceutical composition containing PD1 antibodies.

8. The use according to claim 5, characterized in that: The pharmaceutical composition containing PD1 antibody specifically includes PD1 antibody, gemcitabine and cisplatin.

9. A pharmaceutical composition, characterized in that Contains IGLL5 + B cell inhibitors and anticancer drugs.

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

Images