The inhibition of the CD161-CLEC2D interaction as immune checkpoint blockade in glioblastoma multiforme

多形性胶质母细胞瘤中 CD161-CLEC2D 相互作用的抑制作为免疫检查点阻断

• 批准号: 419826340
• 负责人: Dr. Sascha Marx
• 金额: --
• 依托单位: Department of Cancer Immunology and Virology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/419826340?language=en
• 关键词: inhibition; CD161; CLEC2D; interaction; immune

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Despite standard treatment the mean overall survival is frequently only a little over 1 year. Immunotherapy is not as well developed in GBM compared to other malignancies outside the central nervous system (CNS), because there is lack of knowledge about immunological processes in GBM. The goal of the present project is to close gaps in the understanding of T-cell function in GBM. Preliminary single cell RNA-sequencing data could identify clonally expanded T cell populations based on their TCR chain sequences. In this analysis it was seen that the CD161 (KLRB1) is overexpressed by such T-cells. The CD161 ligand, CLEC2D, is expressed at the cell surface of human GBM cell lines. CD161 is expressed by CD4+ und CD8+ T-cells with an effector gene expression and inactivation of the KLRB1 gene in primary human T cells greatly enhances their effector function in a humanized mouse model of GBM. It is therefore hypothesized that the CD161 – CLEC2D pathway inhibits the anti-tumor function of both CD8 and CD4 effector T cell populations in GBM.The current project aims to study the CD161-CLEC2D interaction in human GBM in more detail to accelerate a translation into the clinic. Initially a genetic approach will be used to study this inhibitory receptor – ligand pair by inactivating the KLRB1 gene in primary T-cells or the CLEC2D gene in GBM cells. Blocking monoclonal antibodies specific for human CD161 and CLEC2D will also be generated to examine the therapeutic potential of antibody-mediated inhibition of this pathway. In vivo experiments will be conducted mainly in humanized mouse models, in which human T-cells (with GBM-specific CAR or TCR) will be injected in immunodeficient mice after stereotactic implantation of GBM-cells into the CNS. Furthermore, the therapeutic potential will be tested in fully immunocompetent mice harboring GBM. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing approaches will be used to define the functional changes in T cell populations when the CD161 – CLEC2D pathway is inhibited.Furthermore analysis of tumor-infiltrating T cells in GBM patients enrolled in a clinical trial (personalized neoantigen peptide vaccination plus PD-1 blockade) will be done. Tumors both at initial surgery and relapse will be investigated in order to determine how expression of these inhibitory receptors and their ligands changes following therapy. CD161 expression in T-cells (as well as other inhibitory receptors) and CLEC2D on tumor cells and myeloid cells (as well as other inhibitory ligands) will be addressed, given that T-cell exhaustion is accompanied with expression of these receptors and ligands. It is hypothesized that CD161 is an important inhibitory receptor for intra-tumoral T-cells in GBM and that inhibition of the CD161-CLEC2D pathway is able to enhance T-cell effector function in GBM.

胶质母细胞瘤多形（GBM）是成年人中最常见的脑肿瘤。尽管有标准处理，平均总生存期通常只有1年以上。与中枢神经系统（CNS）之外的其他疾病相比，GBM的免疫疗法在GBM中的发展不佳，因为GBM中缺乏有关免疫过程的知识。本项目的目的是弥合对GBM中T细胞功能的理解差距。初步的单细胞RNA-sequer-sequing数据可以根据其TCR链序列鉴定克隆扩展的T细胞种群。在此分析中可以看出，这种T细胞过表达CD161（KLRB1）。 CD161配体CLEC2D在人GBM细胞系的细胞表面表达。 CD161由CD4+ UND CD8+ T细胞具有效应子基因表达和在原代人T细胞中的KLRB1基因失活的表达，在GBM的人源性小鼠模型中极大地增强了其效应功能。因此，假设CD161 - CLEC2D途径抑制了GBM中CD8和CD4效应T细胞种群的抗肿瘤功能，目前的项目旨在研究人类GBM中CD161-CLEC2D相互作用，以更详细地加速转化为诊所。最初，一种遗传方法将通过在原代T细胞中灭活KLRB1基因或GBM细胞中的CleC2D基因来研究这种抑制性受体 - 配体对。还将生成针对人CD161和CLEC2D的阻断单克隆抗体，以检查抗体介导的该途径抑制的治疗潜力。体内实验将主要是在人源性小鼠模型中进行的，其中人类T细胞（带有GBM特异性CAR或TCR）将在立体定向植入GBM细胞后将其注入免疫缺陷的小鼠中。此外，将在具有GBM的完全免疫能力的小鼠中测试治疗潜力。 Flow cytometry, immunohistochemistry and single-cell RNA-sequencing approaches will be used to define the functional changes in T cell populations when the CD161 – CLEC2D pathway is inhibited.Furthermore analysis of tumor-infiltrating T cells in GBM patients enrolled in a clinical trial (personalized neoantigen peptide) vaccination plus PD-1 blockade) will be done.初始手术和继电器的肿瘤将进行研究，以确定治疗后这些抑制性受体及其配体的表达如何变化。鉴于这些受体和配体的表达可以实现T细胞的衰竭，将解决T细胞和髓样细胞（以及其他抑制性配体）上T细胞中的CD161表达（以及其他抑制性受体）和CLEC2D（以及其他抑制性配体）。假设CD161是GBM中肿瘤内T细胞的重要抑制受体，并且抑制CD161-CLEC2D途径能够增强GBM中T细胞效应的功能。