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The role of the CD58:CD2 axis in cancer immune evasion and resistance to immunotherapy

CD58:CD2轴在癌症免疫逃避和免疫治疗抵抗中的作用

基本信息

批准号：
10671582
负责人：
Benjamin Izar
金额：
$ 39.94万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10671582
关键词：
Address Adoptive Cell Transfers Antibodies Autologous Binding Proteins Biochemical Biological Assay Biopsy CD28 gene CD58 Antigens CD58 gene CD8-Positive T-Lymphocytes CD8B1 gene CRISPR screen Cells Cellular Indexing of Transcriptomes and Epitopes by Sequencing Clinical Clonal Expansion Co-Immunoprecipitations Coculture Techniques Collection Cytolysis Data Analyses Defect Down-Regulation Ecosystem Engineering Exclusion Flow Cytometry Genes Genetic Genomics Genotype Goals Homologous Gene Immune Immune Evasion Immune checkpoint inhibitor Immunofluorescence Immunologic Immunotherapy Impairment Implant In Vitro Infiltration Lymphocyte Activation Lymphocytic Infiltrate Machine Learning Macrophage Maintenance Malignant Neoplasms Mass Spectrum Analysis Measurement Mediating Melanoma Cell Metastatic Melanoma Methodology Methods Modeling Molecular Monoclonal Antibodies Mus Pathway interactions Patients Peripheral Blood Mononuclear Cell Production Proliferating Proteins RNA Recycling Resistance Resolution Role Sampling Scientist Signal Transduction Skin Cancer Survivors T cell infiltration T-Cell Activation T-Cell Receptor T-Lymphocyte Testing Therapeutic Tissues Tumor Escape Tumor Immunity Up-Regulation Western Blotting Work Yin anti-PD-1 anti-PD1 antibodies anti-PD1 therapy cancer cell cancer immunotherapy cancer infiltrating T cells cell killing cohort cytokine data tools drug development genome-wide humanized mouse immune resistance immunological synapse in vivo in vivo Model innovation melanoma mouse model multimodality neoplasm resource neoplastic cell novel preservation progenitor programmed cell death ligand 1 protein expression reconstitution resistance mechanism responders and non-responders response single-cell RNA sequencing targeted treatment therapeutic development tool tumor virtual

项目摘要

PROJECT SUMMARY Immune checkpoint inhibitors (ICI), such as anti-PD-1 antibodies (aPD1), enable CD8+ T cell-mediated anti- tumor immunity and produce durable responses in a portion of patients with metastatic melanoma. However, most patients do not benefit from ICI and the underlying mechanisms of resistance remain elusive. The overall goal of this work is to elucidate mechanisms of resistance to ICI mediated by cancer cells, and develop novel molecular or immune-based therapies to overcome resistance. Prior work demonstrated that expression of co- stimulatory protein CD28 on CD8+ T cells is required for efficacy of aPD1, however, we show that expression of CD28 is frequently absent in tumor-infiltrating CD8+ T lymphocytes (TILs), including those with preserved progenitor function. In contrast, all TILs maintain expression of CD2, the most potent alternative co-stimulatory protein. Indeed, we find that patient-derived CD28- TILs had preserved capacity for proliferation, clonal expansion, cytokine production and lysis of autologous cancer cells. The ligand of CD2 is CD58, and we discovered that CD58 was expressed on melanoma cells. The role of the CD58:CD2 axis in anti-tumor immunity, and response and resistance to ICI is unexplored. In preliminary studies, we established patient-derived pairs of melanoma cells and autologous TILs. We developed Perturb-CITE-seq, a novel method enabling pooled CRISPR-Cas9 screens with single-cell RNA-seq and protein readouts. We performed a Perturb-CITE-seq screen in patient models, perturbing hundreds of genes with putative roles in ICI resistance, followed by co-culture with TILs and profiled survivor cells that escaped T cell killing. We recovered known mechanisms of cancer immune evasion and novel ones, including loss/downregulation of CD58. In humanized mouse models, loss of CD58 in tumor cells resulted in impaired T cell infiltration and resistance to adoptive cell transfer (ACT). Additionally, we observed compensatory PD-L1 upregulation in CD58 loss. Using genome-scale CRISPR-Cas9 screens and co- immunoprecipitation mass spectrometry screens, we identified a putative regulator of this interaction, CMTM6. Rescue of CD58 re-sensitized cancer cells to T cell killing, increased T cell activation, enhanced T cell infiltration in vivo, and abolished compensatory PD-L1 expression. Based on these findings, we will dissect the role of the CD58:CD2 axis in anti-tumor immunity, cancer immune evasion and resistance ICI resistance in three aims: i) to determine the mechanisms by which CD58 loss confers cancer immune evasion, ii) to determine the role of CMTM6 in regulating compensatory PD-L1 upregulation in CD58 loss, and iii) to determine whether aPD1 resistance is associated with an impaired CD58:CD2 axis in patients. We propose to systematically address these using genetic, biochemical, patient and in vivo models, and multi-modal single-cell genomics analyzed with novel machine-learning tools. We further test two therapies that overcome resistance to ICI due to defects in the CD58:CD2 axis. Upon completion, this work will shed light on the role of the CD58:CD2 pathway in cancer and cancer immunotherapy, and may provide novel avenues for therapeutic development.

项目摘要免疫检查点抑制剂（ICI），如抗PD-1抗体（aPD 1），能够使CD 8 + T细胞介导的抗PD-1抗体（aPD 1）成为可能。肿瘤免疫，并在部分转移性黑色素瘤患者中产生持久的反应。然而，在这方面，大多数患者不能从ICI中获益，并且耐药的潜在机制仍然难以捉摸。整体本工作的目的是阐明癌细胞介导的ICI抗性机制，并开发新的分子或免疫疗法来克服耐药性。先前的工作表明，表达的co- CD 8 + T细胞上刺激性蛋白CD 28是aPD 1功效所必需的，然而，我们显示了 CD 28在肿瘤浸润性CD 8 + T淋巴细胞（TIL）中经常缺失，包括那些保留了CD 28的TIL。祖细胞功能相比之下，所有TIL都维持CD 2的表达，CD 2是最有效的替代共刺激因子。蛋白事实上，我们发现患者来源的CD 28- TILs具有保留的增殖能力，克隆能力，自体癌细胞的扩增、细胞因子产生和裂解。CD 2的配体是CD 58，我们发现CD 58在黑色素瘤细胞上表达。CD 58：CD 2轴在抗肿瘤免疫中的作用，对ICI的反应和抗性尚未探索。在初步研究中，我们建立了患者来源的黑色素瘤细胞和自体TIL。我们开发了Perturb-CITE-seq，这是一种新的方法， CRISPR-Cas9使用单细胞RNA-seq和蛋白质读数进行筛选。我们进行了Perturb-CITE-seq筛选在患者模型中，干扰数百个在ICI抗性中具有假定作用的基因，然后与 TIL和剖析逃脱T细胞杀伤的存活细胞。我们恢复了已知的癌症免疫机制逃避和新的，包括损失/下调的CD 58。在人源化小鼠模型中，肿瘤细胞导致受损的T细胞浸润和对过继细胞转移（ACT）的抗性。另外我们在CD 58损失中观察到代偿性PD-L1上调。使用基因组规模的CRISPR-Cas9筛选和联合通过免疫沉淀质谱筛选，我们确定了这种相互作用的假定调节因子CMTM 6。拯救CD 58使癌细胞对T细胞杀伤重新敏感，增加T细胞活化，增强T细胞浸润在体内，并消除代偿性PD-L1表达。基于这些发现，我们将剖析 CD 58：CD 2轴在抗肿瘤免疫、肿瘤免疫逃避和抗ICI抗性中有三个目的：i）确定CD 58缺失赋予癌症免疫逃避的机制，ii）确定 CMTM 6在调节CD 58损失中的代偿性PD-L1上调中的作用，以及iii）确定aPD 1 耐药与患者中受损的CD 58：CD 2轴相关。我们建议系统地解决这些使用遗传，生物化学，患者和体内模型，以及多模态单细胞基因组学分析 with novel新machine机learning学习tools工具.我们进一步测试了两种克服由于缺陷而对ICI产生耐药性的疗法在CD 58：CD 2轴上。完成后，这项工作将阐明CD 58：CD 2通路在癌症中的作用。和癌症免疫疗法，并可能为治疗开发提供新的途径。