(PQ#3) Novel tumor intrinsic PD-L1 signals direct tumor immune cell infiltration

（PQ

• 批准号: 9307468
• 负责人: Tyler J. Curiel
• 金额: $ 65.08万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9307468
• 关键词: Affect; Antibodies; Autologous; Cell Proliferation; Cell surface; Cells; Cellular biology; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Cytoplasm; Data; Defect; Engineering; FDA approved; FRAP1 gene; Funding; Genes; Growth; Human; Immune; Immune Evasion; Immune response; Immunology; Immunotherapy; In Vitro; Infiltration; Inflammatory Infiltrate; Joints; Knock-out; Knowledge; Lead; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Mediating; Melanoma Cell; Modality; Modeling; Molecular Medicine; Mus; Mutate; Mutation; Outcome; PDCD1LG1 gene; Pathway interactions; Patient-Focused Outcomes; Patients; Pharmacotherapy; Positioning Attribute; Pre-Clinical Model; Publications; Reagent; Regulation; Research; Research Personnel; Resistance; Signal Transduction; Site; Skin graft; Study models; Surface; T-Lymphocyte; Testing; Transplantation; Treatment outcome; Tumor Cell Line; Tumor Immunity; Validation; base; cancer therapy; chemokine; clinical translation; follow-up; human tissue; humanized mouse; immune clearance; improved; in vivo; inhibitor/antagonist; insight; killings; knock-down; mTOR Inhibitor; melanoma; mouse model; neoplasm immunotherapy; neoplastic cell; novel; novel marker; predicting response; predictive of treatment response; response; small hairpin RNA; success; trafficking; transcriptome sequencing; treatment effect; treatment response; tumor; tumor growth; tumor immunology; tumor microenvironment; tumor progression; vector

We respond to PQ3 with our data showing that tumor PD-L1 (CD274, B7-H1) is a major regulator of tumor inflammatory infiltrates. Our preliminary data show that melanoma PD-L1 regulates TIL through several previously unknown tumor-intrinsic and extrinsic mechanisms. We define novel effects of tumor intrinsic PD-L1 signaling on tumor proliferation, sensitivity to immune killing, in vivo growth independent of anti-tumor immunity, and regulation of mTOR signals. We identified intracellular PD-L1, including those whose surface expression is low or negative, and identified interactions with tumor PD-1. We hypothesize that melanoma intrinsic PD-L1-driven signals, particularly mTOR signals, alter tumor progression and treatment responses. The research team is comprised of tumor immunotherapy, tumor immunology and PD-L1 experts at UTHSCSA and Dartmouth. We focus on melanoma for scientific reasons and based on our expertise. Aim 1 Define how tumor PD-L1 alters tumor immune infiltrates and immunotherapy responses. We use control versus PD-L1lo (shRNA) B16 in a novel model to study differential treatment outcomes by tumor PD-L1 status. We generated PD-L1KO B16 by CRISPR for highly detailed follow up mechanistic studies, and to assess if PD-L1 null status differentially affects treatment versus PD-L1lo. Effects will also be tested in transplanted BrafV600E mutated D4M melanoma (PD-L1+) engineered to be PD-L1lo and PD-L1KO, in mice with induced BrafV600E melanomas, and in syngeneic skin grafts of skin from Braf/Pten versus PD-L1KO Braf/Pten mice. Aim 2 Test tumor PD-L1-driven mTOR signal effects on TIL and immunotherapy responses. We will test PD-L1 KO, PD-1 KO and double KO melanoma cells for mTOR signals, TIL and treatment effects. Cells will be engineered for defects in mTORC1/2 for mechanistic studies, complemented with mTOR inhibitor treatments. We will use engineered tumors that express cytoplasm-only versus cell surface-only PD-L1, to define novel, intracellular PD-L1 signals. Constructs with mutations in known PD-1 signal sites will be engineered into these tumors for a complete understanding of PD-L1/PD-1 interactions. Aim 3 Define cell-intrinsic PD-L1 effects in human melanoma. We use well-defined human melanoma lines that are basal PD-L1+ and/or PD-1+ and/or BrafV600E mutated. We will use human vectors to knock down or knock out PD-L1, PD-1 and mTORC1/2 genes. In vitro assessments of effects on proliferation, responses to mTOR inhibitors, αPD-L1 and αPD-1 will be assessed. In vivo effects in NSG mice will be assessed. Primary human melanoma lines will be studied to complement data from long-term lines.

我们回应PQ 3，我们的数据显示肿瘤PD-L1（CD 274，B7-H1）是肿瘤的主要调节因子， 炎性浸润我们的初步数据显示，黑色素瘤PD-L1通过几种途径调节TIL， 以前未知的肿瘤内在和外在机制。我们定义了肿瘤内源性PD-L1的新效应， 对肿瘤增殖的信号传导、对免疫杀伤的敏感性、不依赖于抗肿瘤的体内生长 免疫和调节mTOR信号。我们鉴定了细胞内PD-L1，包括那些表面 表达是低的或阴性的，并且鉴定了与肿瘤PD-1的相互作用。我们假设黑色素瘤 内源性PD-L1驱动信号，特别是mTOR信号，改变肿瘤进展和治疗 应答研究团队由肿瘤免疫治疗、肿瘤免疫学和PD-L1专家组成 和达特茅斯的学生我们专注于黑色素瘤的科学原因和基于我们的专业知识。 目的1明确肿瘤PD-L1如何改变肿瘤免疫浸润和免疫治疗反应。我们使用 在新模型中对照与PD-L1 lo（shRNA）B16，以研究肿瘤PD-L1的差异治疗结果 status.我们通过CRISPR产生PD-L1 KO B16，用于高度详细的后续机制研究，并评估 如果PD-L1无效状态与PD-L1 lo相比对治疗的影响不同。效果也将在移植 BrafV 600 E突变的D4 M黑色素瘤（PD-L1+）在诱导的小鼠中被工程化为PD-L1 lo和PD-L1 KO BrafV 600 E黑色素瘤，以及Braf/Pten与PD-L1 KO Braf/Pten小鼠的同基因皮肤移植物。 目的2检测肿瘤PD-L1驱动的mTOR信号对TIL和免疫治疗应答的影响。我们将测试 PD-L1 KO、PD-1 KO和双KO黑色素瘤细胞的mTOR信号、TIL和治疗效果。细胞将被 针对mTOR 1/2的缺陷进行工程化，以进行机制研究，并辅以mTOR抑制剂治疗。 我们将使用仅表达细胞质与仅表达细胞表面的PD-L1的工程肿瘤来定义新的， 细胞内PD-L1信号。在已知的PD-1信号位点中具有突变的构建体将被工程化到这些细胞中。 PD-L1/PD-1相互作用的完整理解。 目的3明确PD-L1在人黑色素瘤中的细胞内源性作用。我们使用定义明确的人类黑色素瘤细胞系 基础PD-L1+和/或PD-1+和/或BrafV 600 E突变。我们将使用人类载体来击倒或 敲除PD-L1、PD-1和mTORC 1/2基因。体外评估对增殖的影响，对 将评估mTOR抑制剂αPD-L1和αPD-1。将评估NSG小鼠中的体内效应。初级 将研究人黑素瘤细胞系以补充来自长期细胞系的数据。