(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.

我们对PQ3做出了响应，我们的数据表明肿瘤PD-L1（CD274，B7-H1）是肿瘤的主要调节剂 炎症性浸润。我们的初步数据表明，黑色素瘤PD-L1通过几个调节 以前未知的肿瘤内在和外部机制。我们定义了肿瘤内部PD-L1的新作用 对肿瘤增殖的信号传导，对免疫杀死的敏感性，在体内生长与抗肿瘤无关 免疫力和MTOR信号的调节。我们确定了细胞内PD-L1，包括那些表面的 表达是低或阴性的，并确定了与肿瘤PD-1的相互作用。我们假设黑色素瘤 固有的PD-L1驱动信号，尤其是MTOR信号，改变了肿瘤的进展和处理 回答。研究小组完成了肿瘤免疫疗法，肿瘤免疫学和PD-L1专家 在Uthscsa和Dartmouth。出于科学原因和我们的专业知识，我们专注于黑色素瘤。 AIM 1定义肿瘤PD-L1如何改变肿瘤免疫浸润和免疫疗法反应。我们使用 对照与PD-L1LO（SHRNA）B16在一种新型模型中，研究肿瘤PD-L1的差异治疗结果 地位。我们通过CRISPR生成了PD-L1KO B16，用于高度详细的后续机械研究，并评估 如果PD-L1无效状态对治疗与PD-L1LO的影响不同。效果也将在移植中测试 在具有诱导的小鼠中，BRAFV600E突变的D4M黑色素瘤（PD-L1+）设计为PD-L1LO和PD-L1KO BRAFV600E黑色素瘤，以及来自BRAF/PTEN与PD-L1KO BRAF/PTEN小鼠的皮肤的同性皮肤移植物。 AIM 2测试肿瘤PD-L1驱动的MTOR信号对TIL和免疫疗法反应的影响。我们将测试 PD-L1 KO，PD-1 KO和双KO黑色素瘤细胞用于MTOR信号，TIL和治疗效果。细胞将是 通过MTOR抑制剂治疗完成的机械研究的MTORC1/2缺陷设计。 我们将使用表达仅细胞质与仅细胞表面PD-L1的工程肿瘤来定义新颖的肿瘤， 细胞内PD-L1信号。在已知的PD-1信号站点中具有突变的构建体将被设计为这些 肿瘤以完全了解PD-L1/PD-1相互作用。 AIM 3定义了人类黑色素瘤中细胞中性PD-L1的作用。我们使用定义明确的人黑色素瘤系 是基本的PD-L1+和/或PD-1+和/或BRAFV600E突变。我们将使用人类向量击倒或 敲出PD-L1，PD-1和MTORC1/2基因。体外评估对增生的影响，对 将评估MTOR抑制剂，αPD-L1和αPD-1。将评估NSG小鼠的体内效应。基本的 人类黑色素瘤系将研究到长期线的完成数据。