(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的回应是，我们的数据表明，肿瘤PD-L1(CD274，B7-H1)是肿瘤的主要调节因子 炎性浸润物。我们的初步数据显示，黑色素瘤PD-L1通过几种 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的新作用 肿瘤增殖信号、免疫杀伤敏感性、体内生长独立于抗肿瘤 免疫力和mTOR信号的调节。我们鉴定了细胞内的PD-L1，包括那些其表面 低表达或阴性，并发现与肿瘤PD-1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 回应。研究团队由肿瘤免疫疗法、肿瘤免疫学和PD-L1专家组成 在UTHSCSA和达特茅斯。我们专注于黑色素瘤是出于科学原因，并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。我们用 对照与PD-L1lo(ShRNA)B16在研究肿瘤PD-L1差异治疗结果的新模型中的作用 状态。我们由CRISPR生成PD-L1KO B16，用于非常详细的后续机制研究，并评估 如果PD-L1零状态与PD-L1L0相比，对治疗有不同的影响。效果也将在移植后进行测试 BRAFV600E突变的D4M黑色素瘤(PD-L1+)基因工程化为PD-L1LO和PD-L1KO，诱导小鼠 BRAFV600E黑色素瘤，以及BRAF/PTEN与PD-L1KO BRAF/PTEN小鼠的同基因皮肤移植。 目的2检测肿瘤PD-L1驱动的mTOR信号对TIL和免疫治疗反应的影响。我们将测试 PD-L1KO、PD-1KO及双KO黑色素瘤细胞用于mTOR信号、TIL及治疗效果。单元格将是 针对mTORC1/2中的缺陷进行机械研究，并辅以mTOR抑制剂治疗。 我们将使用仅表达细胞质的工程化肿瘤与仅表达细胞表面的PD-L1，来定义新的， 细胞内PD-L1信号。在已知PD-1信号位点有突变的构建物将被工程到这些 以全面了解PD-L1/PD-1相互作用。 目的3明确细胞内源性PD-L1在人黑色素瘤中的作用。我们使用定义明确的人类黑色素瘤株系 碱基Pd-L1+和/或Pd-1+和/或BRAFV600E突变。我们将使用人类媒介来击倒或 敲除PD-L1、PD-1和mTORC1/2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。对NSG小鼠的体内效应将进行评估。主要 将对人类黑色素瘤株进行研究，以补充长期株系的数据。