Develop Conditionally Armored CAR Macrophage Therapy for Pancreatic Cancer

总结/摘要 胰腺导管腺癌（PDAC）是最致命的癌症之一，治疗选择有限。关于 95%的PDAC患者携带致癌突变KRAS（KRAS*），该突变可促进胰腺癌的发生， PDAC维护所需。考虑到KRAS* 现在可以被药物治疗，治疗耐药发生在几种 临床前模型和临床试验，表明需要联合治疗才能实现持久的疾病 控制靶向KRAS* 可导致胰腺、结肠和直肠中肿瘤相关巨噬细胞（TAM）富集。 和肺癌模型，为嵌合抗原受体（CAR）巨噬细胞（Mφ）的治疗提供了可能 和Mφ介导的促炎性细胞因子的癌症特异性递送。然而，移植的Mφ 尽管大多数剩余的Mφ位于肿瘤中，但易于在肝、肺和脾中蓄积， 可能通过条件性基因表达系统解决的日益增加的安全问题。前提是 肿瘤条件性表达的细胞因子装甲CAR Mφs（ca-CAR-Ms）可能与KRAS* 抑制剂协同作用 （KRASi）有效抑制原发性肿瘤生长、转移和肿瘤复发并延长生存期 在PDAC自发转移小鼠模型中。拟定研究的成功结果将产生第一个 ca-CAR-M疗法治疗PDAC，为KRAS* PDAC患者的临床试验铺平道路，并提供 为其他Mφ富集的癌症开发ca-CAR-Ms的适用方法。目标1.优化细胞培养和 用于过继性巨噬细胞治疗的工程平台。原代Mφ在体外是非增殖性的，并且 供体Mφs在健康器官中的蓄积是一种潜在的安全风险。目标1a将实现Mφ的体外扩增 通过遗传或化学筛选Mφ自我更新调节剂。目的1b将限制供体Mφs的肿瘤外活性 通过使用双氧传感开关和鉴定TAM特异性基因与组织驻留Mφ， 启动子/增强子将用于肿瘤特异性基因表达。目标2.将CAR Mφ设计为目标PDAC。 有效抗原特异性激活巨噬细胞和细胞毒作用的CAR综合优化 缺少Mφs。目的2a将设计和优化CAR的组成蛋白结构域，以指导Mφ活性针对 PDAC。目的2b将确定CAR-M作为KRASi辅助治疗在PDAC模型中的肿瘤杀伤作用。 目的2c将剖析CAR激活如何重新编程Mφ转录、分泌和通路的机制 转导以引起杀肿瘤作用。目标3.设计条件性装甲Mφ触发杀肿瘤 免疫力Mφ是肿瘤归巢的，因此通过Mφ递送促炎性细胞因子可以减少全身炎症。 毒性，同时引起强抗肿瘤免疫应答。目的3a将开发安全有效的细胞因子装甲 通过细胞因子筛选和条件性转基因表达系统治疗Mφ。目标3b将评估 通过最佳CAR构建体、最佳候选细胞因子或两者作为靶细胞因子改造的Mφ的肿瘤杀伤活性。 KRASi在各种PDAC模型中辅助治疗。目的3c将描述细胞因子铠装Mφ在细胞凋亡中的作用。 通过单细胞RNA测序和免疫表型分析肿瘤微环境重塑。

SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with limited therapeutic options. About 95% PDAC patients harbor oncogenic mutant KRAS (KRAS*) that promotes pancreatic carcinogenesis and is required for PDAC maintenance. Given that KRAS* can be druggable now, therapy resistance occurs in several pre-clinical models and clinical trials, suggesting that therapy combination is required to achieve durable disease control. Targeting KRAS* results in enrichment of tumor associated macrophages (TAMs) in pancreatic, colon and lung cancer models, offering opportunities for chimeric antigen receptor (CAR) macrophage (Mφ) therapy and for Mφ-mediated cancer-specific delivery of pro-inflammatory cytokines. However, transplanted Mφs were prone to accumulate in liver, lung, and spleen despite that most of the remaining Mφs were localized in tumors, rising safety concerns that might be addressed by conditional gene expression systems. The hypothesis is that tumor-conditionally expressed cytokine armored CAR Mφs (ca-CAR-Ms) may synergize with KRAS* inhibitors (KRASi) to effectively suppress primary tumor growth, metastasis, and tumor recurrence and to prolong survival in PDAC spontaneous metastasis mouse models. A successful outcome of proposed study will generate the first ca-CAR-M therapy for PDAC treatment, pave the way for clinical trials in KRAS* PDAC patients and provide applicable methods to develop ca-CAR-Ms for other Mφ-enriched cancers. Aim 1. Optimize cell culture and engineering platforms for adoptive macrophage therapy. Primary Mφs are non-proliferative in vitro, and accumulation of donor Mφs in healthy organs is a latent safety risk. Aim 1a will enable in vitro expansion of Mφs by genetic or chemical screening of Mφ self-renewal regulators. Aim 1b will limit off-tumor activity of donor Mφs by employing dual oxygen-sensing switch and identifying TAM-specific genes versus tissue-resident Mφs, whose promoter/enhancers will be used for tumor-specific gene expression. Aim 2. Design CAR Mφs to target PDAC. The comprehensive CAR optimization for effective antigen-specific activation of phagocytosis and cytotoxicity in Mφs is lacking. Aim 2a will design and optimize constituent protein domains of CAR to direct Mφ activities against PDAC. Aim 2b will determine the tumoricidal effect of CAR-Ms as an adjuvant therapy of KRASi in PDAC models. Aim 2c will dissect mechanisms how CAR activation reprograms Mφ transcription, secretion, and pathway transduction to elicit tumoricidal effect. Aim 3. Engineer conditionally armored Mφs to trigger tumoricidal immunity. Mφs are tumor-homing, so delivery of pro-inflammatory cytokines by Mφs may reduce systemic toxicity while elicit strong anti-tumor immune response. Aim 3a will develop safe and effective cytokine armored Mφ therapy by cytokine screening and employing conditional transgene expression system. Aim 3b will assess tumoricidal activity of Mφs engineered by the optimal CAR construct, the top cytokine candidate or both as an adjuvant therapy of KRASi in various PDAC models. Aim 3c will delineate the role of cytokine armored Mφs in the tumor microenvironment remodeling by single cell RNA sequencing and immunophenotyping.