Engineering next generation supercharged CAR T-cells against solid tumors

项目摘要 抗肿瘤细胞疗法已经彻底改变了癌症治疗，并具有治愈多种癌症的巨大潜力 类型目前的CAR-T治疗存在多个关键障碍，特别是在实体瘤中，包括T 细胞向癌部位运输，缺乏有效的癌细胞杀伤，毒性严重，免疫抑制强 在肿瘤微环境中，缺乏持久性。克服这些障碍对于实现全面 CAR-T的潜力为了克服这些挑战，重要的是要确定基因靶点， T细胞功能的特征，特别是来自在原代T细胞中进行的完全无偏的遗传筛选。我们有 最近开发了这种基于T细胞CRISPR的筛选方法。这些基因筛选， 严格选择T细胞的特性-包括增强的效力，持久性和体内 浸润/活性-已经鉴定了最大程度地增强所需T细胞特性的基因修饰。 我们在原代T细胞中进行了体外和体内CRISPR筛选的功能敲除和获得， 在动物肿瘤模型中，使用不同的 CAR-T（例如CD 19、CD 22和HER-2汽车）。我们发现了两种特别有效的基因修饰， 增强CART-Ts：1）PRODH 2过表达导致代谢加强和2）PRDM 1（外显子3）缺失 导致表观遗传调节。这一初步研究和方法为进一步增强铺平了道路。 我们的中心假设是，通过PRODH 2过表达的代谢促进来工程化CAR-T细胞， 以及通过PRDM外显子3缺失的表观遗传调控可以增强HER 2 CAR-T细胞性能， 增强体内抗肿瘤功效。为此，我们将利用我们的敲入/敲除（KIKO）方案来引入 CAR和基因修饰序列精确定位在TRAC基因座上，以产生四个HER 2 CAR-T：PRODH 2- OE、PRDM 1-KO、PRODH 2-OE+ PRDM 1-KO和对照CAR-T。体外表征（CAR水平） 表达、增殖、免疫表型、连续杀伤能力）以及体内评价（功效、CAR-1、CAR-2、 将使用至少两种癌细胞进行4种CAR-T中每一种的检测（例如，T浸润和持久性）。 线/小鼠模型。本提案中我们的载体和生产工艺与计划的GMP相同 临床方法，允许顺利过渡到临床开发。 如果成功，这项研究将确定我们的最佳表现HER 2 CART，并提供足够的数据来支持 II期SBIR提交，并允许通往IND和首次人体I期临床试验的路径，因此 为实体瘤患者提供上级CAR-T候选药物。从广义上讲，这项研究的主要目标是 是评估代谢增强和表观遗传重编程在组合时是否会导致更有效的CAR-T 相对于单独的修改。

Project summary Adoptive cell therapy has revolutionized cancer treatment and has immense potential to cure a variety of cancer types. Multiple critical barriers exist for current CAR-T therapy particularly in solid tumor, including insufficient T cells trafficking to the cancer site, lack of effective cancer cell killing, severe toxicity, strong immunosuppression in the tumor microenvironment, and lack of persistence. Overcoming these barriers is critical to achieve the full potential of CAR-T. To overcome these challenges, it is important to identify gene targets that can boost multiple features of T cells function, especially from fully unbiased genetic screens performed in primary T cells. We have recently developed such T cell CRISPR-based screening approaches. These genetic screens, coupled with stringent selection of T cells for properties – including enhanced potency, persistence and in vivo infiltration/activity – have identified gene modifications that maximally enhance desired T cell properties. We conducted knock-out and gain of function in vitro and in vivo CRISPR screening in primary T cells and identified multiple novel negative/positive regulators of T cell function, in animal tumor models using different CAR-Ts (e.g. CD19, CD22 and HER-2 CARs). We identified two especially potent gene modifications that augment CART-Ts: 1) PRODH2 overexpression resulting in metabolic boost and 2) PRDM1 (exon 3) deletion resulting in epigenetic modulation. This initial research and methods pave the way for further enhancements. Our central hypothesis is that engineering CAR-T cells with a metabolic boost from PRODH2 overexpression as well as epigenetic regulation by PRDM exon3 deletion can enhance HER2 CAR-T cell performance and enhance anti-tumor efficacy in vivo. To do this, we will utilize our knock-in/knock-out (KIKO) protocol to introduce CAR and gene modification sequences precisely at the TRAC locus to generate four HER2 CAR-Ts: PRODH2- OE, PRDM1-KO, PRODH2-OE+PRDM1-KO and control CAR-T. The in vitro characterization (level of CAR expression, proliferation, immune phenotyping, serial killing ability) as well as in vivo evaluations (efficacy, CAR- T infiltration and persistence) of each of the 4 CAR-Ts will be performed using at least two cancer cell lines/mouse model. Our vector and manufacturing processes in this proposal are identical to the planned GMP clinical approach, allowing smooth transition to clinical development. If successful, this study will identify our best performing HER2 CART and provide sufficient data to support a Phase II SBIR submission, and allow a path towards an IND and first-in-human Phase 1 clinical trial, thus delivering a superior CAR-T candidate for patients with solid tumors. In broad terms, the major goal of this study is to evaluate if metabolic boost and epigenetic reprograming when combined result in more effective CAR-Ts relative to either modification alone.