Engineering next generation supercharged CAR T-cells against solid tumors

项目总结 采用细胞疗法给癌症治疗带来了革命性的变化，并具有治愈多种癌症的巨大潜力 类型。目前CAR-T治疗存在多种关键障碍，特别是实体瘤，包括T细胞不足 细胞向癌部位转移，缺乏有效的癌细胞杀伤作用，毒性大，免疫抑制强 在肿瘤微环境中，缺乏毅力。克服这些障碍是实现 CAR-T的潜力。为了克服这些挑战，重要的是确定可以促进多个 T细胞功能的特征，特别是在初级T细胞中进行的完全无偏见的遗传筛选。我们有 最近开发了基于CRISPR的此类T细胞筛查方法。这些基因筛选，再加上 严格选择T细胞的特性-包括增强的效力、持久性和体内 渗透/活性-已经确定了最大限度地增强所需T细胞特性的基因修饰。 我们在体外和体内对原代T细胞进行了功能敲除和获得CRISPR筛查 在不同的动物肿瘤模型中，发现了多种新的T细胞功能的负/正调节因子 Car-T(如CD19、CD22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)PRODH2过表达导致代谢增强；2)PRDM1(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改进铺平了道路。 我们的中心假设是，从PRODH2的过度表达中设计出具有代谢促进作用的CAR-T细胞 以及PRDM外显子3缺失的表观遗传调节可以增强HER2 CAR-T细胞的性能和 增强体内抗肿瘤效果。为此，我们将利用我们的敲入/敲出(Kiko)协议来引入 CAR和基因修饰序列精确地位于TRAC基因座以产生四个HER2 CAR-T：PRODH2- OE、PRDM1-KO、PRODH2-OE PRDM1-KO和控制CAR-T。体外表征(CAR水平 表达、增殖、免疫表型、系列杀伤能力)以及体内评估(疗效、CAR-1)。 将使用至少两个癌细胞对4个癌细胞中的每一个进行浸润性和持久性 线条/鼠标模型。本建议书中的载体和制造工艺与计划中的GMP完全相同 临床方法，允许平稳过渡到临床开发。 如果成功，这项研究将确定我们表现最佳的HER2购物车，并提供足够的数据来支持 第二阶段SBIR提交，并允许一条通往IND和第一个人类第一阶段临床试验的道路，因此 为实体瘤患者提供卓越的CAR-T候选方案。从广义上讲，这项研究的主要目标是 是评估新陈代谢促进和表观遗传重新编程结合在一起是否会产生更有效的CAT 相对于任何一个单独的修改。

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.