Optimizing CAR T Cell Therapy

摘要：我们的目标是开发改进的方法来安全地过继转移转基因 T 细胞。而表达抗人 CD19SFv 嵌合抗原受体 (hCD19-CAR) 的 T 细胞的转移 对 CD19 B 淋巴恶性肿瘤具有显着效果，并发症包括长期 B 细胞再生障碍 和致命的严重全身炎症反应综合征。我们建议通过使用来解决这些并发症 一种使用 FDA 批准的亚治疗药物在体内调节 CAR 功能的新方法 浓度。 Tstem 记忆 (Tsm) 细胞由于其自身的作用而具有最高的抗肿瘤反应效力。 体内长寿。扩增的 CAR T 细胞失去了 Tnaive 和 Tsm 表型。我们假设策略 保留或驱动 CAR-T 细胞的 Tsm 状态将改善结果。人类T细胞已被重新编程 转化为可诱导多能干细胞 (IPSC)，可轻松进行基因修饰并重新分化为 成熟的T细胞。我们假设 Tsm 生成的 IPSC 可以提供几乎无限的长期来源 持久的 Tsm CAR T 细胞，并且从 Tsm 细胞状态开始生成 IPSC 将保留表观遗传 最终 CAR-T 细胞产品中 Tsm 细胞的景观和发育可塑性。扩增T细胞 分化为 T 效应细胞或耗尽的 T 细胞，与基因表达的深刻、全局变化相关。 转录因子 (TF) Bcl6 在 CD8 Tsm 中高水平表达，并随着病情进展而降低 分化，而 Prdm1 表达则相反增加。我们假设诱导高 Bcl6 和损失 IPSC T 细胞后代中的 Blimp-1 蛋白将优化 Tsm CAR 功效。额外的控制是由 表观基因组。我们将结合使用 Tsm、IPSC 和 T-IPSC 的表观遗传学和转录分析 后代来鉴定调节 Tsm 与 T-IPSC 后代分化的新 TF。具体目标将 检验假设： 1.药物调节T细胞中的抗hCD19SFv CAR可清除恶性CD19 B细胞 通过将 SFv 受体与含有细胞内信号传导结构域 (1A) 和 CAR 的受体二聚化 持久性并不完全依赖于肿瘤或宿主衍生的 B 细胞抗原信号 (1B)。竞争药物 输注可以通过阻止二聚化来快速终止 CAR 信号传导，从而最大限度地减少副作用 (1C)； 2.人类Tsm 细胞可以被重新编程为 IPSC，以提供自我更新、调节的抗 CD19 SFv CAR-Tsm 细胞 用于过继性肿瘤免疫治疗 (2A)。可以诱导 IPSC 表达 TF 谱（bcl6hi；prdm1lo） 促进再分化、离体扩增的 Tsm CAR T 细胞以保持非衰老状态 (2B)； 3. 体外 Tsm 期间基因表达、表观遗传分析和 TF“足迹”的综合分析 差异化将识别 (3A) 并验证 (3B) Tsm 状态的基本调节器，以优化基于 Tsm 的 CAR疗法。这 3 个目标将为 CAR Tsm T 细胞疗法创造一种“现成”产品，该产品可以克服 CAR 疗法的严重副作用，并识别和验证关键的新型 TF，以指导优化 CAR疗法将在本次PPG中进行未来的临床试验，并建立癌症治疗的新范式。

Abstract: Our goal is to develop improved approaches for safe adoptive transfer of genetically modified T cells. While transfer of anti-human CD19SFv chimeric antigen receptor (hCD19-CAR) expressing T cells have provided dramatic effects in CD19+ B lymphoid malignancies, complications include prolonged B cell aplasia and fatal severe systemic inflammatory response syndrome. We propose to solve these complications by using a novel approach to regulate CAR function in vivo using an FDA approved drug at sub-therapeutic concentrations. Tstem memory (Tsm) cells have the highest potency for anti-tumor responses due to their in vivo longevity. Expanded CAR+ T cells lose Tnaive and Tsm phenotypes. We hypothesize that strategies to retain or drive the Tsm state of CAR-T cells will improve outcomes. Human T cells have been reprogrammed into inducible pluripotent stem cells (IPSCs) that can be readily genetically modified and re-differentiated into mature T cells. We hypothesize that Tsm generated IPSCs can provide a virtually limitless source of long- lasting Tsm CAR T cells and that starting IPSC generation from Tsm cell state will retain the epigenetic landscape and developmental plasticity of Tsm cells in the final CAR-T cell product. Expanded T cells differentiate into Teffectors or exhausted T cells, associated with profound, global changes in gene expression. The transcription factor (TF) Bcl6 is expressed at high levels in CD8 Tsm, decreasing with progressive differentiation, while Prdm1 expression reciprocally increases. We hypothesize that inducing high Bcl6 and loss of Blimp-1 protein in IPSC T cell progeny will optimize Tsm CAR efficacy. Additional control is mediated by the epigenome. We will use a combination of epigenetic and transcription analysis of Tsm, IPSC and T-IPSC progeny to identify novel TFs that regulate the differentiation of Tsm from T-IPSC progeny. Specific aims will test the hypotheses that: 1. Drug regulation of anti-hCD19SFv CAR in T cells can clear malignant CD19 B cells by dimerizing receptors for an SFv with one containing an intracellular signaling domain (1A) and CAR persistence is not fully dependent upon tumor- or host- derived B cell antigenic signals (1B). Competitor drug infusion can rapidly halt CAR signaling by precluding dimerization, minimizing side-effects (1C); 2. Human Tsm cells can be reprogrammed into IPSCs to provide a self-renewable, regulated anti-CD19 SFv CAR-Tsm cells for adoptive tumor immunotherapy (2A). IPSCs can be induced to express a TF profile (bcl6hi; prdm1lo) facilitating re-differentiated, ex vivo expanded Tsm CAR T cells to retain a non-senescent state (2B); 3. Integrated analysis of gene expression, epigenetic analysis and TF “foot-printing” during in vitro Tsm differentiation will identify (3A) and validate (3B) essential regulators of the Tsm state to optimize Tsm based CAR therapy. These 3 aims will create an “off-the-shelf” product for CAR Tsm T cell therapy that can overcome critical side-effects of CAR therapy and identify and validate key, novel TFs that will guide the optimization of CAR therapy for future clinical trials in this PPG and establish a new paradigm of cancer treatment.