Optimizing CAR T Cell Therapy

摘要：我们的目标是为转基因T细胞的安全收养转移开发改进的方法 细胞。而转移抗人CD19SFv嵌合抗原受体(hCD19-CAR)表达的T细胞 在CD19 B淋巴样恶性肿瘤中提供了显著的效果，并发症包括延长B细胞再生障碍性贫血 和致命的严重全身性炎症反应综合征。我们建议使用以下方法来解决这些并发症 在亚治疗阶段使用FDA批准的药物调节体内CAR功能的新方法 浓度。T细胞记忆(TSM)细胞因其自身的功能而具有最高的抗肿瘤反应能力 活体长寿。扩增的CAR T细胞失去Taive和Tsm表型。我们假设战略是为了 保留或驱动CAR-T细胞的TSM状态将改善预后。人类T细胞被重新编程 转化为可诱导的多潜能干细胞(IPSCs)，可以很容易地进行基因改造并重新分化为 成熟的T细胞。我们假设TSM产生的IPSC可以提供一个几乎无限的长期来源- 持续TSM CAR T细胞和从TSM细胞状态开始产生IPSC将保留表观遗传学 最终CAR-T细胞产物中TSM细胞的形态和发育可塑性。扩增的T细胞 分化为T效应细胞或精疲力竭的T细胞，与基因表达的深刻、全球变化有关。 转录因子(Tf)bcl6在CD8型TSM中高水平表达，随病变进展而降低。 分化程度越高，Prdm1表达水平越高。我们假设导致高bcl6和损失 在IPSC T细胞后代中表达Blimp-1蛋白将优化TSM CAR的疗效。额外的控制由 表观基因组。我们将结合TSM、IPSC和T-IPSC的表观遗传学和转录分析 鉴定调控TSM和T-iPSC子代分化的新的转录因子。具体的目标将 验证以下假设：1.药物调节T细胞中抗hCD19SFv CAR可以清除恶性CD19 B细胞 通过将SFV的受体与含有细胞内信号域(1A)和CAR的受体二聚化 持久性并不完全依赖于肿瘤或宿主来源的B细胞抗原信号(1B)。竞争对手药物 输液可以通过阻止二聚化迅速停止CAR信号，将副作用降至最低(1C)；2.人TSM 细胞可以被重新编程为IPSCs，以提供自我更新的、受调节的抗CD19 SFV CAR-TSM细胞 用于过继肿瘤免疫治疗(2A)。诱导IPSCs表达转铁蛋白(bcl6hi；prdm1lo) 促进再分化、体外扩增的TSM CAR T细胞保持非衰老状态(2B)； 体外TSM中基因表达、表观遗传分析和转铁蛋白“足迹”的综合分析 差异化将识别(3A)和验证(3B)TSM状态的基本调节器，以基于 汽车疗法。这3个目标将为CAR TSM T细胞疗法创造一种现成的产品，可以克服 汽车疗法的严重副作用，并确定和验证关键的、新颖的TF，将指导优化 CAR疗法为今后的临床试验奠定了基础，并建立了癌症治疗的新范式。

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.