Optimizing CAR T Cell Therapy

翻译后摘要：我们的目标是开发改进的方法，安全过继转移的转基因T 细胞虽然表达抗人CD 19 SFv嵌合抗原受体（hCD 19-CAR）的T细胞的转移具有显著的免疫抑制作用， 在CD 19 + B淋巴恶性肿瘤中具有显著疗效，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 一种使用FDA批准的药物在亚治疗水平调节CAR体内功能的新方法， 浓度的Tsm细胞具有最高的抗肿瘤反应能力，这是由于它们在肿瘤细胞中的作用。 体内寿命。扩增的CAR+ T细胞失去Tnaive和Tsm表型。我们假设， 保持或驱动CAR-T细胞的Tsm状态将改善结果。人类T细胞已经被重新编程 诱导性多能干细胞（IPSC）可以很容易地进行遗传修饰和再分化成 成熟T细胞我们假设Tsm产生的IPSC可以提供几乎无限的长- 持续的Tsm CAR T细胞和从Tsm细胞状态开始的IPSC产生将保留表观遗传学特征。 Tsm细胞在最终CAR-T细胞产物中的景观和发育可塑性。扩增的T细胞 分化为T效应细胞或耗尽的T细胞，与基因表达的深刻的全球变化有关。 转录因子（TF）Bcl 6在CD 8 Tsm中以高水平表达，随着进行性T细胞增殖而降低。 分化，而Prdm 1表达增加。我们假设，诱导高Bcl 6和损失 IPSC T细胞后代中Blimp-1蛋白的量将优化Tsm CAR功效。额外的控制是由 表观基因组我们将结合表观遗传学和转录分析Tsm、IPSC和T-IPSC T-IPSC子代的Tsm分化，以鉴定调节Tsm从T-IPSC子代分化的新型TF。具体目标将 检验假设：1. T细胞中抗hCD 19 SFv CAR的药物调节可清除恶性CD 19 B细胞 通过将SFv的受体与含有细胞内信号结构域（1A）和CAR的受体二聚化， 持续性不完全依赖于肿瘤或宿主来源的B细胞抗原信号（1 B）。竞争药物 输注可以通过排除二聚化来快速停止CAR信号传导，使副作用最小化（1C）; 2.人Tsm 细胞可以重编程为IPSC，以提供自我更新的、受调节的抗CD 19 SFv CAR-Tsm细胞 用于过继性肿瘤免疫疗法（2A）。IPSC可被诱导表达TF谱（bcl 6 hi; prdm 1 lo） 促进再分化的离体扩增的Tsm CAR T细胞保持非衰老状态（2B）; 3. 体外Tsm过程中基因表达、表观遗传学分析和TF“足迹”的综合分析 区分将识别（3A）和验证（3B）Tsm状态的基本调节器以优化基于Tsm的Tsm。 CAR疗法。这三个目标将为CAR Tsm T细胞疗法创造一种“现成”产品， CAR治疗的关键副作用，并确定和验证关键的，新的TF，将指导优化 CAR疗法可用于未来的PPG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.