BIOPHYSICAL TUNING OF CHIMERIC ANTIGEN RECEPTOR (CAR) SIGNALING FOR SAFE AND EFFECTIVE T CELL IMMUNOTHERAPY

摘要 采用自体T细胞的过继细胞疗法(ACT)是一种新生但具有潜在变革性的治疗方式 癌症免疫疗法。通过体外实验将患者自身的T细胞重定向至靶向肿瘤抗原 慢病毒转导和稳定表达肿瘤靶向嵌合抗原受体(CAR)。当车- T细胞在治疗某些白血病和淋巴瘤、体外扩增、体内扩增方面显示出了希望。 存活率、功能表型和对肿瘤抗原的反应仍然不可预测，有时会导致 治疗失败或严重不良事件--包括危及生命的细胞因子释放综合征(CRS)。我们的 总体目标是将成熟的抗原受体信号转导机制原理应用于 设计更安全的汽车，更可靠地靶向肿瘤抗原。第二代汽车 由细胞外抗体样抗原结合域组成，融合到不同范围的 铰链/间隔区序列，紧随其后的是源于 T细胞共刺激受体和T细胞抗原受体(TCR)Zeta链。虽然众所周知， 垫片的选择会严重影响汽车的性能，目前还没有明确的机械原理来确定 可以解释汽车对间隔物特性的精致敏感性。我们的初步调查显示 众所周知的TCR触发机制，即处于CAR中心的动力学-偏析(K-S)机制 信令，并确定间隔区大小是决定CAR的胞外结构域的关键特征 触发。我们证明，使用不同大小的特征良好的异种间隔物，K-S机制可以 被利用来生物物理地调节CAR-T细胞信号。一系列CAR-T细胞激活表型可以 通过基于大小的调节产生，包括CAR-T细胞，在没有 炎性细胞因子的产生。在这个建议中，我们的目标是利用K-S原理来调整汽车的大小 使用新型人源化间隔物有效识别肿瘤抗原和高效杀灭肿瘤细胞，但 不会产生过多的炎性细胞因子。为了实现这一点，我们将系统地改变 使用新的同基因间隔物识别信号阈值的人性化汽车的外域大小 发挥T细胞的溶细胞功能，而不是细胞因子的释放机制。对于这些面向机制的 研究中，我们将采用T细胞功能的体外生化和共培养分析，以及超 分辨率荧光显微镜。然后，我们将继续测试体内肿瘤的体外调谐汽车设计 使用小鼠液体和固体肿瘤模型的杀伤效率和细胞因子释放谱。我们预料到 这些采用K-S原理设计的新型汽车将有助于提高ACT的安全性和有效性 养生法。

ABSTRACT Adoptive cell therapy (ACT) with autologous T cells is a nascent but potentially transformative modality of cancer immunotherapy. Redirection of the patient’s own T cells to target tumor antigens is achieved by ex vivo lentiviral transduction and stable expression of tumor-targeting chimeric antigen receptors (CAR). While CAR- T cells have shown promise in treating some leukemias and lymphomas, their ex vivo expansion, in vivo survival, functional phenotype, and response to tumor antigens remain unpredictable, sometimes resulting in treatment failures or serious adverse events – including life-threatening cytokine release syndrome (CRS). Our overall objective is to apply well-established mechanistic principles of antigen receptor signal transduction to design CARs with improved safety profiles that more reliably target tumor antigens. Second generation CARs are comprised of an extracellular antibody-like antigen-binding domain, fused to a diverse range of hinge/spacer sequences, followed by transmembrane and cytoplasmic signaling domains that are derived from T cell costimulatory receptors and the T cell antigen receptor (TCR) zeta chain. Although it is well known that the choice of spacer can critically affect CAR function, no clear mechanistic principles have been identified that can account for the exquisite sensitivity of CARs to spacer properties. Our preliminary investigations place a well-understood mechanism of TCR triggering, the kinetic-segregation (K-S) mechanism, at the center of CAR signaling, and establishes that spacer size is the key characteristic of the ectodomain that determines CAR triggering. We show, using well-characterized xenogeneic spacers of varying size, that the K-S mechanism can be exploited to biophysically modulate CAR-T cell signaling. A range of CAR-T cell activation phenotypes can be produced through size-based tuning, including CAR-T cells that retain high cytolytic activity without inflammatory cytokine production. In this proposal, we aim to exploit K-S principles to tune the size of CARs using novel humanized spacers for effective recognition of tumor antigens and high tumor cell killing, but without excessive inflammatory cytokine production. To achieve this, we will systematically vary the ectodomain size of humanized CARs using novel syngeneic spacers to identify signaling thresholds that engage T cell cytolytic function, but not the cytokine release machinery. For these mechanism-oriented investigations, we will employ in vitro biochemical and co-culture assays of T cell function, along with super- resolution fluorescence microscopy. We will then proceed to test in vitro-tuned CAR designs for in vivo tumor killing efficacy and cytokine release profiles using murine liquid and solid tumor models. We anticipate that these novel CARs, designed using K-S principles, will be useful for improving the safety and efficacy of ACT regimens.