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

摘要 使用自体T细胞的免疫细胞疗法（ACT）是一种新生但具有潜在变革性的免疫治疗模式。 癌症免疫疗法患者自身T细胞向靶向肿瘤抗原的重定向通过离体免疫调节来实现。 慢病毒转导和肿瘤靶向嵌合抗原受体（CAR）的稳定表达。虽然汽车- T细胞在治疗一些白血病和淋巴瘤、它们的离体扩增、体内增殖和免疫应答方面已经显示出前景。 存活、功能表型和对肿瘤抗原反应仍然不可预测，有时导致 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 总的目标是将抗原受体信号转导的成熟机制原理应用于 设计安全性更高的汽车，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结构域组成，融合到各种各样的 铰链/间隔区序列，随后是衍生自 T细胞共刺激受体和T细胞抗原受体（TCR）ζ链。尽管众所周知， 间隔区的选择可严重影响CAR功能，但尚未鉴定出明确的机制原理， 可以解释汽车对间隔区性质的灵敏度。我们的初步调查显示 TCR触发的一种众所周知的机制，即CAR中心的动力学分离（K-S）机制， 信号传导，并确定间隔区大小是决定CAR的胞外域的关键特征 触发。我们表明，使用不同大小的良好表征的异种间隔区，K-S机制可以 用于生物药理学调节CAR-T细胞信号传导。一系列CAR-T细胞活化表型可以 通过基于大小的调整产生，包括保留高细胞溶解活性而不 炎性细胞因子产生。在这个建议中，我们的目标是利用K-S原则来调整汽车的大小 使用新的人源化间隔区来有效识别肿瘤抗原和高肿瘤细胞杀伤， 而不产生过多的炎症细胞因子。为了实现这一目标，我们将系统地改变 使用新的同源间隔区来确定人源化汽车的胞外域大小，以鉴定 参与T细胞溶细胞功能，但不参与细胞因子释放机制。对于这些机制导向的 研究中，我们将采用体外生化和共培养试验的T细胞功能，沿着与超级， 分辨率荧光显微镜。然后，我们将继续测试体外调整的CAR设计，用于体内肿瘤 使用鼠液体和实体瘤模型的杀伤效力和细胞因子释放曲线。我们预计 这些新的汽车是根据K-S原理设计的，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.