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

嵌合抗原受体 (CAR) 信号转导的生物物理调节以实现安全有效的 T 细胞免疫治疗

• 批准号: 10284715
• 负责人: Kaushik Choudhuri
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10284715
• 关键词: Adenocarcinoma Cell; Adoption; Adoptive Cell Transfers; Adverse effects; Affect; Antibodies; Antigen Receptors; Antigen Targeting; Antigens; Antitumor Response; Autoimmune; Autoimmune Diseases; Autologous; B lymphoid malignancy; B-Lymphocytes; Biochemical; Biological Assay; Biophysics; CAR T cell therapy; CD19 Antigens; CD19 gene; Carcinoembryonic Antigen; Cell Line; Cell Survival; Cell physiology; Cellular immunotherapy; Characteristics; Clinical; Coculture Techniques; Cytolysis; Cytoplasmic Tail; Cytotoxic T-Lymphocytes; Development; Dimensions; Disease remission; Endothelium; Engineering; Equilibrium; Fluorescence Microscopy; Generations; Goals; Human; Immunity; Immunotherapy; In Vitro; Inflammation; Inflammatory; Investigation; Kinetics; Knowledge; Life; Liquid substance; Macrophage Activation; Malignant Neoplasms; Mediating; Modality; Modeling; Molecular; Mus; Patients; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Production; Property; Proteins; Receptor Signaling; Regimen; Resolution; Rod; Role; Safety; Serious Adverse Event; Signal Transduction; Solid Neoplasm; System; T cell response; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Testing; Treatment Failure; Treatment-related toxicity; Tumor Antigens; antigen binding; base; cancer immunotherapy; cancer therapy; cell killing; chimeric antigen receptor; chimeric antigen receptor T cells; connectin; cytokine; cytokine release syndrome; cytotoxicity; design; effector T cell; experimental study; extracellular; improved; in vitro testing; in vivo; leukemia; leukemia/lymphoma; neoplastic cell; novel; receptor; receptor function; response; segregation; treatment response; trigger point; tumor

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.

抽象的 具有自体T细胞的收养细胞疗法（ACT）是一种新生但潜在的变化方式 癌症免疫疗法。将患者自己的T细胞重定向到靶向肿瘤抗原，通过体内实现 慢病毒转移和靶向肿瘤的嵌合抗原受体（CAR）的稳定表达。而汽车 - T细胞在治疗某些白血病和淋巴瘤时表现出了有望，它们的体内膨胀，体内 生存，功能表型和对肿瘤抗原的反应仍然不可预测，有时导致 治疗失败或严重的不良事件 - 包括威胁生命的细胞因子释放综合征（CRS）。我们的 总体目的是将抗原受体信号转导的完善机理原理应用于 设计具有改进的安全剖面的汽车，更可靠地靶向肿瘤抗原。第二代汽车 累积的细胞外抗体样抗原结合结构域，融合到潜水员的范围 铰链/间隔序列，然后是跨膜和细胞质信号传导域，这些信号域是从源自 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 垫片的选择可以严重影响汽车功能，尚未确定明确的机械原理 可以说明汽车对间隔性能的独家灵敏度。我们的初步调查处 在汽车中心，TCR触发的机制，动力学分离（K-S）机制有充分理解的机制 信号传导，并确定间隔大小是决定汽车的胞外域的关键特征 触发。我们使用尺寸变化良好的特征化异构间隔仪表明，K-S机制可以 可以探索生物物理调节CAR-T细胞信号传导。一系列CAR-T细胞激活表型可以 可以通过基于尺寸的调整生产，包括保留高溶质活性的CAR-T细胞 炎症性细胞因子产生。在此提案中，我们旨在利用K-S原则来调整汽车的大小 使用新型的人源垫片有效识别肿瘤抗原和高肿瘤细胞杀伤，但 没有过多的炎性细胞因子产生。为了实现这一目标，我们将系统地改变 人源化汽车的外域域大小使用新型的同步间隔器来识别信号阈值 参与T细胞细胞溶解功能，但不参与细胞因子释放机械。对于这些机制 研究，我们将采用T细胞功能的体外生化和共培养测定法，以及 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节汽车设计 使用鼠类液体和实体瘤模型杀死效率和细胞因子释放曲线。我们预料到这一点 这些使用K-S原理设计的新型汽车将有助于提高ACT的安全性和效率 方案。