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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的使用自体 T 细胞的过继细胞疗法 (ACT) 是一种新兴但具有潜在变革性的治疗方式癌症免疫疗法通过离体实现将患者自身的 T 细胞重定向至肿瘤抗原。慢病毒转导和肿瘤靶向嵌合抗原受体（CAR）的稳定表达。 T 细胞在治疗某些白血病和淋巴瘤方面显示出前景，它们的体外扩增、体内扩增生存、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）。总体目标是将抗原受体信号转导的既定机制原理应用于设计具有改进的安全性的 CAR，更可靠地靶向肿瘤抗原。由细胞外抗体样抗原结合结构域组成，融合到多种铰链/间隔序列，随后是衍生自的跨膜和细胞质信号传导结构域 T 细胞共刺激受体和 T 细胞抗原受体 (TCR) zeta 链。间隔基的选择可以严重影响 CAR 功能，但尚未确定明确的机制原理可以解释 CAR 对间隔物特性的高度敏感性。众所周知的 TCR 触发机制，即 CAR 核心的动力学分离 (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 机制可以触发。可用于生物物理调节 CAR-T 细胞信号传导。通过基于大小的调整来生产，包括保留高溶细胞活性的 CAR-T 细胞，而无需在本提案中，我们的目标是利用 K-S 原则来调整 CAR 的大小。使用新型人源化间隔区可有效识别肿瘤抗原并实现高肿瘤细胞杀伤，但是为了实现这一目标，我们将系统地改变炎症细胞因子的产生。使用新型同基因间隔区确定人源化 CAR 的胞外域大小，以确定信号阈值参与 T 细胞的溶细胞功能，但不参与细胞因子释放机制。研究中，我们将采用 T 细胞功能的体外生化和共测定培养物，以及超级然后，我们将继续测试针对体内肿瘤的体外调谐 CAR 设计。我们预计，使用鼠液体和实体瘤模型可以实现杀伤功效和细胞因子释放曲线。这些采用 K-S 原理设计的新型 CAR 将有助于提高 ACT 的安全性和有效性治疗方案。