Imaging-guided fine tuning of CAR T cell affinity to limit T cell cytotoxicity to tumor antigen

本研究拟采用定量的方法研究嵌合抗原之间的相互作用 受体（CAR）亲和力和抗原密度对CAR T细胞功效和毒性的影响。除了数量有限的 尽管存在表面表达的肿瘤新抗原，但广泛承认缺乏肿瘤特异性靶标。 选择性地调节CAR对其靶分子的亲和力可以维持其裂解肿瘤细胞的能力，所述肿瘤细胞表现出与CAR的结合。 高密度靶向表达，同时防止这种情况在具有较低基础表达的正常细胞中发生。 然而，我们目前对T细胞活化和细胞毒性阈值的理解是由细胞毒性决定的。 CAR亲和力和抗原密度之间的相互作用仍处于初期阶段。迄今为止的相关研究有 使用具有粗亲和力变化的汽车和缺乏定量， 抗原密度测量。此外，由亲和素介导的全身性肿瘤外毒性的可能性- 调谐的CAR T细胞尚未在临床前环境中进行评估，其中所讨论的CAR可以交叉- 与其同源宿主抗原的天然水平反应，从而严格执行风险评估， 肿瘤外CAR反应性的治疗指数研究。为了系统地检查亲和力调节的CAR， 我们设计了一种特异性靶向细胞间粘附分子（ICAM）-1的CAR， 在多种肿瘤中过度表达但在健康组织中保持弱表达的分子。这 CAR的抗原识别结构域来源于称为淋巴细胞的整合素的插入（I）结构域 功能相关抗原（LFA）-1，我们之前通过引入点突变对其进行了改造，使得 ICAM-1亲和力的逐步变化可以从1 mM到1 nM Kd或106倍的范围导出。人类 LFA-1 I结构域以相当的亲和力结合鼠和人ICAM-1，允许同时在体内 评估CAR对表达人ICAM-1的移植肿瘤组织以及任何 针对鼠ICAM-1的潜在中靶、脱肿瘤反应性/毒性。我们最近还开发了 评估了一种基因报告基因，人生长抑素受体2（SSTR 2）， 使用正电子发射断层摄影-计算机断层摄影实时监测和跟踪T细胞 (PET/CT）和临床批准的成像试剂。在本提案中，我们将1）确定阈值CAR 限制CAR T细胞对具有过表达抗原的肿瘤的细胞毒性的亲和力，和2）检查CAR T细胞对肿瘤的细胞毒性。 CAR亲和力和抗原密度对T细胞扩增和收缩时空动力学的影响， 以及对肿瘤消除、复发和全身毒性的影响。这项研究的结果有可能 通过为CAR T细胞设计提供定量框架，显著影响CAR T细胞的研究， 评估以最大化其治疗指数。

The proposed work is to take quantitative approach to study the effect of interplay between chimeric antigen receptor (CAR) affinity and antigen density on CAR T cell efficacy and toxicity. Aside from a limited number of surface-expressed tumor neoantigens, there is a widely acknowledged paucity of tumor-specific targets. Selectively tuning a CAR’s affinity for its target molecule can maintain its ability to lyse tumor cells exhibiting high density target expression while preventing this occurrence in normal cells with lower, basal expression. However, our current understanding of how the threshold for T cell activation and cytotoxicity is defined by the interplay between CAR affinity and antigen density is still in its infancy. Relevant studies to date have examined this relationship using CARs with crude affinity variations and target cells devoid of quantitative, antigen density measurements. Furthermore, the potential for systemic off-tumor toxicity mediated by affinity- tuned CAR T cells has yet to be evaluated in a pre-clinical environment where the CAR in question can cross- react with natural levels of its cognate host antigen, thereby rigorously performing risk assessment and therapeutic index studies of off-tumor CAR reactivity. To systematically examine the affinity tuned CAR paradigm, we have designed a CAR that specifically targets intercellular adhesion molecule (ICAM)-1, a molecule that is over-expressed in multiple tumors but which retains weak expression in healthy tissue. This CAR’s antigen recognition domain is derived from the inserted (I) domain of an integrin called lymphocyte function-associated antigen (LFA)-1, which we previously adapted by introducing point mutations such that step-wise variations of ICAM-1 affinity could be derived ranging from 1 mM to 1 nM Kd or 106-fold. Human LFA-1 I domain binds murine and human ICAM-1 with comparable affinity, allowing simultaneous in vivo evaluation of CAR reactivity against human ICAM-1 expressing, transplanted tumor tissue alongside any potential on-target, off-tumor reactivity/toxicity against murine ICAM-1. We have also recently developed and evaluated a genetic reporter, human somatostatin receptor 2 (SSTR2), that enables quantitative, non-invasive real-time monitoring and tracking of T cells using positron emission tomography-computed tomography (PET/CT) and clinically approved imaging reagents. In this proposal, we will 1) determine threshold CAR affinity that restricts CAR T cell cytotoxicity to tumors with over-expressed antigens, and 2) examine the influence of CAR affinity and antigen density on spatiotemporal kinetics of T cell expansion and contraction, and on the rate of tumor elimination, relapse, and systemic toxicity. The outcome of this study has the potential to significantly impact the study of CAR T cells by providing a quantitative framework for CAR T cell design and evaluation to maximize their therapeutic index.