SaefCAR: Regulatable CAR-T cells for safe and effective immunotherapy

项目概要： 尽管在某些血液癌症的治疗中取得了显著的成功，但严重不良反应的风险仍然存在。 事件和效力丧失仍然是威胁到嵌合抗体广泛应用的主要问题。 抗原受体（CAR）T细胞疗法用于其他癌症类型，包括实体瘤。所有这些问题， 其受CAR靶向结构域的结合亲和力的影响，是由于 不可预测的过度活化，可能导致潜在致命的全身毒性，如细胞因子释放 综合征（CRS）、肿瘤溶解综合征（TLS）和神经毒性。此外，靶向肿瘤外毒性可 由组成型表达的汽车与健康组织上的肿瘤抗原的不加选择的结合引起， 而由于抗原逃逸和/或T细胞耗竭，可能发生疾病复发。当前和新兴的CAR- 解决这些问题的T细胞策略和设计在响应时间和起效方面往往太慢 的行动。此外，它们过早地和永久地消除“高价值”细胞，并且通常需要 可能限制当前慢病毒和逆转录病毒DNA有效载荷能力的其他蛋白质的表达 向量。因此，迫切需要快速、可逆和可调节的体内可调节CAR-T细胞平台。 并且不会对CAR递送载体施加实质性的DNA负担。本提案的目的是评估 使用免疫调节剂调节CAR T细胞的活化、效应子功能和细胞因子释放的可行性 外源施用的配体，其调节CAR T细胞靶向结构域的亲和力。为此我们 将利用我们专有的通用变构接头和抗体开关模块（UNASMA）技术， 将ON/OFF亲和开关并入充分表征抗体单链可变片段（scFv）中， 针对临床验证的血液学癌症靶标，其随后将被格式化为可切换的 亲和嵌合抗原受体（SaefCAR）。我们假设转导后产生的SaefCAR 将SaefCAR导入T细胞中，将能够通过外源性施用免疫调节剂来体内调节SaefCAR T细胞的活性。 小分子或肽配体，通过以下方式潜在地解决当前CAR-T细胞疗法的若干问题： 1)通过缓慢启动T细胞活化来改善内在毒性，2）防止靶向脱靶， 3）防止肿瘤特异性免疫应答的耗尽， CAR T细胞由紧张性信号传导和其他机制经由CAR的间歇性开/关开关引起。 具体而言，在目标1中，我们将开发可转换形式的scFv，其随后将被格式化为单链抗体。 在Aim 2中，将CAR转化并转导到T细胞中。在目标3中，我们将验证受监管的SaefCAR的概念验证 体外和体内研究中的T细胞。我们的目的是证明，在细胞中的配体可控亲和开关， SaefCAR T细胞的结合结构域可以调节其针对肿瘤的活化和效应子功能。重要的 由于我们的方法的通用性，它提供了一个蓝图，以改善任何基于scFv的CAR T细胞 通过增加调节CAR对肿瘤抗原的亲和力的能力，在未来的治疗中发挥作用。

Project Summary: Despite the remarkable success in the treatment of some hematological cancers, the risk of serious adverse events and loss of potency remain major problems that threaten to curtail widespread application of chimeric antigen receptor (CAR) T-cell therapies to other cancer types including solid tumors. These problems, all of which are influenced by the binding affinity of the CAR targeting domain, result from the propensity for unpredictable hyperactivation that can lead to potentially fatal systemic toxicities such as cytokine release syndrome (CRS), tumor lysis syndrome (TLS) and neurotoxicity. Furthermore, on-target off-tumor toxicities can result from the indiscriminate binding of constitutively expressed CARs to tumor antigens on healthy tissues, whilst disease relapses can occur due to antigen escape and/or T cell exhaustion. Current and emerging CAR- T cell strategies and designs to address these problems are often too slow in terms of response times and onset of action. Furthermore, they prematurely and permanently eliminate the “high value” cells and often require the expression of additional proteins which may constrain the DNA payload capacity of current lenti- and retro- virus vectors. There is thus an urgent unmet need for in-vivo regulatable CAR-T cell platforms that are rapid, reversible and don’t exert a substantial DNA burden on the CAR delivery vector. The objective of this proposal is to assess the feasibility to regulate the activation, effector functions and cytokine release of CAR T-cells using an exogenously administered ligand that modulates the affinity of the CAR T-cell targeting domain. To this end we will utilize our proprietary universal allosteric-linker and switch module for antibodies (UNASMA) technology to incorporate an ON/OFF affinity switch into a well characterized antibody single chain variable fragment (scFv) against a clinically validated hematological cancer target which will subsequently be formatted as a switchable affinity chimeric antigen receptor (SaefCAR). We postulate that the resulting SaefCAR following transduction into a T-cell, will enable the in-vivo regulation of the SaefCAR T-cells’ activity via exogenous administration of a small molecule or peptide ligand, potentially solving several of the problems of current CAR-T cell therapies by: 1) Ameliorating intrinsic toxicities via enablement of a slow start to T-cell activation, 2) Preventing on-target off- tumor toxicities by switching off the CAR-T cell without eliminating it 3) Preventing exhaustion of tumor-specific CAR T cells caused by tonic signaling and other mechanisms via intermittent ON/OFF switching of the CAR. Specifically in Aim 1 we will develop switchable forms of the scFv which will subsequently be formatted into a CAR and transduced into a T-cell in Aim 2. In Aim 3 we will validate proof-of-concept of the regulated SaefCAR T-cells in in-vitro and in-vivo studies. We aim to demonstrate that a ligand-controllable affinity switch in the binding domain of SaefCAR T-cells can regulate it’s activation and effector functions against a tumor. Importantly due to the universal character of our approach, it provides a blueprint to improve any scFv-based CAR T-cell therapy in the future by adding a capability to regulate the CAR affinity for the tumor antigen.