Construction of functional human CAR recognizing live pancreatic beta cells

1型糖尿病（T1 D）是一种自身免疫性儿科疾病，由于自身反应性T细胞过度活化而发生。 淋巴细胞，其破坏胰腺β细胞。调节性T细胞（Tcells）的抑制是一种关键机制， 限制了这种自身反应性。T1 D患者存在定量和功能性TlD缺陷。新的临床试验， 将自体离体扩增的TdR转移到T1 D患者中显示出成功的迹象。 我们的提案旨在对目前的Treg疗法进行重大改进。我们的目标是创建和验证一个 T1 D的新型抗原特异性细胞疗法。因此，我们设计了β细胞特异性嵌合抗原， 受体（汽车），在本文中进行优化和测试。β细胞特异性由以下的scFv片段赋予： 识别独特的人β细胞标志物NTPD酶3的抗体。我们假设β细胞特异性 T细胞上的CAR将诱导适当的CAR信号传导并引起CAR T的活化和扩增 细胞当这种信号传导发生在携带CAR的TcR中时，额外修饰以用于持续的 通过增强TGFRP功能必需转录因子表达的免疫抑制 Foxp 3和Eos，它将使局部持久的抗原依赖性沉默的糖尿病自身免疫。 将在以下具体目标中检验这一假设： 目标1。设计和产生β细胞特异性CAR，优化其结合强度，并询问 它们在体外的信号传导潜力。由于我们的汽车与β细胞抗原的结合太强或太弱， 可能提供不充分的激活，我们将模拟我们的互补位在计算机上设计突变的变体， 最佳抗原结合。将分析优化的突变体scFv-CAR构建体，并汽车 组成型抗原特异性信号传导而没有随后的T细胞耗尽将被优先考虑。 目标2.制定和测试策略，以保持持续稳定和强大 β细胞特异性CAR T细胞的抑制表型。我们将测试我们的创新策略， 来自大量常规CD 4 + T细胞的自体携带CAR诱导的T细胞活化（iT细胞活化）。买那车 盒被设计成提供对TcB发育至关重要的TcB的组成型异位表达， 维持转录因子Foxp 3和/或Eos。CAR iTUNK将在功能上进行测试， 对人β细胞反应性致糖尿病T细胞克隆的抑制能力。 我们的项目的完成将证实β细胞特异性CAR T细胞的可行性，评估其效力， 并为T1 D中CAR Tribunal的未来临床前和临床试验铺平道路。

Type 1 diabetes (T1D), autoimmune pediatric disease, occurs due to hyperactivation of self-reactive T lymphocytes, which destroy pancreatic β cells. Suppression by regulatory T cells (Tregs) is a key mechanism limiting such autoreactivity. T1D patients have quantitative and functional Tregs defects. New clinical trials, transferring autologous ex vivo-expanded Tregs into T1D patients, showed signs of success. Our proposal targets major improvement of the current Treg therapies. We aim to create and validate a novel, antigen-specific cellular therapy for T1D. Hence, we designed β cell-specific Chimeric Antigen Receptors (CARs), to be optimized and tested herein. β cell-specificity is conferred by the scFv fragment of an antibody recognizing unique human β cell marker NTPDase3. We hypothesize that engagement of β cellspecific CAR(s) on T cells will induce proper CAR signaling and cause activation and expansion of CAR T cells. When such signaling occurs in CAR-bearing Tregs, additionally modified for sustained immunosuppression through augmented expression of the essential for Tregs function transcription factors Foxp3 and Eos, it will enable local long-lasting antigen-dependent silencing of diabetogenic autoimmunity. This hypothesis will be tested in the following Specific Aims: Aim 1. Design and produce β cell-specific CAR(s), optimize their binding strength, and interrogate their signaling potential in vitro. As either too strong or weak binding of our CARs to β cell antigen will likely provide inadequate activation, we will model our paratope in silico to design mutated variants with optimal antigen binding. Optimized mutant scFv-CAR constructs will be analyzed and CARs that provide constitutive antigen-specific signaling without subsequent T cells exhaustion will be prioritized. Aim 2. Develop and test strategies allowing for maintenance of continued stable and robust suppressive phenotype of β cell-specific CAR Tregs. We will test our inventive strategy creating autologous CAR-bearing induced T regs (iTregs) from the bulk of conventional CD4+ T cells. For that CAR cassettes are designed to provide constitutive ectopic expression of crucial for Tregs development and maintenance transcription factors Foxp3 and/or Eos. CAR iTregs will be tested functionally for their suppressive abilities against human β cells-reactive diabetogenic T cell clones. Completion of our project will confirm the feasibility of β cell-specific CAR Tregs, evaluate their potency, and pave a road for future pre-clinical and clinical trials of CAR Tregs in T1D.