Construction of functional human CAR recognizing live pancreatic beta cells

1型糖尿病(T1D)是一种自身免疫性儿科疾病，由自身反应性T细胞过度激活引起 淋巴细胞，破坏胰腺β细胞。调节性T细胞(Treg)的抑制是一种关键机制 限制这种自动反应能力。T1D患者存在数量和功能Tregs缺陷。新的临床试验， 将自体体外扩增的Tregs移植到T1D患者身上，显示出成功的迹象。 我们的建议旨在对目前的Treg疗法进行重大改进。我们的目标是创建和验证 针对T1D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(CARS)，在此进行优化和测试。β的单链抗体片段具有细胞特异性 识别独特的人类β细胞标志物NTPDase3的抗体。我们假设β细胞特异性的参与 T细胞上的CAR(S)会诱导适当的CAR信号，导致CAR T的激活和扩张 细胞。当这样的信号出现在载重车中时，额外修改为持续 增强表达Tregs功能必需转录因子的免疫抑制作用 Foxp3和Eos，它将使局部持久的抗原依赖的糖尿病自身免疫沉默。 这一假设将在以下具体目标中得到检验： 目的1.设计制造β细胞专用车(S)，优化其结合强度，并询问 它们在体外的信号潜力。因为我们的CARS与β细胞抗原的结合太强或太弱都会 可能提供不充分的激活，我们将在电子计算机中对我们的对应体进行建模，以设计突变变体 最佳的抗原结合。将分析优化的突变体scFv-car结构，并提供 不会随后耗尽T细胞的结构性抗原特异性信号将被优先考虑。 目标2.制定和测试战略，以维持持续稳定和稳健 β细胞特异性CAR树的抑制表型。我们将测试我们创造性的策略创建 自体车载诱导的T细胞(ITregs)来自大量的传统的CD4+T细胞。买那辆车 盒的设计是为了提供对Tregs发育和Tregs发育至关重要的结构性异位表达 维持转录因子Foxp3和/或Eos。汽车iTregs将进行功能测试，以确定其 对人β细胞的抑制能力-反应性糖尿病T细胞克隆。 我们项目的完成将确认β细胞特异性CAR Tregs的可行性，评估其效力， 为未来CAR Tregs在T1D的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.