Development of a cellular therapy product with single specificity and improved persistence to prevent immunity to biotherapeutics

调节性T细胞（Treg）治疗是一种非常有前途的控制不需要的或致病性免疫的方法 在自身免疫性疾病、移植或抗药物抗体（ADA）形成后， 在血友病A中施用生物治疗剂如凝血因子VIII（FVIII）替代疗法。缺乏 特异性和有限的持久性阻碍了多克隆Treg疗法的临床应用，这是可以克服的 通过表达基于抗体的合成受体如嵌合抗原受体（CAR）或TCR融合体 构建体（TRuC）以重定向抗原特异性。TRuC利用TCR的内部信号传导机制， 重组内源性TCR-CD 3信号传导以响应基于抗体的识别。我们最近在一个 临床前血友病A型FVIII缺陷模型，其通过表达TCRuC的TRuC工程化抗原特异性 通过更忠实地模拟生理TCR信号传导， 内源性睾酮然而，通过合成受体和内源性TCR两者的双重识别可以增加 脱靶抑制作用的可能性，并导致竞争细胞外表达和下游 信号传导介质。 在这个建议中，我们将评估一种新的合成杂交受体，通过取代α和β变量（V） 在一些实施方案中，TCR的VH和VL区与FVIII特异性抗体的VH和VL结构域（FVIII V-swapTCR）结合。单步 CRISPR/Cas介导的FVIII V-swapTCR整合到TCRα恒定（TRAC）基因座中， 内源性TCR表达，从而消除了双重抗原特异性，同时允许更精确地控制 合成受体表达。在目标1中，我们将建立TCR KO FVIII V的特异性和功能性。 swapTCR T细胞。我们将广泛表征TCR KO FVIII所利用的表型和分子途径， V-swapTCR T细胞。通过单特异性工程化的TcR的功能抑制将在以下模型中评估： 血友病A中FVIII替代治疗的ADA形成。在目标2中，我们将探索创新的单链 在一些实施方案中，免疫细胞因子用于驱动TCR KO FVIII V-swapTCR T细胞在体内的选择性增殖，从而改善TCR KO FVIII V-swapTCR T细胞的增殖。 抑制的持久性。本研究的结果将为ADA的有效抑制提供临床前证据 这是一种对生物治疗剂的有效应答，并且能够实现合理的治疗设计和对其他免疫疾病的适用性。 验证TCR KO Vswap TCR的原理将提供原理证明，这将是工程设计的第一步 单抗原特异性“现成”抗原特异性TCR和MHCI KO FVIII V-swap TCR，以克服 同种异体反应性屏障，有助于实现我们的长期目标，即从健康供体中产生普遍适用的THBE。

Regulatory T cell (Treg) therapy is a highly promising approach for controlling unwanted or pathogenic immune responses in autoimmune disease, transplantation or in anti-drug antibody (ADA) formation following the administration of biotherapeutics such as clotting factor VIII (FVIII) replacement therapy in hemophilia A. Lack of specificity and limited persistence impedes clinical application of polyclonal Treg therapy, which can be surmounted by expressing antibody-based synthetic receptors such as a chimeric antigen receptor (CAR) or TCR fusion construct (TRuC) to redirect antigen specificity. TRuCs utilize the internal signaling machinery of a TCR by reconfiguring endogenous TCR-CD3 signaling to respond to antibody-based recognition. We recently showed in a preclinical hemophilia A model of FVIII deficiency that engineering antigen specificity by TRuC expressing Tregs delivers durable suppression of ADA responses by more faithfully mimicking the physiological TCR signaling of endogenous Tregs. However, dual recognition by both the synthetic receptor and endogenous TCR can increase the likelihood of off-target suppressive effects and lead to competition for extracellular expression and downstream signaling mediators. In this proposal, we will evaluate a novel synthetic hybrid receptor generated by replacing the α and β variable (V) regions of the TCR with the VH and VL domains of a FVIII specific antibody (FVIII V-swapTCR). A single-step CRISPR/Cas mediated integration of FVIII V-swapTCR into the TCRα constant (TRAC) locus should abolish endogenous TCR expression, thus eliminating dual antigen specificity, while allowing for more precise control of synthetic receptor expression. In Aim 1, we will establish the specificity, and functionality of TCR KO FVIII V- swapTCR Tregs. We will extensively characterize the phenotype and molecular pathways utilized by TCR KO FVIII V-swapTCR Tregs. Functional suppression by the single-specific engineered Tregs will be assessed in a model of ADA formation to FVIII replacement therapy in hemophilia A. In Aim 2, we will explore an innovative single chain immunocytokine to drive the selective proliferation of TCR KO FVIII V-swapTCR Tregs in vivo, thereby improving durability of suppression. The results of this study will provide pre-clinical evidence for effective suppression of ADA responses to biotherapeutics and enable rational therapeutic design and applicability to other immune disorders. Validating the principles of TCR KO Vswap TCR will provide proof of principle and will be a first step in engineering single-antigen specific “off the shelf” antigen specific TCR and MHCI KO FVIII V-swap Tregs to overcome alloreactive barriers, contributing to our long-term goal to generate universally applicable Tregs from healthy donors.