Enhancing Treg Therapeutic Efficacy in GVHD

摘要：这一更新延续了我们对调节性T细胞(Treg)疗法的优化，以创造移植 预防移植物抗宿主病(GVHD)的耐受性。我们的第一个人类第一阶段CD4胸腺刺激(TTreg)和CD4 诱导性Treg(ITreg)试验显示GVHD减少但没有消除。重要的是，复发率是 不改善和减少移植物抗宿主病但不复发是不够的结果。在这次更新中，我们将建立 基于我们令人兴奋的发现，Tregs可以获得超抑制功能，并通过结合抗CD19单链抗体 Car(CAR19)增加他们的杀伤力。在其他研究中，我们开发出高度抑制cAR19 CD8的基因 有内在杀戮机制的iTregs。比较这些细胞类型的相对效率，单独或在 联合使用，将允许我们使用一种产品启动临床试验，同时抑制GVHD和 矛盾地杀死淋巴瘤，解决了同种异体红细胞移植的两个主要限制。多样性中的共性 获得超抑制功能的途径和Treg亚群是一种显著的代谢 效果。我们发现，靶向选定的细胞表面或细胞内蛋白会导致线粒体融合， 高氧化磷酸化(OXPHOS)和超抑制因子Tregs。极地代谢物分析指出 关键的KEGG途径[丝氨酸-甘氨酸-一碳网络]，由蛋氨酸和叶酸循环连接，反式- 谷胱甘肽和丙氨酸-天冬氨酸-L-谷氨酸的硫化。最终结果是一碳(1C)甲基、甲酰基或 甲基转移用于生物合成、基因表达控制和破坏氧化还原反应。CAR19 表达对hCD19+靶标高度抑制和细胞溶解的CD4 tTregs和人CD8 iTregs将 在GVHD/GVL模型中进行研究。值得注意的是，激活的cAR19 CTL可导致严重的细胞因子释放 综合征(CRS)，我们的初步数据表明，cAR19 CD4 tTregs产生低炎性细胞因子和 请勿导致CRS。Aim 1A验证Tregs的代谢重编程依赖于嘌呤的假设 合成，线粒体融合和OXPHOS，以及氧化还原平衡的谷胱甘肽。AIM 1B利用Tregs 通过4-1BB与CD28和CD2)信号域影响代谢重编程和Treg 实现最佳GVHD/GVL的持久性。目标2的重点是这些Treg子集使用的杀伤机制 来解开目标。为此，我们最近发现了CTL超分子攻击粒子(SMAP)作为一种新的 更持久的杀戮机制。SMAP是一种细胞毒性核壳颗粒，含有颗粒酶B和 穿孔素和在触发免疫突触(T细胞的接触信号点)后分泌 和APC。识别SMAP释放的集成IS信号将提供对Treg杀伤机制的洞察， 辅助目标1.模拟IS的支持脂质双分子层将评估CD2作为IS信号积分器/放大器 通过分析双车特雷格来杀人。我们的中心假设是Tregs在目标1中重新编程为1C-， 嘌呤和谷胱甘肽代谢增强CD2触发的抑制和SMAP释放 集成和放大IS信号，优化GVHD/GVL。

Abstract: This renewal continues our optimization of regulatory T cell (Treg) therapy for creating transplant tolerance to prevent graft-vs-host disease (GVHD). Our first-in-human phase I CD4 thymic Treg (tTreg) and CD4 inducible Treg (iTreg) trials that showed reduction but not elimination of GVHD. Importantly, relapse rates were not improved and decreasing GVHD but not relapse is an insufficient outcome. In this renewal, we will build on our exciting finding that Tregs can acquire super-suppressor function and by incorporating an anti-CD19 scFv CAR (CAR19) increase their potency of killing. In other studies, we developed highly suppressive CAR19 CD8 iTregs that have an intrinsic killing mechanism. Comparing the relative efficacy of these cell types, alone or in combination, will allow us to initiate clinical trials using one product simultaneously suppress GVHD and paradoxically kill lymphoma, solving the 2 major alloHCT limitations. A commonality in the diverse approaches and Treg subsets that acquire super-suppressor function is the presence of a striking metabolic effects. We discovered that targeting selected cell surface or intracellular proteins led to mitochondria fusion, high oxidative phosphorylation (OXPHOS) and super-suppressor Tregs. Polar metabolite analytics pointed to key KEGG pathways [serine-glycine-one carbon network], linked by methionine and folate cycles, trans- sulfuration for glutathione, and alanine-aspartate-L-glutamate. A net result is one-carbon (1C) methyl, formyl, or methyl group transfer for biosynthesis, control of gene expression and damaging redox reactions. CAR19 expressing CD4 tTregs and human CD8 iTregs that are highly suppressive and cytolytic to hCD19+ targets will be studied in GVHD/GVL models. Notably, whereas activated CAR19 CTLs can cause severe cytokine release syndrome (CRS), our preliminary data indicate that CAR19 CD4 tTregs produce low inflammatory cytokines and do not cause CRS. Aim 1A tests the hypothesis that metabolic reprogramming of Tregs depends on purine synthesis, mitochondria fusion and OXPHOS, and glutathione for redox balance. Aim 1B exploits Tregs with 4-1BB vs CD28 and CD2) signaling domains that affect metabolic reprogramming and Treg persistence for optimal GVHD/GVL. Aim 2 is focused on the killing mechanisms that these Treg subsets use to lyse targets. Toward that end, we recently discovered CTL supramolecular attack particles (SMAPs) as a new mechanism for more sustained killing. SMAPs are cytotoxic core-shell particles that contain granzyme B and perforin and are secreted after triggering the immunological synapse (IS), the contact signaling point for T cells and APC. Identifying integrated IS signals for SMAP release will provide insights into Treg killing mechanisms, aiding Aim 1. Supported lipid bilayers simulating the IS will assess CD2 as an IS signal integrator/amplifier of killing by analyzing dual CAR Tregs. Our central hypotheses are that Tregs reprogrammed in aim 1 for 1C-, purine- and glutathione- metabolism have enhanced suppression and SMAP release, triggered by CD2 integrated and amplified IS signals and optimal GVHD/GVL.