LTBR CARs as next-generation therapies for R/R lymphoma

项目摘要 高达50%的弥漫性大B细胞淋巴瘤（DLBCL）患者在一线治疗后复发。嵌合 抗原受体（CAR）T细胞最近已经成为复发性或难治性（R/R）的治愈性疗法， DLBCL。然而，只有35%的用CAR T细胞治疗的R/R DLBCL患者具有持久的应答， 对于未能受益的患者，生存期以月为单位来衡量。迫切需要改善CAR T细胞 以改善结果。最近，我们鉴定了细胞表面的光敏素β受体（LTBR）作为一种阳性的T- 在体外和体内增强CD 19 CAR T细胞功效的细胞调节剂。LTBR通常以子集表示 但在淋巴细胞中不存在;然而，当在T细胞中表达时， 细胞因子释放，并改善抗原特异性CAR T细胞和γδ T细胞应答，没有明显的脱靶 毒性基于这些观察结果，我们假设LTBR可以有效地增强抗肿瘤活性， R/R淋巴瘤T细胞，减少T细胞耗竭的标志物，并优于目前FDA批准的CAR-T 在R/R DLBCL亚型之间。在目的1中，我们表征了T细胞分化的表达差异， 25例初治和25例R/R DLBCL患者中的活化和耗竭标志物以及髓样细胞群 样品为了理解LTBR是否可以类似地改善R/R背景下的CAR-T应答，我们将使用单- 细胞分析和功能测定，以测试自体CD 19+细胞杀伤，有和没有LTBR。在目标2中， 通过将LTBR和CAR T细胞引入小鼠来评估DLBCL亚型对CAR T细胞活性的影响 异种移植多个生发中心B细胞（GCB）和活化B细胞（ABC）细胞系。由于t细胞 激活和动力学进一步受到患者肿瘤负荷的影响，我们还将研究 高肿瘤负荷背景下的LTBR-CAR T细胞疗法和完成后持久免疫记忆的测试 肿瘤消退最近，通过将LBTR的胞内信号传导结构域直接融合到现有的（CD 28和CD 29）中， 4-1BB）汽车，我们已经开发了具有更有效的抗肿瘤应答的新型CAR构建体。在目标3中， 为了对LTBR结构域进行全面的扫描诱变，我们将创建CAR变体文库并测试 它们改善肿瘤杀伤、抗衰竭和细胞因子分泌的能力。我们还将衡量变化 T-B细胞免疫突触和对髓源性抑制细胞免疫抑制的抵抗 在最有前途的LTBR-CAR中的MDSC。该项目首次全面表征T细胞 在初治和R/R DLBCL中评估LTBR作为T细胞活化策略， 在R/R DLBCL中的抗肿瘤活性。我们的工作有很大的潜力，可以作为实验室和 我们的目标是从研究到临床试验，每年帮助40，000名R/R DLBCL和其他B细胞NHL患者。

PROJECT SUMMARY Up to 50% of patients with diffuse large B-cell lymphoma (DLBCL) relapse after first-line treatment. Chimeric antigen receptor (CAR) T-cells have recently emerged as a curative therapy for relapsed or refractory (R/R) DLBCL. However, only 35% of R/R DLBCL patients treated with CAR T-cells have a durable response, and survival is measured in months for patients who fail to benefit. Improvements in CAR T-cells are urgently required to improve outcomes. Recently, we identified the cell surface lymphotoxin beta receptor (LTBR) as a positive T- cell regulator that enhances CD19 CAR T-cell efficacy in vitro and in vivo. LTBR is typically expressed in a subset of myeloid cells but absent in lymphocytes; however when expressed in T-cells, LTBR induces proinflammatory cytokine release, and improves antigen-specific CAR T- and γδ T-cell responses with no appreciable off-target toxicity. Based on these observations, we hypothesize that LTBR can effectively potentiate anti-tumor activity in R/R lymphoma T-cells, reducing markers of T cell exhaustion and outperforming current FDA-approved CAR-Ts across R/R DLBCL subtypes. In Aim 1, we characterize differences in expression of T-cell differentiation, activation, and exhaustion markers and myeloid populations in 25 treatment-naive and 25 R/R DLBCL patient samples. To understand if LTBR can similarly improve CAR-T response in the R/R context, we will use single- cell profiling and functional assays to test autologous CD19+ cell killing, with and without LTBR. In Aim 2 we will evaluate the impact of DLBCL subtype on CAR T-cell activity by introducing LTBR and CAR T-cells into mice xenotransplanted with multiple germinal center B-cell (GCB) and activated B-cell (ABC) cell lines. Since T-cell activation and kinetics are further influenced by patient tumor burden, we will also investigate the efficacy of LTBR-CAR T-cell therapy in a high tumor burden context and test for durable immune memory after complete tumor regression. Recently, by fusing the intracellular signaling domain of LBTR directly to existing (CD28 and 4-1BB) CARs, we have developed a novel CAR construct with more potent antitumor response. In Aim 3 using comprehensive scanning mutagenesis of the LTBR domain, we will create a library of CAR variants and test their ability to improve tumor killing, resistance to exhaustion and cytokine section. We will also measure changes in T-to-B cell immune synapses and resistance to immunosuppression by myeloid-derived suppressor cells (MDSCs) in the most promising LTBR-CARs. This project is the first to comprehensively characterize T cells states in treatment-naive and R/R DLBCL and evaluate LTBR as a T-cell activating strategy to maximize intrinsic anti-tumor activity in R/R DLBCL. There is substantial potential for our work to serve as a bridge from laboratory studies to clinical trials and to help the 40,000 patients per year with R/R DLBCL and other B-cell NHLs.