Improving CAR-T efficacy against solid tumors by expanding lymph node reservoirs of “stem-like” CAR-T cells

项目总结/摘要 使用CAR-T细胞的免疫疗法在血液恶性肿瘤中诱导了显着的反应， 将这一成功扩展到更常见的上皮癌，这导致了最大的死亡率，已经证明了更多 挑战性在我们进行的一项1期试验中，靶向肿瘤相关抗原ROR 1的CAR-T细胞诱导了 慢性淋巴细胞性白血病患者的反应，但最终在乳房和肺部衰竭 癌症患者，这表明需要保留CAR-T功能的策略来实现固体治疗的疗效。 肿瘤的最近的研究表明，PD-1+ Tcf 1+“前体”耗竭细胞（Tpex）的一个子集是至关重要的， 用于介导肿瘤对免疫疗法的成功应答。Tpex保留了类似茎的自我更新能力， 增殖和分化成效应细胞样细胞的能力。有趣的是，Tpex优先共定位 与抗原呈递细胞（APC）一起存在于肿瘤引流淋巴结（tdLN）中， 保护免受肿瘤中驱动终末耗竭的因素的影响，例如更慢性的抗原刺激，mTOR 激活和缺氧。与传统的T细胞不同，CAR-T细胞被表达在细胞表面的完整抗原激活。 肿瘤细胞，而不是由肿瘤细胞和APC上表达的肽/MHC复合物。这种设计限制了CAR- T细胞活化仅限于肿瘤，其中相对于tdLN，干细胞样质量的保留可能受损。 为了验证这一点，我们开发了ROR 1+肺癌的Kras/p53（KP）本地模型， 我们在患者中观察到ROR 1 CAR-T细胞功能障碍。使用这个模型，我们发现ROR 1 CAR-T细胞确实 在tdLN中没有积累，并且与对照组相比，在肿瘤中Tpex的消耗和终末耗竭更快。 肿瘤特异性T细胞（TCR-T），其在输注前同样培养。Tcf 1 + CAR-T细胞表现出更大的 与Tcf 1- CAR-T细胞相比，其增殖能力、干细胞样自我更新和肿瘤控制，表明 保存Tcf 1 + CAR-Ts的策略可以显著提高疗效。有趣的是，tdLN中的CAR-T细胞 形成了一个“预先耗尽”的PD-1-Tcf 1+中央记忆细胞的小水库，我们可以通过 疫苗接种，并且可能能够产生比Tpex更多的多功能效应物，Tpex是表观遗传学上的 致力于产生功能减退的效应器。我们假设CAR-T细胞的活化不足是由于 tdLN中的APC损害了茎样Tpex储库的维持，导致肿瘤控制不太持久， 使用疫苗接种来增加LN中Tcf 1 + CAR-T细胞的储备将克服这一障碍。在这一提议中， 我们将使用KP模型来确定：1）CAR-T是否在维持干细胞样Tcf 1+库中受损 相对于TCR-T细胞，以及这是否是由于APC的活化不足;以及2）是否活化CAR-1， 通过新型疫苗平台在LN中具有内源性APC的T细胞将提高Tcf 1 + CAR-T细胞的数量， 肿瘤控制以及对PD-L1阻断的反应。我们的研究结果将揭示实体瘤中CAR-T耗尽是否 部分由受损的Tpex形成驱动，并将评估一种新的疫苗平台来克服这些缺陷 这可以转化为临床，以增强CAR-T细胞在实体瘤患者中的疗效。

PROJECT SUMMARY/ABSTRACT Immunotherapy using CAR-T cells has induced dramatic responses in hematological malignancies, but extending this success to more common epithelial cancers, which cause the greatest mortality, has proved more challenging. In a phase 1 trial we conducted, CAR-T cells targeting the tumor-associated antigen ROR1 induced responses in patients with chronic lymphocytic leukemia but became terminally exhausted in breast and lung cancer patients, indicating that strategies to preserve CAR-T function are needed to achieve efficacy in solid tumors. Recent work has demonstrated that a subset of PD-1+Tcf1+ “precursor” exhausted cells (Tpex) is critical for mediating successful tumor responses to immunotherapy. Tpex retain a stem-like capacity for self-renewal, proliferation, and the ability to differentiate into effector-like cells. Interestingly, Tpex preferentially co-localize with antigen-presenting cells (APCs) and reside in tumor-draining lymph nodes (tdLN), where they may be protected from factors in tumors that drive terminal exhaustion, such as more chronic antigen stimulation, mTOR activation, and hypoxia. Unlike conventional T cells, CAR-T cells are activated by intact antigen expressed on tumor cells, not by peptide/MHC complexes expressed on both tumor cells and APCs. This design restricts CAR- T activation exclusively to tumors, where preservation of stem-like qualities may be impaired relative to tdLNs. To test this, we developed a Kras/p53 (KP) autochthonous model of ROR1+ lung cancer that recapitulates the dysfunction of ROR1 CAR-T cells we observed in patients. Using this model, we found ROR1 CAR-T cells did not accumulate in tdLNs and showed faster attrition of Tpex and terminal exhaustion in tumors compared to tumor-specific T cells (TCR-T) that were cultured identically prior to infusion. Tcf1+ CAR-T cells showed greater proliferative capacity, stem-like self-renewal, and tumor control compared to Tcf1- CAR-T cells, suggesting that strategies to preserve Tcf1+ CAR-Ts could significantly improve efficacy. Interestingly, CAR-T cells in tdLNs formed a small reservoir of “pre-exhausted” PD-1-Tcf1+ central memory cells that we could increase by vaccination and that may be able to generate more polyfunctional effectors than Tpex, which are epigenetically committed to producing hypofunctional effectors. We hypothesize that insufficient activation of CAR-T cells by APCs in tdLNs impairs maintenance of stem-like Tpex reservoirs, resulting in less durable tumor control, and that using vaccination to increase reserves of Tcf1+ CAR-T cells in LNs will overcome this barrier. In this proposal, we will use the KP model to determine: 1) whether CAR-T are impaired in maintaining a stem-like Tcf1+ reservoir relative to TCR-T cells and whether this is due to insufficient activation by APCs; and 2) whether activating CAR- T cells with endogenous APCs in LNs via a novel vaccine platform will improve numbers of Tcf1+ CAR-T cells, tumor control, and response to PD-L1 blockade. Our results will reveal whether CAR-T exhaustion in solid tumors is driven in part by impaired Tpex formation and will evaluate a novel vaccine platform to overcome these defects that can be translated to the clinic to enhance the efficacy of CAR-T cells in patients with solid tumors.