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

项目摘要/摘要 使用CAR-T细胞的免疫疗法在血液系统恶性肿瘤中引起了戏剧性的反应，但 将这一成功扩展到更常见的上皮性癌症，导致最大的死亡率，事实证明 很有挑战性。在我们进行的一项第一阶段试验中，针对肿瘤相关抗原ROR1的CAR-T细胞诱导 慢性淋巴细胞白血病患者的反应，但在乳房和肺部出现终末期衰竭 癌症患者，表明需要保留CAR-T功能的策略才能在Solid中取得疗效 肿瘤。最近的研究表明，PD-1+Tcf1+“前体”耗竭细胞(TPex)的一个子集是关键的 用于调节肿瘤对免疫治疗的成功反应。TPex保持茎状自我更新能力， 增殖和分化为效应器样细胞的能力。有趣的是，TPex优先地共同定位 与抗原提呈细胞(APC)一起存在，并驻留在肿瘤引流淋巴(TdLN)中，它们可能在那里 防止肿瘤中导致终末衰竭的因素，如更多的慢性抗原刺激，mTOR 激活和缺氧。与传统的T细胞不同，CAR-T细胞是由表达在 肿瘤细胞，而不是通过在肿瘤细胞和APC上表达的肽/MHC复合体。这种设计限制了汽车- T仅对肿瘤激活，与tdlns相比，干细胞样性质的保存可能会受到损害。 为了验证这一点，我们开发了一种Kras/p53(KP)固有的ROR1+肺癌模型，该模型概括了 我们观察了患者ROR1CAR-T细胞功能障碍。使用这个模型，我们发现ROR1CAR-T细胞 未在tdln中蓄积，并显示出与tdln相比，tpex在肿瘤中的消耗和终末耗竭更快 输注前同样培养的肿瘤特异性T细胞(TCR-T)。Tcf1+CAR-T细胞表现为 与Tcf1-CAR-T细胞相比，增殖能力、干细胞样自我更新和肿瘤控制 保留Tcf1+CAR-T的策略可以显著提高疗效。有趣的是，tdlns中的CAR-T细胞 形成了一个小型的预耗尽的PD-1-Tcf1+中央记忆细胞库，我们可以通过 疫苗接种可能比表观遗传的TPex产生更多的多功能效应器 致力于生产功能不全的效应器。我们假设CAR-T细胞激活不足是由于 TdLN中的APC损害了干状TPex储存库的维护，导致更难持久的肿瘤控制，以及 通过接种疫苗来增加LNS中Tcf1+CAR-T细胞的储备将克服这一障碍。在这份提案中， 我们将使用Kp模型来确定：1)CAR-T在维持茎状Tcf1+水库方面是否受到损害 相对于TCR-T细胞，以及这是否由于APC激活不足所致；以及2)激活CAR-T细胞是否 通过一种新的疫苗平台，在LNS中含有内源性APC的T细胞将提高Tcf1+CAR-T细胞的数量， 肿瘤控制，以及对PD-L1阻断的反应。我们的结果将揭示实体瘤中CAR-T的耗竭 部分是由TPex形成受损推动的，并将评估一种新的疫苗平台来克服这些缺陷 这可以转化到临床上，以提高实体瘤患者CAR-T细胞的疗效。

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.