Engineering T cells to overcome inhibitory receptor signals that limit the efficacy of adoptive cell therapy against ovarian cancer

项目摘要 在美国，每年有超过20，000名妇女被诊断患有卵巢癌，其中一半以上将死亡。 五年内在过去20年中，结果几乎没有变化，这突出表明需要采取更有效的措施， 治疗一种有前途的新策略采用免疫T细胞，其被设计为独特地靶向蛋白质 在肿瘤中过表达;这样的T细胞免疫疗法具有控制肿瘤生长而不 对健康组织的毒性。我正在进行的工作针对抗原间皮素（MSLN），这有助于 在卵巢癌的恶性和侵袭性进展中，但在健康细胞中的表达有限。我展示 经工程改造以表达人或小鼠Msln特异性高亲和力T细胞受体（TCRMsln）的T细胞可以 分别杀死人卵巢癌细胞系或鼠ID 8细胞系。在播散性ID 8肿瘤中， 模型中，过继转移的TCRMsln T细胞优先在已建立的肿瘤内积累，延迟转移。 卵巢肿瘤生长和显著延长小鼠存活。然而，数据还显示， 肿瘤微环境（TME）限制了工程化T细胞的持久性和抗癌功效。 肿瘤特异性T细胞在遇到抗原时表达抑制性受体，减少抗肿瘤细胞因子 生产我检测了PD-1、Tim-3和Lag-3抑制性受体（免疫检查点）的配体 在人和ID 8卵巢肿瘤中表达。此外，肿瘤浸润性TCRMsln T细胞表达PD-1，Tim-1，Tim-1，Tim-2和Tim-3。 3和Lag-3，这与减少的细胞因子产生相关。我假设免疫检查点 阻断可以克服抑制性受体连接驱动的工程化T细胞的抑制。所以我 用TCRMsln T细胞加抗PD-1、抗Tim-3和/或抗Lag-3检查点治疗的荷瘤小鼠。 阻断抗体，同时靶向多达三种抑制性受体。三重关卡封锁 显著增加了肿瘤内TCRMsln T细胞的抗肿瘤细胞因子产生，但治疗也 产生了更大的肿瘤外毒性。 我现在建议使用临床前小鼠模型（Aim 1）和一种新的人类切片培养系统（Aim 2）， 询问工程化T细胞和卵巢TME中发生的转录组和功能变化 在检查站封锁后。我计划用T细胞工程敲低内源性抑制受体 在肿瘤特异性T细胞中的表达，并确定在没有免疫- 观察到全身联合检查点阻断的相关毒性。许多实体瘤过度表达 Msln和共享相同的T细胞抑制途径。因此，这些新研究的结果可能会 告知临床相关的T细胞工程策略的发展， 抑制许多恶性肿瘤的实体TME。

PROJECT SUMMARY Over 20,000 women are diagnosed with ovarian cancer in the United States annually, and over half will die within five years. Outcomes have changed little in the last 20 years, highlighting the need for more effective therapies. One promising new strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors; such T cell immunotherapies have the potential to control tumor growth without toxicity to healthy tissues. My ongoing work targets the antigen mesothelin (Msln), which contributes to malignancy and invasive progression in ovarian cancer but has limited expression in healthy cells. I showed that T cells engineered to express a human or mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill human ovarian cancer cell lines or the murine ID8 cell line, respectively. In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated within established tumors, delayed ovarian tumor growth and significantly prolonged mouse survival. However, data also revealed that the ovarian tumor microenvironment (TME) limits engineered T cell persistence and anti-cancer efficacy. Tumor-specific T cells express inhibitory receptors upon encountering antigen, reducing antitumor cytokine production. I detected the ligands for the PD-1, Tim-3 and Lag-3 inhibitory receptors (immune checkpoints) expressed in human and ID8 ovarian tumors. Moreover, tumor-infiltrating TCRMsln T cells expressed PD-1, Tim- 3 and Lag-3, which correlated with reduced cytokine production. I hypothesized that immune checkpoint blockade could overcome inhibitory receptor ligation-driven suppression of engineered T cells. Therefore, I treated tumor-bearing mice with TCRMsln T cells plus anti-PD-1, anti-Tim-3 and/or anti-Lag-3 checkpoint- blocking antibodies, targeting up to three inhibitory receptors simultaneously. Triple checkpoint blockade dramatically increased anti-tumor cytokine production by intratumoral TCRMsln T cells, but treatment also produced greater off-tumor toxicities. I now propose to use preclinical mouse models (Aim 1) and a novel human slice culture system (Aim 2) to interrogate the transcriptomic and functional changes that occur in engineered T cells and the ovarian TME after checkpoint blockade. I plan to use T cell engineering to knock down endogenous inhibitory receptor expression in tumor-specific T cells and determine if anti-tumor function is improved without the immune- related toxicities observed with systemic combination checkpoint blockade. Many solid tumors overexpress Msln and share the same T cell inhibitory pathways. Therefore, the findings from these new studies will likely inform the development of clinically-relevant T cell engineering strategies that are more resistant to immune suppression within the solid TME of many malignancies.