Understanding the influence of bone-metastatic prostate cancer and mesenchymal stromal cells on γδ T cells, in the bone microenvironment.

虽然免疫疗法在治疗其他癌症方面取得了长足进步，但去势抵抗性前列腺癌 （CRPC）在很大程度上仍然没有反应，强调需要新的方法。其中一种方法依赖于 在施用经遗传修饰以表达嵌合抗原受体（CAR）的自体T细胞方面， 识别特定的肿瘤相关抗原。前列腺干细胞抗原（PSCA）在前列腺中广泛表达。 前列腺癌，并且我们先前已经开发了靶向PSCA的CAR，其具有有效的体内功效。到 针对骨转移性CRPC（mCRPC），我们提出操纵称为γδ的特定T细胞亚群， 可以通过用双膦酸盐如唑来膦酸盐（ZOL）的全身治疗被驱动到骨骼， 临床上用于限制骨mCRPC男性中癌症诱导的骨病。重要的是，ZOL治疗 诱导肿瘤细胞中磷酸化抗原的积累，其由γδ T细胞检测。我们的初步 研究结果表明，ZOL可以增强γδ T细胞归巢到骨骼，在那里它们可以通过以下方式阻止癌症生长： CAR和通过内源性T细胞受体（TCR）识别。γδ CAR-T治疗，存在或不存在 ZOL可以减轻癌症引起的骨退化。此外，我们还发现， 骨髓间充质干细胞（MSC）可增强γδ CART细胞的细胞毒活性。 最后，我们发现CAR结构和共刺激部分的选择影响表型， 因此，CAR可以使αβ和γδ T细胞差异地发挥功能，这需要针对γδ T细胞优化汽车的设计。基于这些 初步研究结果，我们假设γδ CAR-T细胞的归巢和细胞毒活性， 骨转移性CRPC的治疗可以通过遗传、药理学和 微环境方法。我们将通过以下方式检验我们的假设：1）定义最佳γδ CAR-T设计 这将显著增强CRPC细胞毒性。我们将测试选择替代品的生物学意义， CAR跨膜和共刺激结构域，特别关注它们调节CAR跨膜和共刺激结构域的能力。 细胞因子受体的表达。我们还将剖析可以控制γδ CAR的特定信号通路， T细胞持久性。最后，我们将鉴定由不同的汽车触发的分子信号通路。 共刺激结构域。2)确定ZOL是否可以驱动γδ CAR-T募集和抗骨mCRPC活性， vivo.我们将使用骨mCRPC的异种移植和PDX模型来表征生物利用度和治疗性。 γδ CAR-T + ZOL的有效性;以及定位磷酸化抗原蓄积位点的免疫活性模型。 3)剖析γδ CAR-T对骨mCRPC微环境的相互作用。我们将分析 γδ CAR-T + ZOL治疗对骨结构和生理学的影响，以及MSC对 γδ CAR-T细胞在体内的表现。根据预期的结果，确定具体的 骨/肿瘤微环境的特性将揭示新的见解，从而提供强有力的理论基础。 用于翻译为消除目前无法治愈的骨mCRPC而定制的免疫疗法。

While immunotherapies have made strides in the treatment of other cancers, castrate resistant prostate cancer (CRPC) remains largely unresponsive, underscoring the need for novel approaches. One such approach relies on the administration of autologous T cells genetically modified to express a chimeric antigen receptor (CAR) that recognize specific tumor-associated antigens. Prostate Stem Cell Antigen (PSCA) is widely expressed in prostate cancer and we have previously developed a CAR targeting PSCA that has potent in vivo efficacy. To target bone metastatic CRPC (mCRPC), we propose to manipulate a specific subset of T cells, called γδ that can be driven to the skeleton via systemic treatment with bisphosphonates such as zoledronate (ZOL) that is clinically used to limit cancer-induced bone disease in men with bone mCRPC. Importantly, ZOL treatment induces accumulation of phosphoantigens in tumor cells, which are detected by γδ T cells. Our preliminary findings show that ZOL can enhance γδ T-cells' homing to bone where they can prevent cancer growth via CAR and via endogenous T-cell receptor (TCR) recognition. γδ CAR-T treatment, in presence or absence of ZOL, can mitigate cancer-induced bone deterioration. Moreover, we found that soluble factors secreted by bone marrow derived mesenchymal stromal cells (MSC) can increase the cytotoxic potential of γδ CART cells. Finally, we found that the choice of CAR structural and costimulatory moieties affects the phenotype and fuction αβ and γδ T cells differentially, requiring the design of CARs optimized for γδ T cells. Based on these preliminary findings we hypothesize that the homing and cytotoxic activity of γδ CAR-T cells for the treatment of bone metastatic CRPC can be greatly enhanced through genetic, pharmacological, and microenvironmental approaches. We will test our hypothesis by; 1) Defining the optimum γδ CAR-T design that will significantly enhance CRPC cytotoxicity. We will test the biological implications of choosing alternative CAR transmembrane and costimulatory domains, with a specific focus on their ability to modulate the expression of cytokine receptors. We will also dissect the specific signaling pathways that can govern γδ CAR- T cell persistence. Finally, we will identify the molecular signaling pathways triggered by CARs with different costimulatory domains. 2) Determining if ZOL can drive γδ CAR-T recruitment and anti-bone mCRPC activity in vivo. We will use xenograft and PDX models of bone mCRPC to characterize the bioavailability and therapeutic efficacy of γδ CAR-T + ZOL; and an immunocompetent model to map sites of phosphoantigen accumulation. 3) Dissecting the reciprocal effects of γδ CAR-T on the bone mCRPC microenvironment. We will analyze the impact of γδ CAR-T + ZOL treatment on the structure and physiology of the bone, and the effects of MSC on the performance of γδ CAR-T cells in vivo. Based on the anticipated results, characterizing the specific properties of the bone/tumor microenvironment will reveal novel insights thereby providing a strong rationale for the translation of immunotherapies tailored to eliminate currently incurable bone mCRPC.