Biomarker Identification to Increase Adjunctive Cryoablation and Checkpoint-Inhibitor Immunotherapy Response

检查点抑制剂(CPI)显著改善了许多肿瘤类型的预后，但仍有 2有很大的改进空间。因此，大量的临床前和临床研究旨在 3评估可能提高CPI应答率的联合治疗方案，并确定 4个指示患者是否将受益于免疫治疗或对其产生抵抗力的生物标记物。 冷冻消融是一种很有前景的辅助策略，可能能够显著提高CPI的反应 6率。冷冻消融单个转移灶会导致坏死性细胞死亡，通过 7个交替的冻结和解冻循环。肿瘤抗原随细胞内炎症一起释放 8目次。这种明显的炎症反应激活树突状细胞来摄取释放的肿瘤抗原。 进而激活适应性免疫系统，攻击身体其他部位的肿瘤。 我开发了一种双植入肿瘤模型，以研究辅助冷冻消融的全身益处。 11并发现，当加入CPI治疗时，冷冻消融显著增加了完全反应 12未消融肿瘤相对于单纯CPI的比率。由于将冷冻消融添加到CPI是最低限度的研究，因此 确定生物标记物以确定哪些肿瘤将从冷冻消融中受益最大的工作仍然意义重大 14加法。为了识别反应生物标记物，我得到了我共同发明的一种独特的调查工具的帮助， 15颗粒酶B正电子发射计算机断层显像。定位于主动免疫介导的肿瘤杀伤部位，颗粒酶B-PET 16使我们能够在肿瘤大小改变之前区分CPI反应和无反应。然后我们就可以 17在免疫反应仍然活跃的时候询问免疫反应，有助于获得独特的见解。 18在这项提议中，在小鼠肿瘤模型中，我将开发一种优化的临床前冷冻消融和CPI方案 19跨多个肿瘤线，反映了CPI的反应范围。在这些模型中，我将使用 20颗粒酶B PET成像有助于识别基因组和非基因组生物标记物的反应和识别 21联合冷冻消融和CPI的抵抗机制。我还将同时领导一个 22项临床试验评估冷冻消融术提高转移性尿路上皮细胞培溴利珠单抗反应的能力 癌23例。从冷冻消融时获得的患者血液和活检样本中，我建议分析 24肿瘤和全身免疫微环境对这些患者的反应生物标志物。这些数据将 25对于指导未来评估冷冻消融能力的优化临床方案的发展是必不可少的 26以提高多种肿瘤类型的系统免疫力。我将在优秀的环境中进行这项研究 27 MGH的研究环境，由免疫学、免疫肿瘤学和 介入放射学28例。他们的指导将有助于确保这个项目的成功和我的成功 29过渡到独立的研究生涯。

1 Checkpoint inhibitors (CPI) have significantly improved outcomes in many tumor types, but there is still 2 immense room for improvement. A tremendous amount of preclinical and clinical research thus is aimed at 3 evaluating combination therapeutic regimens that may increase CPI response rate as well as identifying 4 biomarkers that indicate whether a patient will benefit from or be resistant to immunotherapy. 5 Cryoablation is a promising adjunctive strategy that may be able to significantly increase the CPI response 6 rate. Cryoablation of a single metastatic focus causes necrotic cell death, rupturing cell membranes through 7 alternating freeze and thaw cycles. Tumoral antigens are released along with inflammatory intracellular 8 contents. This markedly inflammatory reaction activates dendritic cells to take up the released tumor antigen 9 which, in turn, activates the adaptive immune system to attack tumor sites elsewhere in the body. 10 I have developed a dual-implanted tumor model to investigate the systemic benefit of adjunctive cryoablation 11 and have found that when added to CPI therapy cryoablation significantly increases the complete response 12 rate of the non-ablated tumor relative to CPI alone. As adding cryoablation to CPI is minimally studied, there 13 remains significant work to identify biomarkers to determine which tumors will benefit most by cryoablation 14 addition. To identify response biomarkers I am helped by a unique investigative tool that I co-invented, 15 granzyme B PET imaging. Localizing to the site of active immune-mediated tumor killing, granzyme B PET 16 allows us to distinguish CPI responders from non-responders prior to changes in tumor size. We can then 17 interrogate the immune-response while it is still active, facilitating unique insights. 18 In this proposal, in a murine tumor model I will develop an optimized preclinical cryoablation and CPI regimen 19 across multiple tumor lines reflective of the range of CPI responsiveness. In these models I will then use 20 granzyme B PET imaging to help identify genomic and non-genomic biomarkers of response and identify 21 mechanisms of resistance to the combination of cryoablation and CPI. I will also simultaneously be leading a 22 clinical trial evaluating the ability of cryoablation to increase pembrolizumab response in metastatic urothelial 23 carcinoma. From patient blood and biopsy specimens obtained at the time of cryoablation I propose to analyze 24 the tumoral and systemic immune microenvironment for response biomarkers in these patients. These data will 25 be essential to guide the development of future optimized clinical protocols evaluating the ability of cryoablation 26 to increase systemic immunity across multiple tumor types. I will conduct this research in the excellent 27 research environment of MGH, guided by mentors expert in the fields of immunology, immuno-oncology, and 28 interventional radiology. Their guidance will help ensure the success of this project and my successful 29 transition to an independent research career.