Investigating PROTAC-mediated antigen degradation as a novel strategy to enhance cytotoxic T-cell responses against cancer in vivo.

摘要 细胞毒性T淋巴细胞(CTL)是一种重要的免疫效应细胞，能在识别抗原后杀伤肿瘤细胞 介绍了主要组织相容性复合体(MHC)I类分子。内源蛋白的蛋白酶体降解 多肽是这一过程的关键，也是MHC I类陈述中的一个限速步骤。癌细胞通常 降低MHC I类演示文稿，以避免CTL的识别和破坏。尽管在以下方面取得了重大进展 癌症免疫治疗，目前还没有专门增强MHC I类分子的治疗策略 癌症相关抗原的呈递。然而，这可能是对现有疗法的补充，从而改变游戏规则。 包括检查站封锁和治疗性癌症疫苗，这些疫苗对大多数癌症患者仍然无效。 该项目提出了一种新型的免疫疗法，利用蛋白水解靶向嵌合体(PROTACs)来增加 肿瘤对CTL介导的杀伤的免疫学可见性通过特异性地促进MHC I类递呈。 PROTAC是将特定的E3连接酶复合体与靶蛋白连接起来的异双功能分子，因此 被泛素/蛋白酶体系统降解。到目前为止，PROTAC仅被设想为治疗 降解必需的肿瘤蛋白(直接抗癌作用)。有趣的是，第一次临床试验显示定制- 设计了针对内源性蛋白的PROTAC，从而为无数新的临床应用打开了大门。 先前的研究表明，PROTAC介导的模型抗原的降解显著增强了它们的MHC 第一类报告。这个项目探索了PROTAC作为一种新的工具来改善体内CTL介导的癌细胞耗竭， 使用临床前黑色素瘤小鼠模型。由于靶向临床相关癌症抗原的PROTAC尚未 它利用最近引入的降解标签(Dtag)系统来降解在线性融合中表达的蛋白质 带有降解标签FKBP12(F12)。小鼠B16F10黑色素瘤细胞的两个新克隆已经产生 对于这些实验：B16F10细胞表达a)F12标记的模型抗原，以及b)临床相关的黑色素瘤 已使用CRISPR/Cas9介导的基因组编辑内源性地标记了降解标签F12的抗原。 针对这些抗原的T细胞受体转基因CTL将用于研究PROTAC介导的对肿瘤生长的影响 并在小鼠身上进行回归。 具体目的是(1)证明PROTACs可以提高癌症特异性CTL反应，从而促进肿瘤 体内回归，(2)表明PROTAC介导的MHC I类递呈的增加可以补充现有的 癌症免疫疗法，如免疫检查点阻断，以及(3)将这些发现转化为人类系统 通过提供证据表明PROTACs可以在体外增强患者来源的CTL对人类黑色素瘤细胞的杀伤作用。 该项目将通过提供PROTAC可以使用的第一个活体证据来影响癌症免疫治疗领域 作为增强MHC-I类分子介导的肿瘤CTL反应的新工具。因此，它扩展了先前在 PROTAC领域走向新的方向和应用，为直接针对PROTAC的设计提供了理论基础 内源性癌症抗原，允许这一新方法的临床翻译。

Abstract Cytotoxic T lymphocytes (CTLs) are key immune effectors and able to kill cancer cells upon recognition of antigens presented on major histocompatibility complex (MHC) class I molecules. Proteasomal degradation of endogenous proteins into peptides is essential for this process and a rate‐limiting step in MHC class I presentation. Cancer cells often downregulate MHC class I presentation to avoid recognition and destruction by CTLs. Despite of significant progress in cancer immunotherapy, there is currently no therapeutic strategy available to specifically enhance the MHC class I presentation of cancer‐associated antigens. This, however, could be game‐changing by complementing current therapies including checkpoint blockade and therapeutic cancer vaccines that remain ineffective in a majority of cancer patient. This project proposes a novel type of immunotherapy utilizing proteolysis targeting chimeras (PROTACs) to increase the immunological visibility of cancer towards CTL‐mediated killing by specifically boosting MHC class I presentation. PROTACs are hetero‐bispecific molecules that link a specific E3 ligase complex with a target protein that is consequently degraded by the ubiquitin/proteasome system. So far, PROTACs have exclusively been envisioned as chemotherapies to degrade essential tumor proteins (direct anti‐cancer effect). Interestingly, first clinical trials show the efficacy of custom‐ designed PROTACs targeting endogenous proteins, thereby opening the door to a myriad of novel clinical applications. Previous studies demonstrate that PROTAC‐mediated degradation of model antigens significantly enhances their MHC class I presentation. This project explores PROTACs as novel tools to improve CTL‐mediated cancer cell depletion in vivo, using preclinical mouse models for melanoma. As PROTACs targeting clinically‐relevant cancer antigens are yet to be developed, it utilizes the recently introduced degradation tag (dTAG) system to degrade proteins expressed in linear fusion with the degradation tag FKBP12 (F12). Two novel clones of murine B16F10 melanoma cells have already been generated for these experiments: B16F10 cells expressing a) an F12‐tagged model antigen, and b) a clinically‐relevant melanoma antigen that has been endogenously tagged with the degradation tag F12 using CRISPR/Cas9‐mediated genome editing. T‐cell receptor transgenic CTLs specific for these antigens will be used to study PROTAC‐mediated effects on cancer growth and regression in mice. Specific aims are (1) to demonstrate that PROTACs can improve cancer‐specific CTL responses and thereby promote tumor regression in vivo, (2) to show that the PROTAC‐mediated increase in MHC class I presentation can complement existing cancer immunotherapies, such as immune checkpoint blockade, and (3) to translate these findings into the human system by providing evidence that PROTACs can enhance the killing of human melanoma cells by patient‐derived CTLs in vitro. This project will impact the field of cancer immunotherapy by providing first in vivo evidence that PROTACs can be used as novel tools to enhance MHC class I‐mediated CTL responses in cancer. It therefore extends previous discoveries in the PROTAC field towards new directions and applications and provides the rationale to design PROTACs directly targeting endogenous cancer antigens, permitting the clinical translation of this novel approach.