Development of novel targeted degradation strategies to modulate immunosuppressive adenosine signaling in cancer

项目摘要 在过去的二十年里，小分子和生物药物一直主导着FDA的抗癌药物审批。 然而，由于它们的占有率驱动的药理学，这些疗法通常容易对癌症产生抗药性。 机械装置。因此，仍然迫切需要开发新的癌症治疗方法来改进 治疗效果和患者存活率。近年来，异型双功能小分子蛋白质降解剂 通过事件驱动的药理学操作已经成为一种替代的癌症治疗策略。这些 针对嵌合体的蛋白分解(PROTAC)等方式已经成功地招募了E3 泛素连接到非天然底物，导致靶标的泛素化和蛋白酶体降解 蛋白。然而，细胞内E3连接酶的募集固有地将PROTAC的靶向范围限制为 胞内蛋白质。为了抵消这一限制，我们的实验室最近开发了一种名为抗体的策略- 基于PROTACs(AbTACs)，这是一种招募膜结合E3连接酶的双特异性抗体构建物， RNF43与一种有效降解溶酶体的细胞表面蛋白结合。虽然取得了成功，但也有一些 我们的方法目前存在局限性。也就是说，(1)我们不能降解多通道膜蛋白 其胞外结合抗体很少，以及(2)RNF43的普遍表达限制了细胞类型 选择性降解。为此，我假设抗体-药物结合物将能够靶向多个- PASS膜蛋白用于E3连接酶介导的降解。此外，我假设非E3连接酶， 例如快速内化受体，可以被增选为细胞类型的选择性靶向。在此，我建议 扩大AbTAC的范围以克服这些限制并调节免疫抑制的腺苷 通过开发两种新的基于抗体的模式在人类癌症中的信号通路。首先，我 已经开发出由抗RNF43抗体组成的抗体-药物结合物，该抗体与一种小分子 分子腺苷2a受体(A2aR)激动剂。我已经确认了这些第一代人的降解能力 MOLT-4来源的T细胞系中的A2aR降解物，并显示出剂量、位点和接头长度依赖性的降解 A2aR。展望未来，我将优化抗体-药物结合物，以最大限度地降解A2aR，这 然后将用于表征降解对促进CD8+的机制和功能后果 T细胞免疫应答。腺苷产生CD39的T调节细胞(Treg)选择性降解物的研制 和CD73，我将在白细胞介素2受体α(IL-2Rα)上进行噬菌体展示，以分离与外部结合的抗体 IL-2表位。我将把这些粘合剂改造成双功能嵌入型抗体，我希望它能有效地 在IL-2刺激和IL-2Rα内化时降解CD39和CD73。最后，我将从功能上描述 CD39/CD73降解以确定其对Treg产生的免疫抑制腺苷的抑制作用。 总的来说，拟议的研究将导致开发两种新的有针对性的降解模式和 提供对降解A2aR、CD39和CD73的功能后果的机械性见解。

Project Summary Small molecule and biologic drugs have dominated FDA cancer drug approvals over the last two decades. However, due to their occupancy-driven pharmacology, these therapies are generally liable to cancer resistance mechanisms. Thus, there remains a critical need for the development of novel cancer therapeutics to improve treatment efficacy and patient survival. Recently, heterobifunctional small molecule protein degraders which operate through event-driven pharmacology have emerged as an alternative cancer treatment strategy. These modalities, such as PROteolysis TArgeting Chimeras (PROTACs), have been successful in recruiting E3 ubiquitin ligases to non-native substrates, leading to the ubiquitination and proteasomal degradation of the target protein. However, recruitment of intracellular E3 ligases inherently limits the target scope of PROTACs to intracellular proteins. To counteract this limitation, our lab has recently developed a strategy termed antibody- based PROTACs (AbTACs), which are bispecific antibody constructs that recruit membrane-bound E3 ligase, RNF43, to a cell surface protein of interest for efficient lysosomal degradation. Though successful, some limitations to our approach currently exist. Namely, (1) we are unable to degrade multi-pass membrane proteins for which few extracellular-binding antibodies exist and (2) the ubiquitous expression of RNF43 limits cell-type selective degradation. To this end, I hypothesize that antibody-drug conjugates will enable targeting of multi- pass membrane proteins for E3 ligase-mediated degradation. Furthermore, I hypothesize that non-E3 ligases, such as rapidly internalizing receptors, can be co-opted for cell-type selective targeting. Here, I propose to expand the scope of AbTACs to overcome these limitations and modulate the immunosuppressive adenosine signaling pathway in human cancers through the development of two novel antibody-based modalities. First, I have developed antibody-drug conjugates that comprise of an anti-RNF43 antibody chemically linked to a small molecule adenosine 2a receptor (A2aR) agonist. I have confirmed the degradation ability of these first-generation A2aR degraders in a MOLT-4 derived T cell line and shown dose-, site-, and linker-length dependent degradation of A2aR. Going forward, I will optimize the antibody-drug conjugate to confer maximal A2aR degradation, which will then be used to characterize the mechanism and functional consequences of degradation on boosting CD8+ T cell immune response. To develop T regulatory cell (Treg)-selective degraders of adenosine-generating CD39 and CD73, I will perform phage display on interleukin-2 receptor α (IL-2Rα) to isolate antibodies that bind outside the IL-2 epitope. I will engineer these binders into bispecific knob-in-hole antibodies, which I expect to efficiently degrade CD39 and CD73 upon IL-2 stimulation and IL-2Rα internalization. Finally, I will functionally characterize CD39/CD73 degradation to determine its effect on inhibiting Treg-generated immunosuppressive adenosine. Collectively, the proposed studies will lead to the development of two novel targeted degradation modalities and provide mechanistic insights into the functional consequences of degrading A2aR, CD39, and CD73.