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，小分子和生物药物主导了FDA癌症药物的批准。 然而，由于其占用驱动的药理学，这些疗法通常容易产生癌症抗性 机制等因此，仍然迫切需要开发新的癌症治疗剂以改善癌症的治疗效果。 治疗效果和患者生存率。最近，异双功能小分子蛋白质降解剂， 通过事件驱动的药理学进行操作已经成为一种替代的癌症治疗策略。这些 诸如PROTEolysis Targeting Chimeras（PROTAC）的方法已经成功地招募了E3。 泛蛋白连接酶与非天然底物连接，导致靶点的泛蛋白化和蛋白酶体降解 蛋白然而，细胞内E3连接酶的募集固有地限制了PROTAC的靶范围， 细胞内蛋白质为了克服这种局限性，我们的实验室最近开发了一种称为抗体的策略- 基于PROTAC（AbTAC）的抗体，其是募集膜结合E3连接酶的双特异性抗体构建体， RNF 43，与感兴趣的细胞表面蛋白质结合，用于有效的溶酶体降解。虽然成功，一些 我们的方法目前存在局限性。即，（1）我们不能降解多通道膜蛋白 对于这些细胞，几乎不存在细胞外结合抗体，以及（2）RNF 43的普遍表达限制了细胞类型 选择性降解为此，我假设抗体-药物缀合物将使得能够靶向多靶向药物。 通过膜蛋白进行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，可用于细胞类型选择性靶向。在此，我提议 扩大AbTAC的范围，以克服这些限制，并调节免疫抑制腺苷 通过开发两种新的基于抗体的模式，研究人类癌症中的信号通路。首先我 已经开发了抗体-药物缀合物，其包含化学连接到小的 分子腺苷2a受体（A2 aR）激动剂。我已经证实了这些第一代的降解能力 在MOLT-4衍生的T细胞系中的A2 aR降解物，并显示出剂量、位点和接头长度依赖性降解 A2aR。展望未来，我将优化抗体-药物缀合物以赋予最大的A2 aR降解， 然后将用于表征降解对增强CD 8+的机制和功能后果 T细胞免疫反应。开发产生腺苷的CD 39的T调节细胞（Treg）选择性降解剂 和CD 73，我将进行噬菌体展示白细胞介素-2受体α（IL-2 R α），以分离抗体， IL-2表位。我将把这些结合剂改造成双特异性的“节中孔”抗体，我希望这种抗体能有效地 在IL-2刺激和IL-2 R α内化后降解CD 39和CD 73。最后，我将从功能上描述 ⑶ 39/⑶ 73降解，以确定其对抑制Treg产生的免疫抑制性腺苷的作用。 总的来说，拟议的研究将导致两种新的有针对性的降解方式的发展， 提供了降解A2 aR，CD 39和CD 73的功能后果的机制见解。

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.