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 de...