PROJECT SUMMARY/ABSTRACT The Problem: Cellular immunotherapies targeting surface proteins are one the most exciting new modalities in cancer. However, most tumors lack surface targets with acceptable efficacy and toxicity profiles. Our Solution to the Problem: In work currently accepted at Nature Cancer, to address this unmet need we developed a new technology called “structural surfaceomics”. This approach is designed to systematically probe an entirely new class of tumor-selective immunotherapy targets: those based on the cancer-specific, 3- dimensional conformation of plasma membrane proteins. To develop this methodology, we used our joint expertise in cell surface proteomics (Wiita) and crosslinking mass spectrometry (Huang). We initially applied this strategy to acute myeloid leukemia (AML), a deadly blood cancer with no known optimal cellular therapy targets. Using structural surfaceomics on AML models followed by patient sample validation, we identified the active conformation of integrin-β2 (aITGB2) as a promising, previously unknown AML target. Via recombinant antibody selection, we developed an engineered T-cell therapy (CAR-T) targeting aITGB2. We found this CAR- T had a highly favorable efficacy vs. toxicity profile compared to other AML targets under clinical investigation. We are now pursuing further preclinical development of this CAR-T as a therapy for AML patients. Overall Hypothesis and Aims: We hypothesize that tumor-specific surface protein conformations are actually widespread, and the structural surfaceomics technology has the potential to uncover these antigens. The objective of our proposal is two-fold: 1) further advance the technical and analytical aspects of the structural surfaceomics technology, to improve our ability to identify cancer-specific surface protein conformations across a broader range of indications; and 2) identify how we can even further improve therapeutic targeting of this conformation-specific surface antigen aITGB2 in AML, even beyond our current approach. To achieve these goals, Aim 1 will define the landscape of surface protein conformations via enhanced structural surfaceomics across several different tumor models, with validation in primary patient samples. This work will ultimately develop a pipeline to move from target discovery to potential therapeutic. Aim 2 will focus on identifying the mechanistic basis for conformation-specific targeting of integrin-β2 in AML with our current cellular therapy. Impact: Findings in Aim 1 will be broadly impactful toward the adoption and dissemination of the structural surfaceomics technology. These results will ideally not only identify new immunotherapy targets in cancer, but open the door to probing surface protein conformations in any biological system. An Aim 2 will inform design of a further optimized cellular therapy for potential clinical translation, directly benefitting AML patients. The Team: The PIs have an uncommon combination of expertise in im...