PROJECT SUMMARY Research Question to be Addressed: The application of immunotherapies, such as immune checkpoint inhibitors (ICIs), in the treatment of cancer has led to significant improvements in long-term patient outcomes. However, existing therapies are not effective in all patients, and our knowledge of the immunological factors that contribute to positive treatment responses and resistance remains limited. This demands targeted research efforts to more comprehensively characterize immune profiles in patients receiving immunotherapies, as a means to uncover biological mechanisms of action and identify more effective biomarkers of treatment response or resistance. Novel tumor- infiltrating lymphocyte (TIL) therapies hold great promise in the clinic for cancer patients that have proven refractory to other treatment strategies. This proposal will take a multi-omic and data-driven approach to deeply characterize immunological signatures in blood and tumor tissues obtained from melanoma patients participating in a clinical trial testing the efficacy of a novel TIL therapy. Our goal is to identify specific biomarkers that associate with treatment outcomes. This effort will result in unique and rich immunological datasets and novel tools and models for clinical outcome prediction and will drive the design of next-generation immunotherapies. Impact of Proposed Team Science: To effectively address the research questions posed, we have assembled a team of researchers with diverse expertise and skill sets. Our team builds from our parent COBRE, drawing on the extensive expertise of MPIs Chesney and Yan, who are world leaders in cancer immunology and clinical research. Both will make critical contributions through administrative oversight. Our research team includes three independent investigators with established research programs in cancer immunology (Yaddanapudi; Aim 1), genomics and immunogenetics (Watson; Aim 2), and bioinformatics and data science (Park; Aim 3). Studies proposed in Aims 1 and 2 are focused on the deep characterization of immunological profiles of melanoma patient tumors, TILs, and blood collected pre- and post-treatment using multi-omics approaches. These aims will delineate immunological variables that vary between tissues and patients in the context of TIL therapy. Given the complex nature of the high-dimensional datasets to be generated in Aims 1 and 2, the activities planned for Aim 3 will be critical to advancing the overall objectives of the proposal. Specifically, studies proposed in Aim 3 will focus on building more streamlined pipelines and tools for processing and analysis of data generated in Aims 1 and 2. In addition, the second major activity of Aim 3 will include the construction of novel machine learning models for predicting therapy outcomes, via the integration of datasets (from Aims 1 and 2) with the rich de-identified clinical data that will be collected as part of the trial. Critically, the completion of the Aims outlined here will lead to a foundational compilation of integrated datasets and accompanying models that can be extended into the later phases of this clinical trial and future efforts. These deliverables will also serve as key preliminary data for pursuing hypothesis-driven multi-PI R01 applications and advancing efforts for obtaining an NIH SPORE award focused on the treatment of melanoma. Goals of Parent COBRE Award: The overarching objective of the parent COBRE award was to establish the Center for Cancer Immunology and Immunotherapy (CCII), a multidisciplinary research center of excellence, focused on advancing discoveries in cancer immunology to improve the development of novel immunotherapies. The motivation for this program was built around the 10-year successes of the UofL-Health-Brown Cancer Center in leading clinical trials of immunotherapeutic agents. Leveraging this long-term track record, the CCII COBRE has focused on more effectively integrating clinical and basic research endeavors across the School of Medicine. The CCII is a unique program, being the only COBRE program exclusively focused on cancer immunology and immunotherapy development. This is evidenced by the fact that, in the initial funding period, all scientific/research objectives led by Junior Faculty and their mentors were centered around applying novel approaches to identifying immunological factors linked to cancer progression and/or treatment outcomes. Through these efforts, our Center Investigators have been able to establish the infrastructure and resources, including critical tools and methodologies, needed for generating large-scale genomic and immunological data in human and animal model systems. This success has laid a foundation for us to orient our research capabilities toward solving key questions in the clinic. Specifically, this Team Science Development Project proposal will be focused on enhancing discoveries from a unique immunotherapy clinical trial currently being conducted at our Cancer Center and seeks to capitalize on our successes by establishing a critically needed research team, composed of COBRE graduates (Drs. Yaddanapudi and Watson) and additional faculty across the School of Medicine (Drs. Park, Rouchka, and Smith).

Decades of research have demonstrated that the immune system can eradicate cancer cells but only if immune evasion mechanisms, such as immune checkpoint proteins, can be overcome. Multiple new immunotherapeutic approaches have recently been developed that can induce durable remissions in patients with advanced cancers – unfortunately, the majority of patients still succumb to their cancers. The goal of this COBRE application is to establish a new Center for Cancer Immunology and Immunotherapy that will focus on understanding immune escape mechanisms, developing new approaches to activate/reinvigorate anticancer immunity, training promising young cancer immunologists and improving the research infrastructure of the University of Louisville.