Optimization of a modified immunotherapeutic LANA antigen for use in treating persons with HIV/AIDS and refractory KSHV-related cancers (Immuno/Microenvironment). ABSTRACT This PA-20-272 administrative supplement to the UPMC Hillman Cancer Center Support Grant (P30CA047904) is directly responsive to the Immunotherapy and Tumor Microenvironment in HIV/AIDS Cancer Patients at NCI-Designated Cancer Centers Administrative Supplement Announcement for P30 Cancer Centers Support Grants (CCSG). It focuses on research to 1) discover and characterize an immunotherapeutic target (KSHV LANA), 2) by developing a new immunotherapeutic approach (protein engineering to enhance LANA peptide presentation), that 3) improves our understanding of the immunosuppressive environment of KSHV tumors. It is 100% aligned with NIH HIV/AIDS Research Priorities as a new and important approach to treat persistent or recurrent AIDS-related Kaposi sarcoma (KS) in patients who have undergone immune reconstitution through antiretroviral therapy. This proposal also addresses targeting KS in the aging KSHV+ HIV/AIDS population who do not currently have KS but can be expected to be at high-risk for developing KS with aging. The purpose of this study is to optimize LANA as an immunotherapeutic by changing the immunologically “cold” LANA protein into an immunologically “hot” CD8+ cytotoxic lymphocytic (CTL) antigen. Our preclinical mouse study, using an ovalbumin T cell peptide reporter, reveals that the LANA central repeat domain 1 (CR1) specifically inhibits LANA proteolytic processing and MHC I peptide presentation to evade cell-mediated immunity. By deleting the CR1 domain and expressing LANADCR1 in murine MC38 cancer cells, we provoke a robust CTL response with specific recognition and cell killing by OT-1 CD8+ cells. When wild-type LANA- expressing MC38 cells are injected into syngeneic, immune-competent C57BL/6 mice, mice develop monotonically-growing tumors that require termination, consistent with LANA-induced CTL immune escape. In contrast, mice injected with MC38 cells expressing LANADCR1 show brief tumor formation and then tumor regression, consistent with an effective anti-tumor CTL response. Both LANA- and LANADCR1-expressing cells generate indistinguishable tumors when injected into Rag1-/- mice demonstrating that this differential response is due to immune surveillance. During this proposal period, we will further optimize the LANADCR1 as an immunotherapeutic antigen by fragmenting the corresponding ORF73 gene, performing codon-optimization and removing additional repetitive peptide regions and the nuclear localization signal that may contribute to reduced CTL responsiveness. We will immunize mice with the optimized antigen and, after an appropriate boosting schedule, challenge the mice with wild-type LANA only-expressing MC38 cells to determine whether optimized antigen can prevent tumor formation through LANA epitope recognition. Successful conclusion of this study wil...