Devising Advanced TCR-T-cells to eradicate OSteosarcoma

Success rates in treatment of Osteosarcoma (OS), an aggressive cancer with many fatalities in affected children and adolescents, have not improved over the last 40 years. In view of the sizable number of OS-associated mutations and opportunities provided by innovation-driven personalized T-cell-based biomedicine with ever-increasing curative potential, such stalemate is no longer acceptable. As a therapeutic anchor point, OS-related tumor antigens (TAs; both tumor-associated and tumor-specific neoantigens) are displayed on tumors via MHC molecules as short peptides for surveillance by TA-specific tumor-infiltrating CD4+ helper and CD8+ cytotoxic T cells (TILs, CTLs). If equipped and unless undercut by tumor immune evasion mechanisms with adequate TCRs, antigen-scanning CTLs are poised to kill targets in the presence of even a single antigenic peptide/MHC complex (pMHC). Considering the complexities characterizing interactions between OS and the T-cell compartment yet also the limits on TCR-reactivity set by negative thymic selection, we predict that achieving game-changing breakthroughs mandates a highly concerted multidisciplinary approach. To this end our team will combine its considerable expertise in cancer biology, T-cell and molecular immunology as well as in biotechnology and systems biology. More specifically, we seek to identify personal OS-associated antigens and their matching TA-specific TCRs as well as T-cell and OS-intrinsic regulatory mechanisms underlying immune evasion to ultimately engineer autologous CTLs with enhanced tumor clearing capacity and find entry points for OS-preconditioning in immunotherapy. For this we will express recombinant tumor-enriched “orphan” TCRs isolated from TILs of OS patients to screen yeast-displayed peptide/MHC (pMHC) libraries with vast epitope coverage for nominal TAs. As a complementary approach we will first classify T-cell epitopes based on their relevance for tumor lysis and then determine cognate TCR matches. Following functional validation of TCR-epitope matches in vitro, in tumor sections and in vivo, we will combine computational, biophysical and protein engineering approaches to derive functionally enhanced patient-specific TCRs. In parallel, to inform the design of TME-resilient T-cells and drug-based OS-preconditioning for effective immunotherapies, we seek to delineate through CRISPR-Cas9- and DNA-barcoding-based screening T-cell- and OS-intrinsic mechanisms underlying immune evasion. Will focus on OS-related strategies to interfere with surface expression and TCR-accessibility of MHC class I. We further intend to identify as of yet unknown T-cell- and OS-intrinsic pathways undermining CTL-antigen sensitivity and effector function for improved therapeutic intervention. We expect to lay the foundation and streamline curative OS treatment in Austria and beyond and to pioneer effective and low-toxicity targeting of other solid tumor entities.