PROJECT SUMMARY Since the 1990s, pediatric oncology has made sustained strides in understanding the biology that drives children’s cancers, but these understandings have not yielded significant therapeutic gains. Approximately 20% of children diagnosed with cancer today are not cured with current standard regimens and cancer remains the leading cause of disease related death in children in the US and other high-income countries. Nearly all current standard treatments are cytotoxic regimens, which are associated with shortened lifespans, high rates of severe late effects and second malignancies and diminished quality of life. There is an urgent need to develop safer and more effective, targeted therapeutics for treatment of children’s cancers. During the last three decades, the Mackall lab has made sustained contributions to a growing body of evidence demonstrating that immunotherapies can selectively target even the most aggressive cancers, raising the prospect that immunotherapies specifically designed for children’s cancers could improve cure rates and diminish toxicity. This program will build upon deep understandings of fundamental cellular and molecular immunology in the Mackall lab and recent progress in defining mechanisms of immune evasion and resistance to current immunotherapies to create next generation immunotherapies for children’s cancer that manifest enhanced safety and potency and potentially enhance access to patients. We will leverage a surfeit of emerging synthetic biology and protein engineering platforms emanating from scientific labs at Stanford University which have been, and continue to be, integrated into the Mackall laboratory. This work will occur within the enabling ecosystem provided by the Stanford Center for Cancer Cell Therapy, which is led by the PI and supports infrastructure to enable rapid forward and reverse translation of promising therapeutics. Areas of focus will include, but are not limited to, developing new approaches to overcome the suppressive tumor microenvironment in pediatric solid tumors, optimizing multi-specific CAR T cells, using combinatorial engineering to create uber potent T cells while enhancing understanding of toxicities that may limit application of these enhancements. We also will enter the promising new arena of in vivo gene delivery to engineer immune populations without the need for cumbersome and expensive ex vivo manufacturing. We have already demonstrated expertise in utilizing CRISPR/Cas9 for screens and therapeutic manipulation and are routinely employing synthetic biology technologies including CRISPR/Cas13 to regulate RNA, base editing to knockout genes without double strand breaks, mutagenesis combined with yeast display to create proteins with unique and specific properties, drug regulatable platforms to control protein expression and viral free cell engineering. Biopharma does not prioritize development of therapeutics for children’s cancers, and thus this Program addresses...