PROJECT SUMMARY/ABSTRACT Immunotherapies like checkpoint blockade therapy (CBT) have been a breakthrough for cancer treatment with durable responses observed in patients with advanced disease. However the majority of patients do not respond and factors leading to resistance remain largely unknown. While clinical data have shown that neoantigen load positively correlates with overall survival and response to CBT, there are patients with apparent neoantigens who show partial or no response. Emerging evidence suggests that the context of neoantigen expression can impact the immune response, where tumors with clonal neoantigens expressed by all tumor cells elicit robust anti-tumor immunity and correlate with CBT response while tumors exhibiting a high degree of intratumoral heterogeneity (ITH), where unique neoantigens are expressed by subclones, display weaker responses. How neoantigen expression patterns can elicit such dissimilar responses is not known. To elucidate how heterogeneously expressed neoantigens can dampen the immune response we created a novel murine tumor model to implant tumors where the same set of neoantigens are expressed homogeneously or heterogeneously. Using this system we have observed that tumors with high ITH are weakly immunogenic compared to homogenous tumors. Intriguingly, our preliminary data also suggest that an earlier and more robust T-cell response against a weakly immunogenic neoantigen can be induced if it is expressed with a very immunogenic neoantigen. This response leads to even more efficient tumor control than that observed in the cell line expressing only the strong neoantigen. These preliminary data led us to hypothesize that weaker anti-tumor T- cells responses against heterogeneously expressed neoantigens are mediated by a reduction in dendritic cell- mediated T cell priming. To address this question I propose to: 1. Determine the impact of heterogeneous neoantigen expression on anti-tumor T-cell responses. 2. Determine the mechanistic basis for weaker anti-tumor immune response in heterogeneous tumors. By addressing these aims we will gain critical insight into fundamental factors necessary for a productive anti- tumor immune response which would have significant impact on immunotherapeutic design, particularly for treatment of patients with high ITH. Work on this project will foster my development as an independent scientist as I gain additional experience performing hypothesis-driven science at the Koch Institute at MIT. I will interact with, receive feedback from and collaborate with current and future leaders in cancer biology research here. Furthermore, this environment stresses integration of basic cancer research and bioengineering to drive treatment discovery which will provide me with opportunities to learn how I can translate my discoveries, especially as I further develop my collaborations. Ultimately, this project will train me in the overall approach to asking and addressing fundamental ques...