PROJECT SUMMARY/ABSTRACT A major breakthrough in cancer immunotherapy in the last decade is the development of chimeric antigen receptor (CAR) T cell therapy. Although anti-B cell maturation antigen (BCMA) directed CART cell therapy demonstrated unprecedented initial responses in patients with relapse/refractory B-cell malignancies or multiple myeloma (MM), respectively, durable responses are limited. The mechanisms of relapse after CART cell therapy are not fully unveiled yet. The long-term goal of my proposal is to uncover mechanisms of resistance to CART cell therapy and to develop an independent research career focused on targeting the tumor microenvironment simultaneously with CART cells. The unifying objective of this application is to design novel strategies for the treatment and prevention of tumor immunoescape based on my clinically relevant preliminary data, using BCMA CART cell as a model. The central hypothesis is cancer-associated fibroblast (CAFs) induce CART dysfunction through complex mechanisms and that dual targeting of tumor cells and CAFs is safe and enhances the efficacy of CART cell therapy. To test this hypothesis, I have designed three specific aims: Aim #1) Determine the mechanisms of CAF-induced BCMA CART cell dysfunction in MM patients; Aim #2) Study the potential toxicity of dual targeting CART cells in syngeneic humanized MM-tumor microenvironment (TME) models; Aim #3) Utilize samples from patients who were treated with BCMA CART cell therapy to study the interactions between CAFs and CART cells. The rationale for this proposal is that in my established MM-TME model, CART cell trafficking to the tumor site is significantly inhibited. My preliminary data also showed that CAFs suppressed CART cell functions by secreting TGF-β and inhibitory cytokines as well as altering the PD-1/PD-L1 axis. Dual targeting tumor cells and CAFs significantly improved CART cell functions. This research will be significant because it will contribute depth (of understanding the CART cell resistance) and breadth (of novel potentially curative therapy) to the immunotherapeutic strategy against not just MM but also solid tumors. Ultimately, such discoveries have the potential to vertically advance the field of CART cell immunotherapy as well as other targeted immunotherapies. This project is innovative because it combines high throughput screening techniques to study the possible mechanisms of resistance after immunotherapy and, therefore, to generate novel treatments. The proposed research activities are crucial to the development of the applicant as an independently funded scientist with a focus on cellular immunotherapy. I will receive further training in molecular biology, translational medicine, and immunology from my mentors and training in cancer biology and bioinformatics from experienced collaborators at the Mayo Clinic. Therefore, at the conclusion of the training period, I will have acquired a unique set of intellectual and technical skills that will allow me to promote myself to become an independent translational researcher.

PROJECT NARRATIVE The tumor microenvironment strongly induces chimeric antigen receptor T (CART) cell inhibition in most malignancies. Our studies indicate that cancer-associated fibroblasts (CAFs) specifically inhibit CART cells in multiple myeloma and that dual targeting CAFs and cancer cells with CART cell has a potential to overcome this resistance. I will study the mechanisms of CART cell inhibition by CAFs, potential toxicities of dual CART cell, and the impact of CAFs in patients receiving CART cell therapy.