Uncovering mechanisms of adoptive cellular therapy dysfunction in rhabdomyosarcoma
Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, carries a dismal 5-year overall survival rate of 12-30% for metastatic disease. Cellular immunotherapies, including chimeric antigen receptor (CAR) T cells, have emerged as a method to overcome the immune “desert” of the RMS tumor microenvironment (TME) by directing T cells to recognize sarcoma antigens and effect clearance of these tumors. Early CART trials have highlighted the potential of this approach, with evidence of some RMS patients demonstrating complete response. However, in most RMS patients, the efficacy of CART therapy is limited by poor expansion and dysfunction of CARTs within the TME. There is a gap in knowledge about the mechanisms limiting CART function within the RMS TME, significantly hampering our ability to design rational combination therapies to effectively reverse CART dysfunction and cure RMS patients. To move this powerful therapy forward, we must identify ways to uncover and address RMS-mediated CART inhibition.
Data suggest that CARTs create inflammation in the RMS TME, which can contribute to T cell exhaustion and dysfunction. Further, targeting the immune checkpoint B7-H3, for which a CAR is in clinical trials, changes the immunosuppressive makeup of RMS tumors. With the RMS TME fundamentally altered by CART infiltration, these data highlight the need to study the TME post-CART therapy to identify mechanisms of CART inhibition. However, with limited RMS patients in clinical trials and few opportunities to obtain tumor samples after CART therapy, modeling the TME post-CARTs will be critical. I will use an immunocompetent murine model of RMS using the M39M cell line along with murine B7-H3 CART cells to model CART therapy. My first goal will be to test the combination of CART therapy with inhibitory receptor (IR) blockade against PD1 and Lag3. IR blockade has previously shown little benefit in pediatric sarcomas, likely due to low levels of inflammation and exhaustion in the RMS TME at baseline. However, the inflammation created by CART infiltration should drive IR-mediated CART cell exhaustion in the RMS TME, rendering the CARTs sensitive to IR blockade. Further, targeting B7-H3 should promote an influx of anti-RMS T cells similarly susceptible to IR-mediated exhaustion. Therefore, I hypothesize that IR blockade will synergize with B7-H3 CART therapy in RMS. In the second aim, I will use this murine RMS model to further characterize the changes in the RMS TME post-CART therapy. Single cell RNA/ATAC sequencing will identify changes in the intra-tumoral immune populations post-CART therapy, permitting design of new combination strategies to boost CART function by targeting the dominant suppressive cells in the TME. In all, the aims in this proposal will significantly advance our understanding of CART therapy in RMS, providing rationale to design new clinical trials that make use of optimal combination therapy strategies.