Clinical strategies to enhance the efficacy of tumor-infiltrating lymphocyte (TIL) therapy using preclinical models

Advanced metastatic melanoma refractory to immune checkpoint blockade (ICB) remains exceedingly difficult to treat, highlighting the need for alternative immunotherapeutic strategies. Due to the immunosuppressive mechanisms within the tumor microenvironment (TME), the infiltration and effector function of tumor-associated antigen (TAA) and neoantigen-specific CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) are limited, allowing the tumor to escape immune surveillance and hinder the endogenous immune response. Adoptive cell therapy (ACT) of autologous, ex-vivo expanded TILs was recently approved as a second line of therapy for advanced metastatic melanoma patients refractory to ICB. Despite positive responses observed in a subset of patients, many fail to respond, highlighting the urgent, unmet need to identify strategies which improve TIL-ACT efficacy. We have shown that a lower precursor frequency of self-antigen-specific CD8+ T cells enhances the quality of the antitumor response, whereas self-antigen-specific CD4+ T cells are susceptible to exhaustion at the same frequencies. These preliminary findings suggest that clonal abundance plays a critical role in determining T cell functional state-an unexplored area in the context of TIL therapy efficacy. ICB reinvigorates exhausted T cells under certain conditions, however, whether ICB can also reprogram exhaustion that arises as a consequence of dysregulated precursor frequency or subset composition is unknown. We hypothesize the efficacy of TIL therapy is determined by antigen-specific precursor frequency and T cell subset composition, and combining TIL-ACT with ICB will reprogram the exhausted T cells to sustain the hostile TME. To investigate this, we transferred varied precursor frequencies of neoantigen and tumor associated antigen-specific CD4+ and CD8+ T cells into tumor-bearing mice. Preliminarily, we have found antigen-specific CD4+ T cells to modestly enhance the anti-tumor response to ACT, however, this effect is not mediated through effector function and tumor control. This suggests that antigen-specific CD4+ T cells provide a helper role in ACT products. Next, we have shown upregulated immune checkpoint expression on ex vivo expanded TILs using flow cytometry and aim to assess combinatorial strategies with ICB to improve anti-tumor immune response. Further, we aim to investigate how precursor frequency and CD8+/CD4+ T cell ratio impacts TIL-ACT response by spiking in exogenous transgenic, antigen-specific T cells in the TIL product at varied frequencies.