Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Shari Pilon-Thomas, Ph.D.

Committee Member

James J. Mulé, Ph.D.

Committee Member

José Conejo-Garcia, Ph.D.

Committee Member

Kenneth L. Wright, Ph.D.

Committee Member

Eric Tran, Ph.D.

Keywords

TIL, ACT, PD-1, 4-1BB, OX40, Immunotherapy

Abstract

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) is a promising immunotherapeutic approach for patients with advanced solid tumors. Metastatic melanoma is a tumor type amenable to immunotherapy in part due to the presence of antigen-specific TIL. These T cells can be activated and expanded for ACT, which has resulted in relatively high rates of clinical responses. While numerous advances have been made, the inclusion of neoantigen-specific CD4+ T cells within TIL infusion products remains underexplored and therefore offers a significant opportunity for progress. Similarly, immune checkpoint inhibitors, specifically PD-1 blocking antibodies, augment antitumor immunity and may improve the outcomes for patients in combination with ACT with TIL. Combination therapy has the potential to augment overall treatment efficacy and has been demonstrated as a successful means to reduce patient attrition, effectively allowing greater patient access to TIL therapy. We further hypothesized that anti-PD-1 treatment may increase the influx of tumor-reactive T cells into tumors prior to surgical resection and improve TIL function and persistence following ACT. Chapter Two discusses our Phase I clinical trial for metastatic melanoma patients who received ACT with TIL generated with anti-4-1BB agonistic antibody in vitro in combination with nivolumab infusions. Stage III/IV metastatic melanoma patients with unresectable disease who were anti-PD-1 treatment-naïve were enrolled. TIL were generated in the presence of anti-4-1BB antibody in vitro and expanded for ACT. Patients in Cohort 1 received TIL infusion followed by nivolumab. Patients in Cohort 2 also received nivolumab prior to surgical harvest and during TIL production. Four out of 11 patients achieved a partial response (36% overall response rate) with median progression-free survival (PFS) of 5 months. Responses to therapy were durable and were characterized by relatively high persistence of infused TIL. Expanded TIL were primarily CD8+ T cells with potent antitumor reactivity detected in vitro. Two non-responders who developed new metastatic lesions were analyzed to determine potential mechanisms of therapeutic resistance, which included TIL clonal divergence and intrinsic TIL dysfunction. Additional research specimens suggested that metastatic melanoma tumors from patients who progressed after anti-PD-1 therapy yielded reduced expansion of TIL, which was rescued by supplemental agonistic anti-4-1BB antibody during TIL production. Overall, combination therapy with TIL and nivolumab was safe and feasible for metastatic melanoma patients and provides important insights for future therapeutic developments in ACT with TIL. While the majority of immunotherapeutic approaches aim to augment the CD8+ T cell response, additional opportunities for improved efficacy are evident. CD4+ T cells are present within the tumor microenvironment and TIL infusion products, yet their role in the anti-tumor immune response is poorly defined. As a plastic and polyfunctional population, CD4+ TIL are capable of tumor recognition and consequent effector function; however, their precise role in ACT remains understudied. In Chapter Three, we analyzed TIL from metastatic melanoma patients previously treated with ACT for the presence of neoantigen-specific CD4+ T cells. In both responders and non-responders, we discovered CD4+ TIL clones that recognized neoantigens through an effector T cell response and demonstrated cytotoxicity toward autologous tumor in an MHC Class II dependent manner. Neoantigen-specific CD4+ TIL persisted in vivo following ACT, which has been previously correlated with patient response. These results were validated by paired TCR and single cell RNA sequencing, which elucidated distinct profiles of neoantigen-specific CD4+ TIL. This study supports the identification of neoantigen-specific CD4+ T cells as a potent source of tumor-specific effectors, and advocates for their inclusion in ACT with TIL. In totality, we have explored multiple methodologies to augment ACT with TIL at each phase of TIL generation and activity. While this therapy has demonstrated substantial efficacy, we suggest that a thorough examination of the individual components may enhance our understanding of the limitations and promote improved therapeutic results for patients.

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