ACTIVATING THE 4-1BB PATHWAY FOR THE EXPANSION OF TUMOR-INFILTRATING LYMPHOCYTES FOR ADOPTIVE T-CELL THERAPY FOR METASTATIC MELANOMA PATIENTS
This dissertation project focused on improving the quality of the tumor-infiltrating lymphocytes (TIL) used in Adoptive T-cell therapy by understanding the role of 4-1BB/CD137 co-stimulation during the expansion of the tumor-infiltrating lymphocytes (TIL). Adoptive T-cell therapy using TIL is a promising therapy for late stage melanoma patients, resulting in a 50% response rate and durable long-term survival in over 20% of patients. Current research is aiming at improving the quality of the expanded cells and their persistence in vivo after adoptive transfer to further boost response rates. The specific focus of this dissertation project is the testing of agonistic anti-4-1BB antibodies at different stages of TIL expansion from tumors for its effects on modulating the phenotype and anti-tumor activity of the cells. The expansion of TIL from the melanoma tumor occurs in 2 stages: The first stage involves the initial expansion of the TIL from small cut 3-5 mm2 fragments of viable tumor (12-24 fragments/tumor) with Interleukin-2 (IL-2) over a 4-5 week period; TIL isolated after this stage are referred to as ‘pre-REP’ TIL. The second stage involves the pre-REP TIL undergoing a secondary expansion referred to as the rapid expansion protocol (REP) for a period of 2 weeks in which the TIL are activated through the T-cell receptor (TCR) and also provided IL-2 to trigger rapid cell division. The TIL are then referred to as ‘post-REP’ TIL after this expansion. We have found that 4-1BB is expressed on freshly isolated T cells that are within melanoma tumor fragments, as well as expressed on pre- and post- REP TIL. This observation prompted us to investigate what the role of 4-1BB ligation would play during the expansion of the TIL. We demonstrated that providing co-stimulation to the TIL during the initial expansion stage and during the secondary expansion using an agonist anti-4-1BB antibody facilitated an increased expansion of CD8+ TIL with increased cytolytic function and a phenotype of memory T cells with enhanced cell survival gene expression. TIL receiving 4-1BB co-stimulation during expansion also exhibited longer persistence and increased anti-tumor activity in an in vivo human TIL adoptive transfer model using NOD-SCID-gamma chain-/- (NSG) mice xenografted with HLA-A-matched melanoma cells. The post-REP TIL also exhibited improved responses to antigenic re-stimulation when the anti-4-1BB antibody was added during expansion.
We also investigated the role of 4-1BB ligation in the tumor microenvironment ex vivo in the tumor fragments used as the source of the expanded TIL. We found that in addition to 4-1BB being expressed on T cells in these fragments 4-1BB was also expressed on dendritic cells within the melanoma tumor fragments. Addition of agonist anti-4-1BB increased activation and NFκB (a key marker of 4-1BB signaling) in these dendritic cells and T cells, that was associated with the increased proliferation and activation state of the CD8+ T cells growing out of these fragments. Moreover, 4-1BB co-stimulation in these early tumor fragment cultures also significantly enriched the tumor specificity of the TIL, as found by an increase in the frequency of tumor-specific CD8+ T cells in single cell and bulk anti-tumor reactivity assays.
In conclusion, our results demonstrate that enhancing 4-1BB co-stimulation at different stages of melanoma TIL expansion ex vivo increases the CD8+ TIL yield, greatly increases tumor specificity, and enhances the effector-memory phenotype of the cells conducive to improved persistence and anti-tumor activity in vivo during adoptive cell therapy. Our results indicate that addition of an anti-4-1BB antibody during the initial and/or secondary expansion of the TIL in the clinic we will result in a significantly enhanced TIL product than with currently used expansion protocols that will boost clinical response rates and durable long-term survival in treated patients.