Activating The 4-1Bb Pathway For The Expansion of Tumor-Infiltrating Lymphocytes For Adoptive T-Cell Therapy For Metastatic Melanoma Patients

<p>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 <em>in vivo</em> 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 <em>in vivo</em> 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.</p> <p>We also investigated the role of 4-1BB ligation in the tumor microenvironment <em>ex vivo</em> 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.</p> <p>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 <em>in vivo</em> 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.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2014

Author dc:creator

• Chacon, Jessica Ann

Contributors dc:contributor

• Laszlo Radvanyi
• Dean Lee
• Greg Lizee

• Immunotherapy
• melanoma
• CD8
• tumor infitrating lymphocyte
• CD137
• 4-1BB
• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/440

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1479