Tumor infiltrating lymphocyte (TIL) therapy represents a promising approach for the treatment of patients whose disease does not benefit from conventional immunotherapies. While TIL resulted in tumor shrinkage or disappearance in approximately one third of patients with advanced melanoma, it has become increasingly important to develop novel strategies for TIL ex vivo expansion to develop superior TIL in greater numbers and anti-tumor activity that when adoptively transferred into patients will provide improved rates of response and enhanced clinical benefit. Current standards for TIL therapy include surgical excision of patient’s tumor typically from the most accessible metastatic sites, growth of TIL outside of patient’s body in cultures supported by Interleukin-2 (aka T cell growth factor; TCGF) and subsequent infusion of these expanded TIL into patients after undergoing chemotherapy. The first part of the project evaluatesif the quality of TIL can be improved through addition of a combination costmulatory CD40L-4-1BB antibody into ex vivo TIL cultures. We hypothesize that CD40L-4-1BB will increase number of TILs and result in production of better quality of TIL which is characterized by higher percentage of tumor specific so called “reactive” TIL. Adding additional growth molecules during growth of TIL outside of body hasthe potential to improve TIL characteristics and translate to improved responses to TIL therapy and survival of patients with melanoma. The second part of this project investigates whether TIL can successfully growth from core biopsy samples and evaluates what unique clinical, pathological, and immunological characteristics of tumor tissue impact the quality of the TIL product to inform best anatomic sites for initial TIL harvest. Current standard of performing surgery of tumors to growth TIL is mainly based on targeting either skin or lymph node samples. Tumors are heterogenous and with this strategy visceral metastases are often not used to grow TIL, indeed, those metastatic sites also appear to determine disease outcome. We postulate that obtaining tumor cores from visceral metastatic locations such as lung, liver will result in superior generation of tumor-reactive TIL outgrowth and effector functionality. We will obtain additional cores “passes” from biopsies which were ordered as standard of care procedure for generation of TIL and study how the TME of core biopsies impacts the quality of expanded TIL. The studies will involve morphological and cellular interrogation of tumor sections and profiling of the TIL product. If successful, these studies will establish a workflow for effective outgrowth of TIL from core biopsies which can avoid the need for additional surgical procedures for patients with advanced melanoma. Furthermore, this strategy will open a new avenue for patients those tumor metastatic locations would otherwise disqualify them from access to receiving TIL therapy due to their “unresectable” tumors.