Abstract 3193: Developing non-exhausted tumor infiltrating lymphocytes (TILs) as a therapy for glioblastoma
Authors: Hotchkiss Kelly M. , Noldner Pamela , Singh Kirit , Patel Anoop , Fecci Peter , Antonia Scott , Shaz Beth H. , Khasraw Mustafa 
Published: April 2023

Abstract
Glioblastoma is the most prevalent primary brain malignancy, with a median survival of less than 21 months despite treatment with surgery, radiation, and chemotherapy. Significant intra-tumoral heterogeneity, combined with the immunosuppressive micro-environment and the limited immune population present within the privileged CNS has resulted in the failure of immunotherapy approaches. We seek to address these challenges by isolating Tumor Infiltrating Lymphocytes (TILs) which are already neoantigen specific and expanding them ex vivo before re-administering them to the patient. This allows us to greatly increase the available pool of T cells which are specific for a diversity of tumor neoantigens in the patient, while also reversing exhaustion by the addition of stimulatory co-culturing agents. We report the successful expansion of glioblastoma associated TILs from both surgically resected tumors (5 of 7 samples, 71%) and liquid aspirate from the surgical field collected in Lukens traps (4 of 6 samples, 66%). TILs were expanded with high-dose IL2 initially in the presence of tumor digest (PreREP) followed by activation and expansion with CD3/CD28 nanobeads (REP). Flow cytometry performed on PreREP TILs revealed that 50-90% of the population consisted of effector memory T cells (CCR7-CD45RA-) with 5-40% consisting of central memory T cells (CCR7+CD45RA-). The addition of stimulatory agents such as anti-4-1BB (Urelumab) during the PreREP phase increased cell yields, as well as skewing the population towards a non-exhausted CD8+ phenotype (reduced expression of PD1, LAG3, TIM3). These cells have demonstrated tumor antigen specific activation based on IFNγ and TNFα secretion following stimulation with autologous tumor digests and patient matched neurosphere cultures. TIL expansion with Urelumab doubled CD8% in the product (16% to 31%) and reduced CD4% (78% to 65%). Expanded TILs had more potent cytotoxic CD8+ T cells responses characterized by significantly increased numbers of tumor antigen specific IFNγ+ (std: 1000, Ure: 6000) and TNFα+ (std:2000, Ure:20,000))cells compared to traditionally expanded TILs. We have demonstrated that TIL isolation from resected clinical glioma is feasible and can generate large quantities of immune cells whose functional status can be altered by the addition of stimulatory agents. And most importantly these TILs are reactive and specific for tumor antigens. Following additional assessment of in vitro cytotoxicity against matched tumor neurospheres in vitro we will evaluate safety and feasibility of our novel TIL approach in patients with glioblastoma.
Citation Format: Kelly M. Hotchkiss, Pamela Noldner, Kirit Singh, Anoop Patel, Peter Fecci, Scott Antonia, Beth H. Shaz, Mustafa Khasraw. Developing non-exhausted tumor infiltrating lymphocytes (TILs) as a therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3193.