Abstract 1638: Improved outcomes with drug-resistant immunotherapy in a human xenograft model of glioblastoma multiforme
Authors: Langford Samantha B. , Spencer Harold T. , Dasgupta Anindya , Gillespie George Y. , Sutton Kathryn , Pereboeva Larisa , Lamb Lawrence S. 
Published: July 2017

Abstract
INTRODUCTION: Conventional treatment strategies for high-grade gliomas have been uniformly dismal. We have previously shown that both primary and Temozolomide (TMZ)-resistant glioblastoma (GBM) cell lines upregulate stress-associated NKG2D ligands (NKG2DL) during the first several hours following exposure to TMZ, thereby creating an opportunity for NKG2DL-directed cell therapy, particularly γδ T cells that directly recognize these stress-associated antigens. Using a human/mouse patient-derived tumor xenograft (PDTX) model, we combined TMZ chemotherapy and TMZ-resistant ex vivo expanded/activated γδ T cells as Drug Resistant Immunotherapy (DRI). Drug resistance in this example is conferred by O-6-methylguanine-DNA-methyltransferase (MGMT) gene transfer, thereby enabling cytotoxic lymphocyte function in a chemotherapy-rich environment when the tumor is maximally stressed.
METHODS: A total of five GBM PDTX were examined, three parent (X12P, X22P, X59P) and two TMZ-resistant (X12T, X22T). Tumor NKG2DL expression and cytotoxicity of DRI were assessed using flow cytometry with cultured human astrocytes as controls. Intracranial (IC) glioma xenografts were established using either an unmodified (P) or a TMZ resistant clone (T) of human GBM explants passaged exclusively in immunodeficient mice. Tumor-bearing mice received intraperitoneal 60mg/kg TMZ on days 6, 8,13, and 15 and received IC injection of 1.5 x 106 DRI 4 hours following TMZ injection. Control mice received DRI T cells alone, TMZ alone or no therapy. Survival was assessed using Kaplan-Meier analysis.
RESULTS: All xenografts were found to constitutionally express NKG2DL which were upregulated upon exposure to TMZ. DRI T cells were cytotoxic to all tumors in vitro, showing 65%-80% specific lysis at an E:T ratio of 20:1 with no evidence of toxicity against cultured human astrocytes. Median survival (MS) for all groups of untreated mice was approximately 25 days, and γδ T cell therapy alone did not improve survival in the absence of TMZ. For the parent tumors, TMZ therapy significantly improved MS over untreated controls for both X12P and X59P (29 vs. 59 days and 20 vs 51 days respectively, p =0.0001) and eliminated tumors in X22P. DRI γδ T cells + TMZ significantly increased median survival additionally over TMZ alone with 80% of animals in both X12P and X59P surviving long-term (p=0.0001 and 0.05 respectively). DRI had no clear effect over TMZ for X22T (p=0.46), however, for X12T, DRI + TMZ significantly increased median survival from 22 to 38 days (72.7%) over TMZ alone (p = 0.04).
CONCLUSIONS: Combined TMZ chemotherapy and drug-resistance modified γδ T cell therapy can produce a significant increase in time to progression and improvement in median and overall survival for both primary and TMZ-resistant GBM using a strategy is readily adaptable to the clinical setting.
Citation Format: Samantha B. Langford, Harold T. Spencer, Anindya Dasgupta, George Y. Gillespie, Kathryn Sutton, Larisa Pereboeva, Lawrence S. Lamb. Improved outcomes with drug-resistant immunotherapy in a human xenograft model of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1638. doi:10.1158/1538-7445.AM2017-1638