Improved Outcomes Following Drug-Resistant Immunotherapy in a Hunan Xenograft Model of Temozolomide-Resistant Glioblastoma Multiforme

INTRODUCTION: Conventional treatment strategies for Temozolomide (TMZ)-resistant high-grade gliomas have been uniformly dismal. Our previous studies have shown that TMZ-resistant tumors 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 ex vivo expanded/activated γδ T cells and NK cells that directly recognize these stress-associated antigens. Using a human/mouse primary xenograft model, we report improved survival using a combination of TMZ chemotherapy and gene modified TMZ-resistant γδ T cells + NK cells which we term Drug Resistant Immunotherapy (DRI). Drug resistance is conferred by lentivector transfer of methylguanine methyltransferase (MGMT), enabling cytotoxic lymphocyte function in a chemotherapy-rich environment at a time when the tumor is maximally stressed and regulatory T cells are depleted.

METHODS: Tumor NKG2DL expression and cytotoxicity of DRI were assessed using flow cytometry. Intracranial (IC) glioma xenografts were established in immunodeficient mice using either an unmodified (P) or a TMZ-resistant clone (T) of human GBM-X12 primary explants. Tumor-bearing mice were treated with an intraperitoneal 60mg/kg dose of TMZ on days 6, 8,13, and 15 post-tumor placement and received IC injection of 1.5 x 10⁶DRI 1x/week x 2weeks on post-injection days 7 and 14 or 2x/week x 2 weeks following each TMZ dose by 4h. Control mice received non-modified cells, TMZ alone or no therapy. Survival was assessed using Kaplan-Meier analysis.

RESULTS: Both GBM-X12P and X12T express stress antigens MIC-A, MIC-B, and ULBP-4. DRI cells tested for potency in vitro killed both tumors at approximately 80% at an effector: target ratio of 20:1 and showed no evidence of toxicity against cultured human astrocytes. Cell therapy alone did not improve survival beyond that of untreated mice for either tumor.  For the unmodified parent tumor X12P, both TMZ therapy and TMZ + DRI significantly improved survival over untreated controls (p < 0.0001), and the combined therapy increased median survival from 57 to 75 days over TMZ alone. Combined therapy with TMZ + DRI also marginally improved survival for X12T (p = 0.0147) extending median survival over TMZ alone from 22 to 27 days (p = 0.0966).  Keeping the TMZ dosing schedule constant, intensification of DRI therapy from 1x/week to 2x/week increased median survival from 22 days to 38 days over untreated animals (p = 0.0004) and signiicantly improved medan survival over TMZ-treated animals from 27 to 38 days (p = 0.0017) with 10% of animals showing long-term survival.

CONCLUSIONS: The combination of chemotherapy-induced tumor stress and targeted DRI significantly increases time to progression and improves survival in immunodeficient mice bearing otherwise impervious temozolomide-resistant human xenograft tumors.