Introduction: Recent evidence supports the crucial contribution of innate immunity to chemotherapy-based cancer treatments. Our previous in vitro modeling shows improved killing of glioblastoma (GBM) cell lines when γδ T cells are combined with alkylating agents such as Temozolomide (TMZ), which cause DNA damage but are also lymphodepleting. We now report findings from combined TMZ therapy with MGMT-modified γδ T cells (Drug Resistant Immunotherapy - DRI) in a xenograft (PDXT) mouse model of primary and recurrent GBM.

Methods: Effectorγδ T cells were manufactured from apheresis product in media containing 5mM Zoledronic Acid and 50U/mL rhIL-12. Cultures were transduced with a P140K-MGMT lentivector, maintained for up to 14 days, harvested, and evaluated in vitro for PDXT lysis +PD-1, CTLA-4 and PD-L1 by flow cytometry. Primary (P) and recurrent TMZ-resistant (T) GBM were modeled orthotopically using classical JX12P and mesenchymal JX59P intracranial (IC) xenografts versus JX12T, JX22T and JX59T. After placement of IC xenografts, mice received concurrent intraperitoneal TMZ 60mg/kg and 1 x 106IC DRI γδ T cells on post-tumor days 6, 8,13 and 15. Control mice received either γδ T cells, TMZ, or no therapy. Survival was assessed by Kaplan-Meier analysis.NKG2DL and PD-L1 expression was assessed on FFPE sections of tumor during therapy and at termination.

Results: DRI-treated JX12P mice showed significant improvement in median survival (MS) over TMZ alone (p=0.0001) with 80% tumor-free survivors. DRI also improved survival for JX59P-bearing mice with 60% survivors as compared to 30% for mice receiving TMZ alone (p=0.044). TMZ reduced tumor growth in primary but not TMZ-resistant PDXT-bearing mice while γδ T cells alone had no survival benefit over untreated controls for any model. For JX12T, DRI improved MS from 27 to 38 days (+41%) over TMZ alone (p=0.017). JX22T and JX59T tumors were resistant to DRI (p=0.0966 and 0.1390). DRI γδ T cells upregulated PD-1 and CTLA-4 over baseline and showed improved function against TMZ-resistant xenolines after blockade of PD-1 and/or CTLA-4 by as much as 200%. TMZ induced upregulation of NKG2DL (MIC-A/B, ULBP-4), and PD-L1 on both primary and TMZ-resistant PDXT following TMZ injection and for several days thereafter.

Conclusions: Primary GBM PDXT are significantly more sensitive to DRI than TMZ-resistant GBM suggesting that DRI would be more effective if administered as adjuvant therapy in the TMZ-maintenance phase following primary resection and chemo/radiotherapy. Additionally, TMZ increases GBM immunogenicity by upregulating NKG2DL expression but also PD-L1. Checkpoint blockade improves γδ T cell function. These findings show the potential to augment the effect of DRI and improve immune effectiveness against more resistant tumors.

Citation Format: Sailesh Pillai, George Y. Gillespie, Louis B. Nabors, Samantha Langford, Catherine P. Langford, Lawrence S. Lamb. Chemotherapy, checkpoint inhibition, and MGMT-modified adoptive gamma-delta (γδ) T cell-based therapy to treat post-resection, primary glioblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2262.