While recent advances in immunotherapies have shown promise in extracranial tumors, Glioblastoma Multiforme (GBM) has remained impervious to these advances with a consistent median survival of 15 months. We have developed a novel approach to the treatment of primary GBM by combining simultaneous intracranial administration of gene-modified γδ T cells and standard temozolomide (TMZ) maintenance chemotherapy. These γδ T cells are transduced with O-6-Methylguanine-DNA Methyltransferase (MGMT), conveying resistance to alkylating chemotherapies, thereby allowing effector function at therapeutic concentrations of TMZ when tumor NKG2DL expression is significantly elevated. We modified γδ T cells with a lentivector construct expressing a chimeric antigen receptor (CAR) with a chlorotoxin binding domain (CLTX-CAR) to improve GBM targeting. CLTX is a small natural peptide derived from scorpion venom that specifically targets altered expression of matrix metalloproteinase 2 (MMP2) and chloride channel CLCN3 on GBM. MGMTp140k was co-expressed within the same CLTX-CAR vector to confer TMZ resistance to the CAR-T cells. The CLTX-CAR vectors contain a CD8α signal peptide, a mono or dual CLTX binding domain, a Myc-Tag peptide, a CD8α hinge domain, a CD28 transmembrane domain, and a costimulatory domain followed by a CD3ζ activation domain. We used P2A peptide to co-express MGMTp140k with the CAR. Initially, we demonstrated efficient transduction of the CLTX-CAR with a dual-CLTX construct as the binding domain (dCLTX-CAR) in Jurkat T cells. Cell surface localization of the dCLTX-CAR was verified by flow cytometry. When compared with the mono-CLTX-CARs (mCLTX-CARs) transduced Jurkat cells, the dCLTX-CARs demonstrated increased CD69 activation (MFI= 4873 vs 1078) and greater cytotoxicity against tumor cells in vitro. The modified T cells also demonstrated enhanced cytotoxicity against tumor cells under TMZ exposure in vitro. Since γδ T cells recognize and kill tumors through NKG2DL stress antigen recognition, we hypothesized that a CLTX-CAR without an activation signal may be sufficient to enhance recognition and cytotoxicity of γδ T cells to tumor cells and mitigate CAR-T activation-induced cell death. We then developed dCLTX-CAR constructs without the CD3ζ domain (dCLTX- Δz-CARs). As expected, the dCLTX-Δz-CAR lentivirus transduced Jurkat cells demonstrated enhanced cell-cell binding compared to the full dCLTX-CAR but no CD69 expression when co-cultured with GBM cells. Overall, we were able to generate dCLTX-CAR T cells with resistance to TMZ and showed improved activation and cytotoxicity against GBM cells under TMZ exposure. Our approach of combining the dCLTX-CAR and TMZ resistance will be further validated in animal model experiments and could be a potential candidate for clinical development for GBM.
Citation Format: Lei Ding, Lawrence S. Lamb. Dual chlorotoxin and methylguanine methyltransferase γδ-T cells for drug resistant immunotherapy of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1490.