Abstract 4202: Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy

Abstract

Abstract High-grade gliomas are the most aggressive and refractory of brain tumors. Historical approaches to treating glioblastoma (GBM) using chimeric antigen receptor modified T cells (CAR-T) have been challenging, without a lasting benefit to patients, often due to tumor heterogeneity and a lack of tumor specific antigens. We report preliminary in vitro design and optimization of a γδ T cell-based CAR-T using Chlorotoxin (CLTX-CAR), a peptide isolated from the venom of the Israeli Deathstalker Scorpion that efficiently binds to GBM and other tumor cells with limited cross reactivity to normal tissues. This CLTX-CAR (CTLX-CAR-noZ) omits a CD3z signaling domain to maintain the natural innate targeting and tumor recognition properties of the γδ T cell through its endogenous T cell and NKG2D receptors, and CD16-based antibody-dependent cellular cytotoxicity (ADCC). This construct also includes our Drug Resistant Immunotherapy (DRI) platform that incorporates an additional p140K-MGMT encoding gene into the construct. This confers resistance to alkylating agents such as temozolomide (TMZ) that allows our DRI γδ T cells to remain viable and functional when combined with chemotherapy to force high-expression of natural killer group D ligands (NKG2DL) on the surface of tumor cells. As expected, the non-signaling, CLTX-CAR-noZ modified Jurkat cells did not result in T cell activation, as compared to the highly activated CD69 corresponding to signaling CLTX-CAR-CD3z transduced Jurkat cells. Conversely, CTLX-CAR-noZ transduced γδ T cells demonstrated higher cytotoxicity against U87MG and U251MG GBM cell lines over unmodified control γδ T cells. Dual-CLTX-CAR constructs lacking CD3z and using 2x CLTX peptides as the targeting domain (dCLTX-CAR-noZ) exhibited up to 90% lentiviral transduction efficiency into γδ T cells. When co-cultured in suspension with GBM cells in vitro, dCLTX-CAR-noZ γδ T cells demonstrated an average cytotoxicity of 62.1% against U87MG-GFP cells at an E:T ratio of 4:1 in comparison to 35.4% with non-transduced γδ T control cells. Time-lapsed microscopic recording over a period of 15hrs also verified killing of U251MG-GFP cells by dCLTX-CAR-noZ γδ T cells when cocultured with GBM cells at an E:T ratio of 2:1. Serial-killing of tumor cells by γδ T cells was documented. We are advancing this approach to xenograft animal models and as a clinical development candidate (INB-300) for both GBM and other extracranial tumors. Citation Format: Lei Ding, Yanjie Li, Evan Klumpp, Lawrence Lamb. Dual chlorotoxin and methylguanine-DNA methyltransferase (MGMT) γδ-T cells for drug resistant immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4202.