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Background The complex, immunosuppressive microenvironment of solid tumors necessitates innovative approaches to refining chimeric antigen receptor (CAR) T cell therapy. In the context of glioblastoma (GBM) — a highly aggressive and infiltrative type of brain cancer with a poor prognosis, even with standard-of-care treatment — designing a highly specific CAR that maintains prolonged and potent anti-tumor activity in spite of high interpatient and intratumoral heterogeneity is an ongoing priority. Chlorotoxin (CLTX) — a naturally-occurring peptide found in the venom of the death stalker scorpion ( Leiurus quinquestriatus) — has demonstrated high specificity for GBM tissue and minimal off-tumor toxicity, thus representing a promising candidate as the targeting domain of a novel CAR construct.1 Characterization of the binding dynamics of this CLTX-CAR construct with GBM tissue suggested that tumor expression of membrane-bound matrix metalloproteinase 2 (MMP2) is required for CLTX-CAR T cell binding and activation.1 Methods As part of an ongoing phase I clinical trial evaluating the safety and feasibility of this CLTX-CAR construct in patients with recurrent or progressive MMP2+ GBM, different tissue samples — tumor, both tumor cavity and cerebrospinal fluid, leukapheresis PBMCs and patient-derived engineered CLTX-CAR T cells (referred to as ‘product’) — were collected from 20 patients, 17 of which with known outcomes (stable disease, n = 7, or progression disease, n = 10), and processed using the 10X Genomics Chromium platform. Single-cell multi-omic profiling of these samples enabled characterization of gene expression, T cell receptor clonality, and CLTX-CAR construct expression.Results Analysis of 173,311 product cells yielded 14 distinct clusters and revealed varying degrees of CLTX-CAR expression across clusters, with 36% of cells having at least one sequence read mapping to the construct. In a comparative analysis between CLTX-CAR T products from patients with stable and progressive disease, we observe an enrichment of specific CLTX-CAR+ cytotoxic T cell clusters in patients with stable disease. Parallel analysis of 95,305 leukapheresis PBMCs will enable examination of CLTX-CAR T cell fitness with respect to the characteristics of the population from which they were derived.Conclusions These analyses, together with a single-cell multi-omic and spatial interrogation of a variety of biosamples, will guide the future development of CAR T and other therapies to overcome the immunosuppressive nature of GBM and other tumors.Reference Wang D, Starr R, Chang WC, Aguilar B, Alizadeh D, Wright SL, Yang X, Brito A, Sarkissian A, Ostberg JR, Li L, Shi Y, Gutova M, Aboody K, Badie B, Forman SJ, Barish ME, Brown CE. Chlorotoxin-directed CAR T cells for specific and effective targeting of glioblastoma. Sci Transl Med. 2020 Mar 4;12(533)Ethics Approval The trial ( NCT04214392) was conducted in accordance with the Institutional Review Board, Data Safety Monitoring Committee and Independent Ethics Committee at The City of Hope (COH) National Medical Center as well as the US Food and Drug Administration.