Chlorotoxin, a peptide toxin component of scorpion venom, binds selectively to glioblastoma and other neuroepithelial tumors, with minimal binding to non-malignant cells. We have recently developed a CAR T cell incorporating chlorotoxin (CLTX) as its target recognition domain, and CLTX-CAR T cells are now in clinical trial for recurrent glioblastoma (NCT04214392). We determined in preclinical studies that surface MMP-2 was required for CLTX-CAR T cell killing. However, the precise composition and structure of the cell surface complex recognized by CLTX-CAR T cells remains an unresolved question of increasing importance. Previous investigations have variously proposed matrix metalloprotease-2 (MMP-2), ClC-3 chloride channels, regulators of MMP-2, annexin A2, and neuropilin-1, as receptors, or components of receptors, for CLTX. To approach this question, we have visualized bound Cy5.5-conjugated CLTX peptide (CLTX.Cy5.5) or biotin-conjugated CLTX peptide (CLTX.biotin) on tumor cells in fixed sections of patient resections, on tumor cells in organotypic cultures of patient resections, and on cells of cultured patient-derived glioblastoma tumor lines. At tissue- and cell-level spatial resolution, we saw good correlation of CLTX binding with MMP-2 expression in patient tumor samples and on cultured GBM cells, and between CLTX binding and tumor cell death (cleaved caspase-3) in organotypic GBM cultures. However, at subcellular resolution, surface binding of CLTX was related to, but not precisely overlapping, with MMP-2 or other putative receptors. Rather, on fixed PBT003, PBT030 and PBT106 cells in monolayer cultures, MMP-2 staining was clustered, with diffuse loosely associated CLTX.biotin staining. Diffuse distribution of CLTX.Cy5.5 was also seen in fixed xenograft sections of PBT003-4, PBT1206, PBT030-2, PBT051 and PBT138 tumor cells, and not obviously associated with more discrete staining for IL13Rα2 and EGFR. Ongoing experiments are further examining association of CLTX with other putative components of CLTX receptor complexes, and their redistribution during binding by CLTX and CLTX-CAR T cells.
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