We have shown that antigen escape tumor cell variants prevail in glioblastoma (GBM) recurring after treatment with chimeric antigen receptor (CAR) T cells with a single specificity. Recurrent tumors preserve alternative non-targeted tumor associated antigens. We thus hypothesize that a bispecific CAR will mitigate antigen escape, enhancing the antitumor activity of T cells. Two validated glioma-associated antigens, HER2 and IL13Rα2 are currently targeted in phase I GBM trials using CAR T cells. We used computational modeling to design and create a bispecific CAR molecule with a HER2-specific scFv joined in tandem to an IL13Rα2-binding moiety in the CAR exodomain (Tandem CAR) and a CD28.ζ signaling endodomain. GBM patients' Tandem CAR T cells showed distinct binding to soluble HER2 and IL13Rα2 and killed primary autologous GBM cells. Tandem CAR T cells exhibit significantly enhanced activation dynamics when compared to conventional HER2 or IL13Rα2 CAR T cells and better controlled established GBM xenografts in an orthotopic murine model by offsetting both HER2 and IL13Rα2 escape. To investigate if the Tandem CAR T cells were ‘bispecific’ in nature, we determined the localization of the tumor antigens at the CAR T-cell/GBM contact point, also referred to as the immunological synapse (IS). Three-dimensional reconstitution and quantification of confocal images of the Tandem CAR T-cell/tumor interface revealed enhanced bi-functional IS compared to conventional CARs. Further interrogation of the tumor antigen co-clustering at the IS using Stimulated Emission Depletion (STED) microscopy revealed the size resolution for the dual clusters to be at a 100–200 nm range, suggesting their co-docking with the Tandem CAR receptor at the IS. (Figure 1, Figure 2)
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