A bispecific chimeric antigen receptor molecule enhances T cell activation through dual immunological synapse formation and offsets antigen escape in glioblastoma

Background Antigen escape tumor cell variants prevail in tumors recurring after treatment with chimeric antigen receptor (CAR) T cells with a single specificity. Recurrent tumors preserve alternative non-targeted tumor associated antigens. Hypothesis A bispecific CAR will mitigate antigen escape enhancing the antitumor activity of T cells. Methods and results HER2 and IL13Rα2 are currently targeted in Phase I glioblastoma (GBM) trials using CAR T cells. We created 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. We used computational modeling to interrogate this design. GBM patients' Tandem CAR T cells showed distinct binding to soluble HER2 and IL13Rα2 and killed primary autologous GBM cells. Three-dimensional reconstitution and quantification of confocal images of the Tandem CAR T cell/tumor interface revealed enhanced bifunctional immunological synapses compared to conventional CARs. Further, Tandem CAR T cells exhibited significantly enhanced inexhaustible activation dynamics when compared to conventional HER2 or IL13Rα2 CAR T cells and better controlled established GBM in an orthotopic murine model by offsetting both HER2 and IL13Rα2 escape. Conclusion Tandem chimeric antigen receptors enhance T cell activation and mitigate antigen escape through bifunctional immunological synapse formation in GBM

TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy

Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor'the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment