We describe the development of targeted immunotherapy using a bispecific chimeric antigen receptor (CAR) modified T-cell directed against the tumor associated antigens, human epidermal growth factor receptor 2 (HER2) and the disialoganglioside GD2, surface expressed in a large proportion of melanoma. Melanoma remains the most aggressive of skin cancers with a 5-year survival rate less than 20% in those with metastatic disease. For these patients, immunotherapy has offered an alternative promising approach in the recent years. We designed bispecific CAR molecule ectodomains (n=8) using different permutations of single chain variable fragments (scFv) recognizing HER2 and GD2 in tandem arrangements. HER2 recognizing scFvs were derived from FRP5 and 4D5 (Trastuzumab), and GD2 recognizing scFvs were derived from 14G2a and humanized 3F8. The ectodomain containing the tandem arrangements of two distinct scFvs was joined to a signaling endodomain of the co-stimulatory molecules, CD28 and OX40, and a T-cell receptor zeta-chain. Primary human T cells were successfully transduced using a retroviral system achieving 37-73% efficiency of surface expression among various bispecific CAR constructs, as determined by flow cytometry. The function of bispecific CAR T cells were evaluated in a series of immunoassays against melanoma cell lines with variable levels of HER2 and GD2 expression. T cells expressing CARs with single specificity (HER2 or GD2) and non-transduced T cells from the same donor were used as controls. T cells transduced with bispecific TanCAR molecules proliferated after co-culture with melanoma cells. Upon evaluation of the cytokine release after encountering melanoma cells, 14G2a/FRP5 derived bispecific CAR T cells demonstrated the highest level of interferon-γ release with 14G2a-FRP5 (distal-proximal) tandem arrangement exhibiting the most robust activation. Further, consistent cytotoxicity against melanoma cells was observed in 4-hour 51Cr-release assay across donors for T cells expressing CAR ectodomain with 4D5-14G2a, 14G2a-FRP5, FRP5-14G2a and hu3F8-FRP5 tandem arrangements. Of these, T cells expressing 14G2a-FRP5, FRP5-14G2a and hu3F8-FRP5 tandem ectodomain arrangements demonstrated long-term tumor control in an impedance based in vitro tumor cell killing assay. In conclusion, we have rationally designed and generated HER2/GD2 bispecific CAR T cells with the intent of targeting the antigenic heterogeneity in melanoma. We have successfully screened multiple iterations of bispecific CAR ectodomains against melanoma demonstrating that the scFv as well as their sequence of tandem arrangement influences the antitumor function of modified T cells. The antitumor function of the top three candidate bispecific ectodomains identified will be further evaluated in pre-clinical animal models to determine the optimal construct for clinical development.

Citation Format: Sujith K. Joseph, Shoba A. Navai, Zakaria Grada, Kristen Fousek, Pretty R. Mathew, Ankita Shree, Amanda Wakefield, Nabil Ahmed, Meenakshi Hegde. Functional screening of bispecific CAR ectodomains targeting HER2 and GD2 in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6605.