Abstract LB-199: A rationally designed bispecific chimeric antigen receptor molecule that simultaneously redirects T cells to target HER2 and GD2 in osteosarcoma
Authors: Hegde Meenakshi G. , Corder Amanda , Byrd Tiara T. , Fousek Kristen , Heslop Helen E. , Gottschalk Stephen , Yvon Eric , Dotti Gianpietro , Ahmed Nabil 
Published: May 2015

Tumor immunotherapy with chimeric antigen receptor (CAR)-modified T cells has shown substantial efficacy in early phase clinical trials. Targeting a single tumor antigen could however result in creation of antigen escape variants. One strategy to overcome this immune evasion mechanism is to simultaneously target two or more tumor restricted antigens (TAAs). We have created a novel bispecific CAR molecule that can effectively target HER2 and GD2 in osteosarcoma (OS), using in silico modeling. The extracellular domain of the bispecific CAR consists of two distinct antigen-recognition domains linked by a Gly-Ser linker; one for HER2 (scFv derived from monoclonal antibody FRP5) and the other for GD2 (scFv derived from monoclonal antibody 14g2a), joined in tandem (TanCAR). This extracellular-domain was assembled on Clone Manager®, then cloned into the Gateway® entry vector pDONR™221, sequence-verified and cloned inline with a signaling domain of the co-stimulatory molecule, CD28 and T-cell receptor zeta-chain. Healthy donor and patients’ T cells were transduced with HER2/GD2 TanCAR using a retroviral system. Surface expression of the TanCAR extracellular domain in its entirety was confirmed by flowcytometry using a HER2-scFv and another GD2-scFv specific method. We studied the varying expression pattern of HER2 and GD2 in 12 OS cell lines; e.g. LM7 was HER2/GD2 +/+, HOS HER2/GD2 +/- and IOR/OS9 was HER2/GD2 -/+. Burkitt's lymphoma cell line Raji, was used as a HER2/GD2 -/- control. TanCAR T cells released immunostimulatory cytokines (IL-2, IFN-γ) in co-cultures with tumor cells as detected by ELISA. In standard 51Cr cytotoxicity assays, TanCAR T cells distinctly recognized and killed HER2 and GD2 expressing OS cells as well as tumor cells expressing either HER2 or GD2 only. Importantly, bispecific TanCAR T cells attained full activation only upon encounter of both target antigens simultaneously yet they sustained their anti-tumor activity at low T-cell to tumor cell ratios and at modest or absent expression of either. CONCLUSION: The described bispecific single chimeric antigen receptor molecule (TanCAR) could widen the therapeutic application of a T-cell product by recognition of either target antigens (the Boolean OR), offsetting antigen escape. Importantly, if could improve selective full T cell activation (the Boolean AND) to maintain safety by minimizing off-target effects.

Citation Format: Meenakshi G. Hegde, Amanda Corder, Tiara T. Byrd, Kristen Fousek, Helen E. Heslop, Stephen Gottschalk, Eric Yvon, Gianpietro Dotti, Nabil Ahmed. A rationally designed bispecific chimeric antigen receptor molecule that simultaneously redirects T cells to target HER2 and GD2 in osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-199. doi:10.1158/1538-7445.AM2014-LB-199