T-cells genetically modified to express a chimeric antigen receptor (CAR) against a tumor associated antigen are attractive anti-cancer therapeutic agents. The 5T4 cell surface oncofoetal antigen is an attractive target antigen for cancer immunotherapy as it is expressed by a wide spectrum of cancers including gastric, ovarian and colorectal while showing limited expression in normal adult tissues. Previously it was shown that human and murine T-cells engineered to express human 5T4 specific CAR can specifically lyse human 5T4-expressing tumor targets in vitro and in vivo, respectively. This study aimed to isolate single chain variable fragments (scFvs) specific for murine 5T4 (m5T4) and to examine their efficacy in the context of CAR in a fully autologous model. Screening four novel hybridoma cell-lines producing anti-m5T4 monoclonal antibodies to clone the scFv yielded one functional m5T4-specific scFv from the hybridoma cell-line P1C9. The P1C9 scFv expressed as a fusion protein with the Fc domain of human IgG significantly labels m5T4-expressing targets. Murine T-cells modified to express the P1C9 scFv fused to the CD3? molecule can specifically lyse target cells in vitro and result in IFN-? cytokine release, while T-cells expressing the non-signalling CAR derivative; P1C9 scFv fused to the murine MHC-I transmembrane domain, were unable to lyse m5T4-expressing tumor targets. On-going experiments aim to test the efficacy and assess the toxicity of m5T4-specific CAR against m5T4-expressing tumor models in vivo. This model will allow further understanding of how gene-modified T-cells function in an autologous setting with the aim to improve human T-cell based cancer immunotherapy.


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