IMST-50. DEVELOPMENT AND EXPRESSION OF A T CELL ENGAGER TARGETED TO GLIOBLASTOMA BY CHLOROTOXIN

Abstract

Bispecific T cell activating molecules are potent inducers of T cell destruction against target cells of interest and are commonly composed of two single chain variable fragments from distinct antibodies held together by a short peptide linker. We have developed a bispecific fusion protein (anti-CD3/ Ctx) composed of the variable fragment of an antibody targeting mouse CD3 linked to chlorotoxin for the purpose of activating T cells specifically against glioblastoma cells in vivo. After bringing T cells into close contact with glioma cells, anti-CD3/Ctx is theorized to promote the formation of an immunological synapse that will allow for perforin and granzyme-mediated destruction of tumor cells. By activating T cells with anti-CD3, the need for MHC presentation of antigens on the tumor cells is circumvented, an obstacle that is difficult to overcome with other immunotherapies. We have cloned the sequence encoding His-tagged anti-CD3/Ctx into a tobamovirus-based plant expression vector (ICON Genetics, Germany) for the transient expression of anti-CD3/Ctx in the tobacco relative Nicotiana benthamiana. Peak expression of anti-CD3/Ctx was found to occur five days post infiltration of plants with agrobacteria harboring the expression vector. The fusion protein was purified from leaf extracts by a 50-80% ammonium sulfate fractionation followed by TALON affinity chromatography. Expression level and purity were analyzed by SDS-PAGE and immunoblotting. Biochemical characterization is ongoing.