ITOC2 – 024. Selective lysis of biphenotypic leukaemia cells is mediated by dual-targeting triplebody 33-3-19 treatment

We recently reported that triplebody 19-3-19 can engage and activate T cells as cytolytic effectors via a central OKT3-derived single chain variable fragment (scFv) domain (Roskopf et al., 2014). Antibody-derivatives in the triplebody format can simultaneously target two different tumour cell antigens (“dual-targeting”), while concomitantly recruiting immune effector cells. To take full advantage of this dual-targeting capacity, a triplebody 33-3-19, designed for lysis of CD19+ CD33+ biphenotypic leukaemia cells with enhanced selectivity, was also constructed and analysed. 33-3-19 was produced in suspension-adapted 293F cells, purified and tested using standard molecular biology and flow cytometric techniques. 33-3-19 activated T cells as efficiently as 19-3-19 and induced the specific lysis of established myeloid (MOLM13, THP-1) as well as B cell lines (SEM, Raji) and of primary patient cells at picomolar concentrations. However, 33-3-19 achieved a lower maximal specific lysis of the B lymphoid cell lines (35–55 %) than 19-3-19 (55–90 %) in 3 h. This may in part be due to its monovalent affinity for CD19. Importantly, 33-3-19 induced the preferential lysis of double- over single-positive leukaemia cells (i.e. BV173 versus SEM cells) in a target cell mixture. Finally, 33-3-19 led to the elimination of >95% colony-forming leukaemia-initiating cells. These results highlight the potential of dual-targeting triplebodies for efficient and selective immune-intervention. We recently reported that triplebody 19-3-19 can engage and activate T cells as cytolytic effectors via a central OKT3-derived single chain variable fragment (scFv) domain (Roskopf et al., 2014). Antibody-derivatives in the triplebody format can simultaneously target two different tumour cell antigens (“dual-targeting”), while concomitantly recruiting immune effector cells. To take full advantage of this dual-targeting capacity, a triplebody 33-3-19, designed for lysis of CD19+ CD33+ biphenotypic leukaemia cells with enhanced selectivity, was also constructed and analysed. 33-3-19 was produced in suspension-adapted 293F cells, purified and tested using standard molecular biology and flow cytometric techniques. 33-3-19 activated T cells as efficiently as 19-3-19 and induced the specific lysis of established myeloid (MOLM13, THP-1) as well as B cell lines (SEM, Raji) and of primary patient cells at picomolar concentrations. However, 33-3-19 achieved a lower maximal specific lysis of the B lymphoid cell lines (35–55 %) than 19-3-19 (55–90 %) in 3 h. This may in part be due to its monovalent affinity for CD19. Importantly, 33-3-19 induced the preferential lysis of double- over single-positive leukaemia cells (i.e. BV173 versus SEM cells) in a target cell mixture. Finally, 33-3-19 led to the elimination of >95% colony-forming leukaemia-initiating cells. These results highlight the potential of dual-targeting triplebodies for efficient and selective immune-intervention.