P23. Efficient ex vivo lysis of acute myeloid leukaemic (AML) cells mediated by triplebodies with dual-targeting capability in conjunction with natural killer cells as effectors

Materials and methods Triplebodies are a new class of antibody derivatives developed by our team, which consist of 3 antigen-binding domains carried in a single polypeptide chain. The distal domains bind 2 targets on the same cancer cell, the central domain binds a trigger molecule on an effector cell. If the 2 targets on the cancer cell are different, the triplebody is said to be ‘dual-targeting’. The key new capability resulting from dual-targeting is the elimination of cancer cells with ‘increased selectivity’ (Schubert 2013; MAbs. PMID 24135631). Here the dual-targeting triplebody 33-16-123 (SPM-2) was developed and tested in redirected lysis assays in vitro. It carries binding domains for the surface antigens CD33 and CD123 on AML cells and for CD16, the Fc gamma RIII receptor on Natural Killer (NK) cells. This pair of target antigens is expressed on the blasts of a majority of AML patients and is present in increased surface density on AML-leukaemia stem cells (LSCs) relative to bulk AML cells and healthy hematopoietic stem cells. This combination offers the possibility of preferential targeting of AML-LSCs, and thus of minimal residual disease (MRD) cells. Human AML cell-lines and primary cells freshly drawn from AML patients were used in redirected lysis experiments. Target cells were labeled with Calcein AM. Effector cells were either ex vivo expanded mononuclear cells from healthy donors, or patient’s autologous NK cells enriched by immunomagnetic beads. Reactions proceeded for 4 hrs, and specific lysis was measured by release of fluorescent Calcein using an ELISA plate reader. Multicolor cytofluorimetry with fluorescent-labeled antibodies was used in addition to study elimination of subsets of AML cells.