Abstract
Monoclonal antibodies are being considered as biopharmaceuticals for the in vivo targeting of acute myeloid leukemia. Here we describe the generation and characterization of a fully-human monoclonal antibody specific to CD123, a surface marker which is overexpressed in a variety of hematological disorders, including acute myeloid leukemia. The cloning and expression of the extracellular portion of CD123 as recombinant Fc fusion allowed the selection and affinity maturation of a human antibody, called H9, which specifically recognized the cognate antigen in biochemical assays and on leukemic cells. The H9 antibody and a previously-described anti-CD123 antibody (CSL362) were reformatted into full immunoglobulin human IgG1 formats, including a variant bearing S293D and I332E mutations to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). The two antibodies recognized different epitopes on the surface of the N-terminal domain of CD123, as revealed by crystallography and SPOT analysis. Both H9 and CSL362 in full immunoglobulin format were able to selectively kill leukemic cells in in vitro ADCC assays, performed both with cell lines and with patient-derived AML blasts. Further, the two antibodies, when reformatted as bispecific BiTE™ reagents by fusion with the anti-CD3 scFv(OKT3) antibody fragment, induced selective killing of AML blasts by patient-derived, autologous T-cells in an in vitro setting, but BiTE(CSL362/OKT3) exhibited a 10-fold higher potency compared to BiTE(H9/OKT3). The availability of two classes of CD123-specific biopharmaceuticals, capable of redirecting the cytolytic activity of NK cells and T cells against AML blasts, may enable novel interventional strategies and combination opportunities for the treatment of AML.
Hutmacher, Cornelia