A Recombinant Human Antibody to EphA3 with Pro-Apoptotic and Enhanced ADCC Activity Shows Selective Cytotoxicity against Myeloid Leukemia Cells and CD123-Positive Leukemic Stem Cells.

Abstract 1728

Poster Board I-754

Members of the Eph family of receptor tyrosine kinases play important roles in embryonic development but have restricted tissue distribution and activity in adult tissues. The EphA3 receptor is an oncofetal antigen expressed at high levels on the surface of several solid tumor types and certain leukemias. KaloBios is in pre-clinical development with KB004, a high-affinity recombinant human antibody, derived from a monoclonal anti-EphA3 antibody by Antibody Humaneering^TM Technology. KB004 binds EphA3 and stimulates apoptosis in primary cells from myeloid leukemia patients. The cell-surface expression of EphA3 was analyzed by flow cytometry on primary cells from chronic myeloid leukemia (CML) [n=10], acute myeloid leukemia (AML) of various sub-types [n=29], and myelodysplastic syndromes (MDS) [n=7]. EphA3 surface expression was detected in at least 50% of patient samples from each of the diseases analyzed. CD123-positive leukemia stem cells (CD34⁺ CD38^- CD123⁺) also displayed surface expression of EphA3 whereas normal bone marrow CD34+ stem/ progenitor cells lacked detectable EphA3. KB004 was shown to stimulate apoptosis in EphA3⁺ primary leukemia cells, including CD123⁺ leukemic stem cells with no activity in EphA3-negative specimens, including normal CD34⁺ bone marrow cells. Cross-linking of the antibody was not required for induction of apoptosis. KB004 also inhibited myeloid leukemia colony formation (CFU-L) from primary AML samples in methylcellulose colony assays without affecting normal hematopoietic colony formation. In addition to its direct pro-apoptotic effect, KB004 induced potent antibody-dependent cellular cytotoxicity (ADCC) activity against EphA3-positive cells, mediated by CD16-expressing effector cells. CD16-dependent ADCC activity was further enhanced by expression of the antibody in CHO cells that lack a1,6 fucosyl transferase, generating afucosylated antibody with higher affinity for CD16a. The data support the development of an anti-EphA3 antibody for the treatment of hematologic malignancies in addition to solid tumors. In particular, the selective activity against leukemic stem cells supports a novel strategy for therapeutic targeting of leukemia-initiating cells that merits clinical investigation.