Abstract 1758: Targeting EphB4 with a novel antibody in acute leukemia
Authors: Kim Miriam Y. , Jorapur Aparna , McManus Amy R. , Liu Ren , Krasnoperov Valery , Naga Kranthi , Gill Parkash S. , Merchant Akil 
Published: May 2015

Abstract
Introduction: EphB4, a receptor tyrosine kinase, binds to ephrinB2, a cell surface ligand, resulting in bidirectional signaling through both receptor and ligand. EphB4 stimulates cell proliferation and migration through the phosphatidylinositol 3-kinase (PI3K) and Akt signaling pathway. MAb131 is a novel anti-EphB4 monoclonal antibody that induces degradation of EphB4 through receptor endocytosis. We investigated the use of MAb131 as a therapeutic agent for acute leukemia.
Methods: Peripheral blood or bone marrow specimens from patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) were obtained after informed consent. EphB4 and ephrinB2 expression in cells were profiled using flow cytometry. Cell lines and patient samples were treated with MAb131 at 0, 10 and 100ug/ml concentrations. Blocking experiment was performed with 200ug/ml sEphB4-HSA in combination with 10ug/ml MAb131. Downregulation of EphB4 was evaluated by flow cytometry. Phosphorylated AKT (pAKT) levels were measured by Western blot. Cell viability was measured by trypan blue exclusion.
Results: EphB4 was highly expressed on the cell surface in the majority of leukemia cell lines of both myeloid (K562, Molm14, U937) and lymphoid (REH, RS4;11, Jurkat) origin; only one leukemia cell line evaluated, KG-1, did not express EphB4. In contrast, none of the lymphoma cell lines (BJAB, Ramos, Namwala) expressed EphB4. Of 9 AML patient samples evaluated, 4 expressed high levels of EphB4, and 5 expressed low levels of EphB4. Of 7 ALL patient samples evaluated, 2 expressed high levels of EphB4, 3 had low expression and 2 did not express EphB4. None of the cells showed robust expression of ephrinB2. Treatment of ALL and AML cell lines with MAb131 induced downregulation of EphB4, decreased levels of pAKT, and &gt;90% cell death after 72h. Primary AML and ALL samples also showed downregulation of surface EphB4 and increased cell death with MAb131 treatment. A decoy receptor comprised of the soluble extracellular domain of EphB4 fused to albumin (sEphB4-HSA) was used to block MAb131 binding to EphB4 and confirm specificity of MAb131 activity.
Conclusion: We present the first report that EphB4 signaling is active in acute leukemia and can be targeted therapeutically. MAb131, a novel anti-EphB4 antibody, has potent anti-leukemic activity as a single agent in vitro and is likely to sensitize leukemia to cytotoxic chemotherapy through downregulation of AKT signaling. Based on these results, clinical studies exploring the efficacy of EphB4/ephrinB2 antagonists in patients with acute leukemia should be pursued.
Citation Format: Miriam Y. Kim, Aparna Jorapur, Amy R. McManus, Ren Liu, Valery Krasnoperov, Kranthi Naga, Parkash S. Gill, Akil Merchant. Targeting EphB4 with a novel antibody in acute leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1758. doi:10.1158/1538-7445.AM2014-1758