Abstract 3834: scFv antibodies against EphA2 receptor as a therapeutic strategy for AML

Abstract

Abstract The ephrin (Eph) receptors have been sought as therapeutic targets in liquid and solid tumors. Eph receptors are a subcategory of receptor tyrosine kinases and play vital parts in cell survival and function. Recently, monoclonal antibodies have been designed to bind to the ligand-binding cavity, that block ephrin to bind to ephrin type-A receptor 2 (EphA2). The potential clinical importance has been demonstrated in lymphomas, and the ability of the antibody to reduce proliferation and induce apoptosis (Goldgur et. al 2014). Acute myelogenous leukemia (AML) is a disease with dismal outcomes and new therapeutic targets have been explored. EphA2 has been described to be critical for the survival in EphA2-positive mouse leukemia models and not essential for normal hematopoiesis. Thus, we examined cell surface expression of EphA2 in peripheral blood, and bone marrow samples from AML patients. We screened 30 primary AML samples and 5 normal samples and assessed their expression of EphA2 using flow cytometry using the lymphoma cell line (Raji) as a positive control, as reported to express EphA2. The EphA2 expression was evaluated under culture conditions as well as without culture. Prior to culture, 22 (73.3%) of 30 samples were EphA2 positive in more than 90% of blasts. The mean cell surface expression of EphA2 in blasts was 95.4% (range: 64.4-99.7%) with a mean fluorescence intensity (MFI) of 201.5 (range: 18.4-675.0) with a MFI of 4.4 for isotype control. Interestingly, we observed 13.3% increase in EphA2 expression post-culture, bringing the proportion of EphA2-high samples from 73.3% to 86.6% of total samples. In addition to the increase of EphA2-high population, the average MFI of this cohort was also increased by 47.6% post-culture relative to pre-culture. Furthermore, we saw a more significant increase in the CD34+ population and little or no change in the CD34- population. These data shows that many AML express EphA2, and that culturing with cytokines can induce robust EphA2 expression. After validating EphA2 expression in leukemia cells, we have tested a set of single-chain antibodies (scFv) against EphA2 that have shown activity against Raji cells (Goldgur et. al 2014). We tested two single-chain variable fragments (G2 or D2) on samples with low or high expression of EphA2. The samples were incubated in the serum free IMDM medium supplemented with cytokines. The scFv antibodies were tested at 0.5ug and 1.0ug and evaluated after 24hr- or 72hr-culture. We found that exposure to D2 resulted in a significant decrease in cell number after 72 hr-culture, 52.6% and 99.3% relative to vehicle control treatment for 0.5ug and 1.0ug respectively in EphA2-high AML cells. These data suggests that the scFv antibodies against EphA2 are lethal to AML cells that express high level of EphA2. Taken together, our data suggests that more than 70% of AML cells highly express EphA2 and can be targeted using scFv antibodies specific to EphA2. Citation Format: Michael Albert, Mayumi Sugita, Hongliang Zong, Juha Himanen, Monica L. Guzman. scFv antibodies against EphA2 receptor as a therapeutic strategy for AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3834.