Antibody-dependent cell-mediated cytotoxicity effector-enhanced EphA2 agonist monoclonal antibody demonstrates potent activity against human tumors

Abstract

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

Figure 1

Characterization of 3F2-3M. (A) The EphA2 agonist activity of 3F2-3M was determined by evaluating EphA2 phosphorylation in MDA-MB-231 cells at concentrations from 0.1 to 50 µg/ml similar to that previously described [21]. (B) The binding of 3F2-3M to human blood cell subpopulations was determined using healthy human PBMCs and characterizing cell subpopulations as described in Materials and Methods. Binding was demonstrated to NK cells and monocytes where a statistically significant 100- to 250-fold increase in binding to NK cells was observed with 3F2-3M compared to 3F2-WT (P = .002). Error bars, SD.

Figure 2

EphA2 antibodies elicit ADCC activity in vitro. The ADCC activity of 3F2-3M was assessed using healthy donor PBMCs as effectors and SKOV3 ovarian cancer (A), A549 NSCLC (B) and MDA-MB-231 breast cancer (C) cell lines as targets as described in Materials and Methods. Cytotoxicity was observed in a dose-dependent and effector-to-target ratio manner. Concentrations of 3F2-3M or isotype control antibodies used were 5 ng/ml (diamonds), 50 ng/ml (hatched lines), or 500 ng/ml (solid bars). Little to no cytotoxicity was observed when 3F2-3M was evaluated in the SKMel28, a non-EphA2-expressing melanoma cell line (D). Error bars, SEM. Data are representative of three independent experiments.

Figure 3

NK cells are required for ADCC activity in vitro. The ADCC activity of 3F2-3M was evaluated in purified NK and non-NK cells isolated from healthy donors as described in Materials and Methods. Potent and reproducible ADCC activity against MDA-MB-231 cells (A) and A549 NSCLC cells (B) in the presence of human purified NK cells but not purified NK-depleted PBMCs. Error bars, SEM. Antibody concentrations tested were 5 ng/ml (diamonds), 50 ng/ml (hatched lines), and 500 ng/ml (solid bars) at an effector-to-target ratio of 10:1 for the MDA-MB-231 study and 5:1 for the A549 study. Data are representative of two independent experiments.

Figure 4

CD107a expression is increased on NK cells in the presence of EphA2 antibodies. The expression of the activation marker CD107a was evaluated on the surface of healthy donor NK cells by flow cytometry. Isolated NK or non-NK cells were incubated in the presence of tumor cells expressing or lacking EphA2, EphA2 antibodies, or isotype control antibodies. When EphA2-expressing tumor cells are present, a dose-responsive increase in CD107a protein occurs with both the 3F2-WT (open squares) and 3F2-3M (solid squares) antibodies, where 3F2-3M > 3F2-WT. At high concentrations of antibody, CD107a expression increases in a nonspecific fashion as observed with the isotype control antibody (open circles) and with EphA2-negative cell lines (data not shown). Error bars, SEM. 3F2-3M was statistically different from both 3F2-WT and isotype control antibody at all concentrations greater than 0.010 µg/ml (P < .001).

Figure 5

Impact of FcγRIIIA polymorphisms on ADCC activity. Natural killer cells isolated from healthy human donors expressing the FcγRIIIA 158 V/F, F/F, or V/V polymorphisms were evaluated in the LDH release cytotoxicity assay using A549 NSCLC cells as targets (E/T = 2:1). 3F2-3M (solid triangles) elicited robust cytotoxicity independent of the FcγRIIIa allotype. The 3F2-WT (solid squares) antibody demonstrated higher cytotoxicity using the V/V allotype compared with either the V/F or the F/F allotype: (A) V/F, (B) V/V, (C) F/F. Isotype control antibody is represented as open squares. Net cell lysis across independent experiments is represented in (D) where *P < .0022, comparing 3M to WT for each allotype.

Figure 6

3F2-3M is efficacious in vivo. The in vivo efficacy of 3F2-WT and 3F2-3M was evaluated in nude, SCID, and SCID/NOD mice. 3F2-3M demonstrated significant suppression of MDA-MB-231 tumor growth when administered twice per week in the nude mouse mammary fat pad model (A) (P < .009): isotype control (solid triangles), 3F2-3M (solid upside-down triangles), taxol positive control (solid diamonds). In addition, the role of NK cells on antitumor activity was evaluated using SCID and SCID/NOD mouse models (B). Significant suppression of tumor growth was observed with the 3F2-3M-treated SCID mice compared with the 3F2-3M-treated SCID/NOD mice and control-treated animals (P < .001). The 3F2-WT-treated SCID mice also showed significant tumor suppression compared with the 3F2-WT-treated SCID/NOD mice (P < .031). In addition, there was a significant difference between the 3F2-3M- and 3F2-WT-treated SCID mice (P <.001): isotype control (open circles), 3F2-WT (open triangles), and 3F2-3M (open squares). In SCID/NOD animals: isotype control (solid circles), 3F2-WT (solid triangles), and 3F2-3M (solid squares). Data are representative of two independent experiments. Error bars, SEM.