NeuroMab™ Anti-EPHA2 BBB Shuttle Antibody(NRZP-1022-ZP3853)

Figure 1 EphA2 Eph099B-233.152 antibody promotes EphA2 tyrosine phosphorylation and EphA2 degradation in MDA-MB-231 cells.

MDA-MB-231 cell monolayers were incubated at 37°C in the presence of a single dose of 5 μg/ml Eph099B-233.152. Cell lysates were then immunoprecipitated with D7 (an EphA2-specific antibody), resolved by SDS-PAGE and subjected to western blot analysis with (A) phosphotyrosine-specific antibody or (B) EphA2-specific antibody.

Figure 2A EphA2 Eph099B-233.152 antibody inhibits tumor cell growth in vivo.

Tumor growth. Tumor growth was assessed and expressed as the ratio of tumor volume divided by initial tumor volume (100 mm3). Control mice are indicated by circles and Eph099B-233.152-treated mice are indicated by squares. Arrows indicate the time of Eph099B-233.152 or PBS administration.

Figure 2B EphA2 Eph099B-233.152 antibody inhibits tumor cell growth in vivo.

Survive. Tumors were allowed to grow until the tumor volume reached 1000 mm 3 . Mice survival was assessed by scoring the percentage of mice surviving each day after treatment. Control mice are shown in gray and Eph099B-233.152-treated mice are shown in black.

Figure 3 EphA2 antibodies EA2, Eph099B-208.261 and Eph099B-233.152 inhibit tumor cell growth in vivo.

MDA-MB-231 breast cancer cells were implanted (A) orthotopically or (B) subcutaneously into athymic mice. (C) A549 lung cancer cells were implanted subcutaneously in athymic mice. After tumor growth to an average volume of 100 mm3, mice were administered 6 mg/kg of the indicated antibody or negative control (PBS or 1A7 antibody) intraperitoneally twice weekly for 3 weeks. Tumor growth was assessed and expressed as the ratio of tumor volume divided by initial tumor volume (100 mm3).

Figure 4 EphA2 antibodies EA2, Eph099B-208.261 and Eph099B-233.152 inhibit tumor cell growth in vivo.

MDA-MB-231 breast cancer cells were implanted subcutaneously in athymic mice. After tumor growth to an average volume of 100mm3, mice were dosed intraperitoneally with 6 mg/kg of the indicated antibody or negative control twice a week for 3 weeks. Total tumor volume was determined after sacrifice. The negative control is black, EA2 is white, Eph099B-208.261 is dark gray, and Eph099B-233.152 is light gray.

Figure 5 Kinetic analysis of EphA2 monoclonal antibodies.

The BIACORE™ (surface plasmon resonance-based) assay was used to determine the kinetics of binding of EphA2 mAb to immobilized EphA2-Fc. Eph099B-208.261 is represented by a solid line, Eph099B-233.152 is represented by a dotted line, EA2 is represented by a dotted line, and the negative control is represented by squares.

Figure 6A The EphA2 EA2 epitope is distinct from the Eph099B-233.152 epitope and ligand binding site.

(A) EphA2-Fc was incubated with and bound to immobilized ephrin A1-Fc. Labeled ephrin A1-Fc (black), EA2 (white) or Eph099B-233.152 (grey) was incubated with EphA2-ephrin A1-Fc complex and the amount of binding measured.

Figure 6B The EphA2 EA2 epitope is distinct from the Eph099B-233.152 epitope and ligand binding site.

(B) EphA2-Fc incubated with and bound to immobilized Ephrin A1-Fc. Labeled EA2 was then incubated with the EphA2-Ephrin A1 complex. Unlabeled competitors were incubated with the indicated amounts of EphA2-Ephrin A1-EA2 complexes. Competitors are Ephrin A1-Fc (black), EA2 (white) or Eph099B-233.152 (gray).