64 CuCl 2 in 50 µl 0.1N NaOAc buffer, and 20 µg of each DOTA-antibody conjugate in 40 µl

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1 Number of DOTA per antibody The average number of DOTA chelators per antibody was measured using a reported procedure with modifications (1,2). Briefly, nonradioactive CuCl 2 (80-fold excess of DOTA antibodies) in 20 µl 0.1N sodium acetate (NaOAc) buffer (ph 5.5) was added to approximately 1.0 mci 64 CuCl 2 in 50 µl 0.1N NaOAc buffer, and 20 µg of each DOTA-antibody conjugate in 40 µl 0.1N NaOAc buffer were added to the above carrier-added 64 CuCl 2 solution. The reaction mixture was incubated with constant shaking at 40 C for 1 h. The 64 Cu-DOTA-antibodies were loaded onto a PD-10 column and eluted with 1 PBS. Eluent ( ml) was collected. The number of DOTA per antibody was calculated using the following equation: number of DOTA per antibody = moles (Cu 2+ ) activity ( ml)/moles (DOTA-antibody)/total activity (loaded for each labeling). The activities in the equation were all decay-corrected to the same time point. The results were expressed as mean ± SD (n = 3). Supplemental Table 1. Number of DOTA per Conjugate DOTA conjugates DOTA number in each conjugate DOTA-Cys-hAb ± 0.34 DOTA-Lys-hAb ± 0.23 DOTA-Sug-hAb ± 0.18 DOTA-Lys-hIgG 2.24 ± 0.15 DOTA-Lys-hAb ± 0.23 FPLC of antibodies and DOTA conjugates The ion exchange chromatography by fast protein liquid chromatography (FPLC) using a Sephacryl S-200 column (GE Healthcare) was performed on a Beckman Coulter Gold chromatography system. The mobile phase was 0.2 M sodium phosphate buffer (ph 7.0), and the flow rate was 0.1 ml/min.

2 A B Supplemental Figure 1. The size exclusion chromatograms of hab47 (A) and DOTA-LyshAb47 (B).

3 A B Supplemental Figure 2. The size exclusion chromatograms of hab131 (A) and (B) DOTA-LyshAb131.

4 A B Supplemental Figure 3. The size exclusion chromatograms of higg (A) and DOTA-Lys-hIgG (B). Tumor tissue staining After fixing and blocking, tumor sections were incubated with anti-ephb4 (hab131) and anti- CD31 primary antibody overnight at 4 C, followed by corresponding secondary antibody for 1 h at room temperature. Subsequently, the slides were covered with VECTASHIELD Mounting Medium with DAPI, and images were obtained with Nikon Eclipse 80i fluorescence microscope. Secondary antibody goat anti-human Alexa Fluor 568 and goat anti-rat Alexa Fluor 488 were used to detect EphB4 and CD31 respectively.

5 A. HT29 B. MDA MB 231 EphB4 CD31 DAPI Merge Supplemental Figure 4. Costaining of EphB4 and CD31 on HT29 and MDA-MB-231 tumor tissues. The scale bar is 100µm. Supplemental Figure 5. Schematic illustration of EphB4 receptor. G = ephrin-binding globular domain; C = Cys-rich domain; F1/F2 = fibronectin type III-like domain 1/2; PM = plasma membrane; EC = extracellular; IC = intracellular. Biodistribution of 64 Cu-DOTA-Lys-hAb47 and 64 Cu-DOTA-Sug-hAb47 Animal procedures were performed according to a protocol approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC). For biodistribution studies, the normal athymic mice (n = 3 for each group) were injected with approximate 3.7 MBq (100 μci) of 64 Cu-DOTA-Lys-hAb47 or 64 Cu-DOTA-Sug-hAb47 through the tail vein and sacrificed at 2.0 h after injection. The normal organs of interest were removed and wet-weighed, and their radioactivity was measured in a gamma-counter. The radioactivity uptakes in the tumor and normal organs were expressed as a percentage of the injected radioactive dose per gram of tissue (% ID/g).

6 Cu-DOTA-Lys-hAb47 64 Cu-DOTA-Sug-hAb47 %ID/g Blood Liver Kidneys Muscle Heart Spleen Lung Intestine Supplemental Figure 6. Biodistribution of 64 Cu-DOTA-Lys-hAb47 or 64 Cu-DOTA-Sug-hAb47 in athymic nude mice at 2 h after injection. Data are expressed as normalized accumulation of activity in %ID/g ± SD (n = 3).

7 References 1. Cai W, Wu Y, Chen K, Cao Q, Tice DA, Chen X. In vitro and in vivo characterization of 64 Cu-labeled Abegrin, a humanized monoclonal antibody against integrin α v β 3. Cancer Res. 2006;66: Meares CF, McCall MJ, Reardan DT, Goodwin DA, Diamanti CI, McTigue M. Conjugation of antibodies with bifunctional chelating agents: isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions. Anal Biochem. 1984;142:68 78.