Anti-Human CD51+CD61 Recombinant Antibody (Etaracizumab) (CAT#: TAB-025)

Figure 1 Expression of αvβ3 in ovarian cancer cell lines.

Recognition of the αvβ3 heterodimer was assessed first by flow cytometry, using the murine monoclonal antibody clone LM609 as primary antibody (A). This is the clone from which the etaracizumab antibody was derived and shares identical Fab′ regions. There is a high expression of αvβ3 in the SKOV3ip1 and HeyA8 lines, and expression in a smaller population in A2780ip2. LM609 does not bind to αvβ3 in murine endothelial cells. Modified immunoprecipitation was then performed, where cells were exposed to biotin in culture, immunoprecipitated with anti-αv, anti-β3, or control IgG, then separated by SDS-PAGE electrophoresis (B). Bands at the expected location of the αv and β3 integrin subunits (125 and 105 kDa, respectively) are noted when either primary antibody was used in SKOV3ip1 and HeyA8. Expression in A2780ip2 was not high enough to be detected by this method.

Landen, C. N., Kim, T. J., Lin, Y. G., Merritt, W. M., Kamat, A. A., Han, L. Y.,... & Tice, D. (2008). Tumor-selective response to antibody-mediated targeting of αvβ3 integrin in ovarian cancer. Neoplasia, 10(11), 1259-1267.

Figure 2 Effects of etaracizumab on the AKT pathway.

To explore an explanation for the differential effects seen in vivo in SKOV3ip1 and HeyA8 despite αvβ3 expression, we examined effects of exposure to etaracizumab on cells plated on vitronectin. Cell lysate from cells exposed for 24 hours was collected and tested for phosphorylated Akt (p-Akt), total Akt2 (tot Akt2), total Akt1 (tot
Akt1), phosphorylated PI3 kinase 85-kDa subunit (p-PI3K-85), phosphorylated PI3 kinase 55-kDa subunit (p-PI3K-55), phosphorylated mTOR (p-mTOR), and β-actin (A). Band intensity was quantified with Scion Image and normalized with β-actin band intensity, and the change noted with etaracizumab is shown (B). At 24 hours in the SKOV3ip1 cell line, phosphorylated Akt, total Akt1, and total Akt2 were all decreased with treatment, as was phosphorylated mTOR. Interestingly, phosphorylated PI3K–55-kDa subunit was increased with treatment. No treatment effect was seen in HeyA8 at 24 hours, although the pathway was shown to be intact by higher phosphorylated Akt (in both treatment groups) at 5 minutes, likely a transient effect due to a change to low-serum media. Phosphorylated mTOR was not measurable (NM) in HeyA8 at 5 minutes and
24 hours.

Landen, C. N., Kim, T. J., Lin, Y. G., Merritt, W. M., Kamat, A. A., Han, L. Y.,... & Tice, D. (2008). Tumor-selective response to antibody-mediated targeting of αvβ3 integrin in ovarian cancer. Neoplasia, 10(11), 1259-1267.

Figure 3 Mechanisms of etaracizumab effects on ovarian cancer.

In an MTT viability assay, cells were allowed to grow on uncoated wells or wells precoated with vitronectin, with increasing concentrations of etaracizumab (A). At low concentrations of etaracizumab, vitronectin promoted cell viability. However, at higher concentrations of etaracizumab, viability was inhibited on cells grown on vitronectin,
the αvβ3 receptor. Etaracizumab did not affect viability when wells were not precoated with vitronectin. Data shown is in SKOV3ip1, with HeyA8 giving similar results. To assess in vivo effects on tumor biology, tumors were collected at the conclusion of the therapy experiments described in Figure 2 and tested for proliferation index as determined by PCNA positivity (B) and microvessel density as determined by number of CD31-positive lumens (C). The mean number of PCNA-positive cells or vessels in five randomly selected fields were counted and compared between groups. Consistent with in vitro proliferation data, etaracizumab (eta) reduced the proliferation index compared to IgG-exposed tumors, though there was no significant difference between IgG/paclitaxel– (IgG + pac) and etaracizumab/paclitaxel– (eta + pac) treated tumors. However, all four groups were similar in microvessel density, which may be a reflection of the lack of recognition of the murine αvβ3 integrin by etaracizumab. Finally, using the MICS, cell invasion through a human defined matrix in SKOV3ip1 and HeyA8 were examined with and without etaracizumab (D). Blockade of αvβ3 leads to a dose-dependent inhibition of invasion in both cell lines.

Landen, C. N., Kim, T. J., Lin, Y. G., Merritt, W. M., Kamat, A. A., Han, L. Y.,... & Tice, D. (2008). Tumor-selective response to antibody-mediated targeting of αvβ3 integrin in ovarian cancer. Neoplasia, 10(11), 1259-1267.