Anti- EGFR/ErbB3 DVD-Ig protein internalization.

<p>(A) A431 and (B) FaDu cells were treated with 10nM pHRodo-labeled anti-EGFR mAb alone, the bsAb, or anti-EGFR/ErbB3 DVD-Ig proteins on ice or at 37°C for 2 hours in PBS. Fluorescence intensity was measured via FACS. The error bars indicate standard deviation from the mean. p value was calculated via student T-test. *p<0.05.</p

Anti- EGFR/ErbB3 DVD-Ig proteins induce apoptosis and arrest cell cycle.

<p>An apoptosis assay was used to analyze (A) A431 or (B) FaDu cells after DVD-Ig proteins, mAbs, or combination treatment. Apoptotic (Annexin V-positive) cells were quantitated via FACS. A BrdU-incorporation assay was used to analyze (C) A431 or (D) FaDu cells after DVD-Ig proteins, mAbs, or combination treatment. BrdU-positive cells were quantitated via FACS. Three independent experiments with triplicates were performed. The error bars indicate standard deviation from the mean. p value was calculated via student T-test. *p<0.05.</p

DVD-Ig proteins inhibit HRG binding to FaDu cells.

<p>Serial dilutions of HRG were incubated with 100 nM anti-EGFR and anti-ErbB3 mAbs alone, anti-EGFR and anti-ErbB3 mAbs in combination, the bsAb, or anti-EGFR/ErbB3 DVD-Ig proteins. HRG binding affinity to FaDu cells was measured via FACS. MFI: median fluorescence intensity. The error bars indicate standard deviation from the mean.</p

Characterization of EGFR and ErbB3 binding of anti-EGFR-ErbB3 DVD-Igs.

<p>Characterization of EGFR and ErbB3 binding of anti-EGFR-ErbB3 DVD-Igs.</p

Anti- EGFR/ErbB3 DVD-Ig proteins in cell signaling assay.

<p>A431 and FaDu cells were cultured in medium containing 3nM HRG and 1% serum were treated with 100nM anti-EGFR and anti-ErbB3 mAbs alone, anti-EGFR and anti-ErbB3 mAbs in combination, the bsAb or anti-EGFR/ErbB3 DVD-Ig proteins for 24 hours. Cells were then harvested and lysed for Western blot. * indicates that 10% of sample lysates were used for A431 cells.</p

DVD687 and DVD688 show different mechanism of actions (MOAs).

<p>Apoptosis assay was used to analyze (A) N87 or (C) Calu-3 cells after DVD-Ig proteins, mAbs, or combination treatment. BrdU-incorporation assay was used to analyze (B) N87 or (D) Calu-3 cells after DVD-Ig proteins, mAbs, or combination treatment. Three independent experiments with triplicates were performed. One representative experiment is shown here. The error bars indicate standard deviation from the mean. p value was calculated via student T-test.</p

ErbB receptor expression.

<p>(A) EGFR, (B) ErbB2, and (C) ErbB3 protein expression was analyzed in Calu-3, N87, and MDA-MB175 VII cells. Log-phase proliferating subconfluent cells were lysed, proteins were separated on SDS-PAGE gels and analyzed via western blot. Three independent experiments were performed. One representative experiment is shown here.</p

Anti-ErbB2 DVD-Ig proteins in cell signaling assay.

<p>(A) Calu-3 and (B) N87 cells were cultured in medium containing 1% FBS were treated with 100nM antibodies or DVD-Ig proteins for 30 minutes. Cells were lysed, proteins were separated on SDS-PAGE gels and analyzed via western blot. Three independent experiments were performed. One representative experiment is shown here.</p

Generation of anti-ErbB2 DVD-Ig molecules.

<p>Generation of anti-ErbB2 DVD-Ig molecules.</p

Half DVD687 loses agonist effect.

<p>N87 cells were treated with indicated dosages of antibodies, DVD687, half DVD687, DVD37, or DVD38. After 10 days, cells were stained with crystal violet (A) and then quantitated (B). Three independent experiments with triplicates were performed. One representative experiment is shown here. The error bars indicate standard deviation from the mean.</p