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Identification of Anti-EGFR and Anti-ErbB3 Dual Variable Domains Immunoglobulin (DVD-Ig) Proteins with Unique Activities.

Gu J, Yang J, Chang Q, Liu Z, Ghayur T, Gu J - PLoS ONE (2015)

Bottom Line: Interestingly, in the presence of β-Heregulin (HRG), the DVD-Ig proteins show synergies with respect to inhibiting cell proliferation.The DVD-Ig proteins downregulate EGFR protein expression in the presence of HRG, which may be due to receptor internalization.Furthermore, the DVD-Ig proteins remarkably disrupt β-Heregulin binding to FaDu cells.

View Article: PubMed Central - PubMed

Affiliation: AbbVie Bioresearch Center, R&D, Worcester, Massachusetts, 01605, United States of America.

ABSTRACT
Epidermal growth factor receptor (EGFR) and receptor tyrosine-protein kinase 3 (ErbB3) are two well-established targets in cancer therapy. There is significant crosstalk among these two receptors and others. To block signaling from both EGFR and ErbB3, we generated anti-EGFR and anti-ErbB3 DVD-Ig proteins. Two DVD-Ig proteins maintained the functions of the combination of the two parental antibodies. The DVD-Ig proteins inhibit cell signaling and proliferation in A431 and FaDu cells while half DVD-Ig proteins lost proliferation inhibition function. Interestingly, in the presence of β-Heregulin (HRG), the DVD-Ig proteins show synergies with respect to inhibiting cell proliferation. The DVD-Ig proteins downregulate EGFR protein expression in the presence of HRG, which may be due to receptor internalization. Furthermore, the DVD-Ig proteins remarkably disrupt β-Heregulin binding to FaDu cells.

No MeSH data available.


Related in: MedlinePlus

Anti- EGFR/ErbB3 DVD-Ig proteins in cell signaling assay.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.
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pone.0124135.g003: Anti- EGFR/ErbB3 DVD-Ig proteins in cell signaling assay.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.

Mentions: We then tested whether the anti-EGFR/ErbB3 DVD-Ig proteins could inhibit cell signaling. EGFR and ErbB3 form heterodimers on the cell surface upon ligand binding to induce downstream signaling of multiple pathways including PI3K/AKT and Ras/MAPK. A431 or FaDu cells were treated with the anti-EGFR and anti-ErbB3 single antibodies or anti-EGFR/ErbB3 DVD-Ig proteins in the presence of HRG for 24 hours. The results showed that DVD1 and DVD2 proteins significantly inhibit cell signaling, especially pEGFR, pErbB2, pErbB3, and pAkt, in both A431 and FaDu cells (Fig 3). In another assay, we serum starved A431 or FaDu cells for 24 hours and then pretreated with the anti-EGFR and anti-ErbB3 single antibodies or anti-EGFR/ErbB3 DVD-Ig proteins. After HRG stimulation for 10 minutes, cells were harvested. Under this transient stimulation condition, DVD1 and DVD2 proteins inhibit cell signaling at the similar level as to anti-EGFR and anti-ErbB3 single antibodies, combination, and the bsAb (S2 Fig). Taken together, these results suggest that DVD1 and DVD2 proteins have longer cell signaling inhibition effects compared to the anti-EGFR and anti-ErbB3 single antibodies, combination, and the bsAb.


Identification of Anti-EGFR and Anti-ErbB3 Dual Variable Domains Immunoglobulin (DVD-Ig) Proteins with Unique Activities.

Gu J, Yang J, Chang Q, Liu Z, Ghayur T, Gu J - PLoS ONE (2015)

Anti- EGFR/ErbB3 DVD-Ig proteins in cell signaling assay.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440733&req=5

pone.0124135.g003: Anti- EGFR/ErbB3 DVD-Ig proteins in cell signaling assay.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.
Mentions: We then tested whether the anti-EGFR/ErbB3 DVD-Ig proteins could inhibit cell signaling. EGFR and ErbB3 form heterodimers on the cell surface upon ligand binding to induce downstream signaling of multiple pathways including PI3K/AKT and Ras/MAPK. A431 or FaDu cells were treated with the anti-EGFR and anti-ErbB3 single antibodies or anti-EGFR/ErbB3 DVD-Ig proteins in the presence of HRG for 24 hours. The results showed that DVD1 and DVD2 proteins significantly inhibit cell signaling, especially pEGFR, pErbB2, pErbB3, and pAkt, in both A431 and FaDu cells (Fig 3). In another assay, we serum starved A431 or FaDu cells for 24 hours and then pretreated with the anti-EGFR and anti-ErbB3 single antibodies or anti-EGFR/ErbB3 DVD-Ig proteins. After HRG stimulation for 10 minutes, cells were harvested. Under this transient stimulation condition, DVD1 and DVD2 proteins inhibit cell signaling at the similar level as to anti-EGFR and anti-ErbB3 single antibodies, combination, and the bsAb (S2 Fig). Taken together, these results suggest that DVD1 and DVD2 proteins have longer cell signaling inhibition effects compared to the anti-EGFR and anti-ErbB3 single antibodies, combination, and the bsAb.

Bottom Line: Interestingly, in the presence of β-Heregulin (HRG), the DVD-Ig proteins show synergies with respect to inhibiting cell proliferation.The DVD-Ig proteins downregulate EGFR protein expression in the presence of HRG, which may be due to receptor internalization.Furthermore, the DVD-Ig proteins remarkably disrupt β-Heregulin binding to FaDu cells.

View Article: PubMed Central - PubMed

Affiliation: AbbVie Bioresearch Center, R&D, Worcester, Massachusetts, 01605, United States of America.

ABSTRACT
Epidermal growth factor receptor (EGFR) and receptor tyrosine-protein kinase 3 (ErbB3) are two well-established targets in cancer therapy. There is significant crosstalk among these two receptors and others. To block signaling from both EGFR and ErbB3, we generated anti-EGFR and anti-ErbB3 DVD-Ig proteins. Two DVD-Ig proteins maintained the functions of the combination of the two parental antibodies. The DVD-Ig proteins inhibit cell signaling and proliferation in A431 and FaDu cells while half DVD-Ig proteins lost proliferation inhibition function. Interestingly, in the presence of β-Heregulin (HRG), the DVD-Ig proteins show synergies with respect to inhibiting cell proliferation. The DVD-Ig proteins downregulate EGFR protein expression in the presence of HRG, which may be due to receptor internalization. Furthermore, the DVD-Ig proteins remarkably disrupt β-Heregulin binding to FaDu cells.

No MeSH data available.


Related in: MedlinePlus