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Comparative Study
. Mar-Apr 2014;6(2):340-53.
doi: 10.4161/mabs.27658. Epub 2013 Dec 26.

Engineering Multivalent Antibodies to Target Heregulin-Induced HER3 Signaling in Breast Cancer Cells

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Free PMC article
Comparative Study

Engineering Multivalent Antibodies to Target Heregulin-Induced HER3 Signaling in Breast Cancer Cells

Jeffrey C Kang et al. MAbs. .
Free PMC article

Abstract

The use of antibodies in therapy and diagnosis has undergone an unprecedented expansion during the past two decades. This is due in part to innovations in antibody engineering that now offer opportunities for the production of "second generation" antibodies with multiple specificities or altered valencies. The targeting of individual components of the human epidermal growth factor receptor (HER)3-PI3K signaling axis, including the preferred heterodimerization partner HER2, is known to have limited anti-tumor effects. The efficacy of antibodies or small molecule tyrosine kinase inhibitors (TKIs) in targeting this axis is further reduced by the presence of the HER3 ligand, heregulin. To address these shortcomings, we performed a comparative analysis of two distinct approaches toward reducing the proliferation and signaling in HER2 overexpressing tumor cells in the presence of heregulin. These strategies both involve the use of engineered antibodies in combination with the epidermal growth factor receptor (EGFR)/HER2 specific TKI, lapatinib. In the first approach, we generated a bispecific anti-HER2/HER3 antibody that, in the presence of lapatinib, is designed to sequester HER3 into inactive HER2-HER3 dimers that restrain HER3 interactions with other possible dimerization partners. The second approach involves the use of a tetravalent anti-HER3 antibody with the goal of inducing efficient HER3 internalization and degradation. In combination with lapatinib, we demonstrate that although the multivalent HER3 antibody is more effective than its bivalent counterpart in reducing heregulin-mediated signaling and growth, the bispecific HER2/HER3 antibody has increased inhibitory activity. Collectively, these observations provide support for the therapeutic use of bispecifics in combination with TKIs to recruit HER3 into complexes that are functionally inert.

Keywords: HER2; HER3; antibody engineering; bispecific antibody; receptor internalization.

Figures

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Figure 1. Schematic representation of the bispecific antibody (TAb6) comprising trastuzumab and a scFv derived from the anti-HER3 antibody, Ab6, used in the current study.
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Figure 2. Effects of antibodies specific for HER2 and/or HER3 on HER2-overexpressing breast cancer cells. (A) Cells were incubated with 50 nM anti-HER3 (Ab6), tetrameric anti-HER3 (Ab6tet), trastuzumab (T), pertuzumab (P), trastuzumab or pertuzumab and Ab6 (T + Ab6 or P + Ab6), bispecific trastuzumab with anti-HER3 Ab6 scFv (TAb6), or bispecific pertuzumab with anti-HER3 Ab6 scFv (PAb6) for 5 d. Proliferative responses were assessed using the MTS reagent and were normalized against the proliferation of cells incubated in medium (Med) only. Data shown are means of triplicates ± standard deviation. The symbols * and ** indicate significantly lower or higher proliferative responses, respectively, between cells treated with antibody and PBS vehicle (Student t-test; P < 0.05). (B) SK-BR-3 or BT-474 cells were treated with anti-HER2/HER3 antibodies (50 nM) for 1 or 24 h, and cell lysates analyzed by immunoblotting. Data shown are representative of at least two independent experiments.
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Figure 3. Ab6tet internalizes into SK-BR-3 cells more rapidly than Ab6. Cells were pulsed with 50 nM anti-HER3 (Ab6), tetrameric anti-HER3 (Ab6tet) or bispecific trastuzumab with anti-HER3 Ab6 scFv (TAb6) for 5 min at 37οC, chased for 0, 10 or 20 min, fixed, permeabilized and stained with anti-EEA-1 antibody. The combined pulse plus chase times are indicated on the left margin. Anti-HER3 or HER2/HER3 antibodies were detected with Alexa 555-labeled secondary antibody (pseudocolored red in overlay) and anti-EEA-1 antibody with Alexa 647-labeled secondary antibody (pseudocolored green in overlay). Yellow arrows in the images for Ab6tet-treated cells indicate examples of internalized Ab6tet that is associated with EEA-1 positive endosomes. Scale bars = 2 μm.
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Figure 4. Antibodies specific for HER2 and/or HER3 have reduced efficacy in inhibiting proliferation and PI3K/Akt signaling in the presence of heregulin. (A) Cells were incubated with heregulin (HRG, 6.25 nM) and 50 nM anti-HER3 (Ab6), tetrameric anti-HER3 (Ab6tet), trastuzumab (T), trastuzumab and Ab6 (T + Ab6) or bispecific trastuzumab with anti-HER3 Ab6 scFv (TAb6) for 5 d. Proliferative responses were assessed using the MTS reagent and were normalized against the proliferation of cells incubated in medium (Med) only. Data shown are means of triplicates ± standard deviation. * indicates statistically significant differences between proliferative responses for cells treated with antibody in the presence of heregulin and cells treated with heregulin only (Student t-test; P < 0.05). (B) SK-BR-3 or BT-474 cells were treated with anti-HER2/HER3 antibodies (50 nM) in the presence of 6.25 nM heregulin for 1 or 24 h, and cell lysates analyzed by immunoblotting. Data shown are representative of at least two independent experiments.
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Figure 5. Heregulin treatment reverses the anti-proliferative effects of lapatinib in HER2 overexpressing cell lines. (A) Cells were incubated with different concentrations of lapatinib (L) in the presence and absence of heregulin (HRG; 6.25 nM) for 5 d. Proliferative responses were assessed using the MTS reagent and were normalized against the proliferation of cells incubated in medium (Med) only. Data shown are means of triplicates ± standard deviation. * indicates statistically significant differences between proliferative responses for cells treated with lapatinib and DMSO vehicle (Student t-test; P < 0.001). (B) SK-BR-3 or BT-474 cells were treated with 1 μM lapatinib (Lap) in the presence and absence of heregulin (6.25 nM) for 1 or 24 h, and cell lysates analyzed by immunoblotting. Data shown are representative of at least two independent experiments.
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Figure 6. The bispecific anti-HER2/HER3 antibody, TAb6, has the highest activity in reducing cell proliferation and PI3K/Akt signaling in the presence of heregulin and lapatinib. (A) Cells were incubated with 1 μM lapatinib (L) in the presence of heregulin (HRG; 6.25 nM) and treated with 50 nM anti-HER3 (Ab6), tetrameric anti-HER3 (Ab6tet), trastuzumab (T), trastuzumab and Ab6 (T + Ab6) or bispecific trastuzumab with anti-HER3 Ab6 scFv (TAb6) for 5 d. Proliferative responses were assessed using the MTS reagent and were normalized against the proliferation of cells incubated in medium (Med) only. Data shown are means of triplicates ± standard deviation. * indicates statistically significant differences between proliferative responses for cells treated with antibodies vs. vehicle in the presence of lapatinib and heregulin (Student t-test; P < 0.05). ** indicates statistically significant differences between proliferative responses for the pairwise comparison of the two indicated treatments (horizontal bars). (B) SK-BR-3 or BT-474 cells were treated with lapatinib (Lap), heregulin (6.25 nM) and anti-HER2/HER3 antibodies (50 nM) as indicated for 1 or 24 h, and cell lysates analyzed by immunoblotting. Data shown are representative of at least two independent experiments.

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