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Found 1 result for PubMed for id: 119265
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. 2013 Oct 1;73(19):6024-35.
doi: 10.1158/0008-5472.CAN-13-1198. Epub 2013 Aug 8.

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin

Andrew P Garner  1 , Carl U Bialucha, Elizabeth R Sprague, Joan T Garrett, Qing Sheng, Sharon Li, Olga Sineshchekova, Parmita Saxena, Cammie R Sutton, Dongshu Chen, Yan Chen, Huiqin Wang, Jinsheng Liang, Rita Das, Rebecca Mosher, Jian Gu, Alan Huang, Nicole Haubst, Carolin Zehetmeier, Manuela Haberl, Winfried Elis, Christian Kunz, Analeah B Heidt, Kara Herlihy, Joshua Murtie, Alwin Schuller, Carlos L Arteaga, William R Sellers, Seth A Ettenberg
Affiliations

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin

Andrew P Garner et al. Cancer Res. .

Abstract

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

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Figures

Figure 1
Figure 1. Identification of LJM716, a HER3 binding monoclonal antibody capable of blocking ligand-dependent and –independent HER3 signaling and cell proliferation
(A) SKBR-3 were treated with increasing concentrations of either IgG, MAB348 or AF234 for 5 days followed by cell viability assessment using the CTG assay. (B) SKBR-3 cells were treated with 10 µg/ml IgG, MAB3481 and AF234 for 1 or 24 hours. Cell lysates were harvested and immuno-blotted with antibodies directed against the indicated proteins. (C) HER3-targeted antibodies were profiled for their ability to inhibit HER3 tyrosine phosphorylation and cell growth in HER2 amplified SKBR-3 cells. Maximal growth inhibition was calculated relative to that achieved with AF234. pHER3 was measured via MSD assay and Family 15 members are highlighted in red. (D) MCF7 cells were treated with HER3-targeted antibodies prior to stimulation with NRG1 (50 ng/ml) and their impact upon HER3 phosphorylation and proliferation determined. Maximal growth inhibition was calculated relative to that achieved with AF234. pHER3 was measured via MSD assay and Family 15 members are highlighted in red. (E) SKBR-3 cells were treated with LJM716 (red) or IgG (black) for one hour and the level of pHER3 quantified via MSD assay. (F) MCF7 cells were treated with LJM716 (red) or IgG (black) for 30 minutes prior to NRG1 stimulation for 10 minutes. pHER3 levels were measured by MSD assay. (G) SKBR-3 cells grown in full serum were treated with LJM716 (red) or IgG (black) for 5 days and cell viability determined by CTG. (H) Serum starved MCF7 cells were treated with LJM716 (red) or IgG (black), stimulated with NRG1 (50 ng/ml) and cell viability determined after 5 days using CTG. Cell proliferation values relative to unstimulated cells are plotted.
Figure 2
Figure 2. LJM716 inhibits HER3 signaling in vivo and is efficacious in multiple ligand-dependent and independent xenograft tumor models
(A) BT474 xenografts were dosed intravenously with a single 20 mg/kg dose of LJM716 and tumors were harvested at 0, 4 or 24 hours followed by analysis of lysates for pHER3 and pAKT levels by MSD assay. (B) BxPC-3 xenografts were dosed intravenously with a single 20 mg/kg dose of LJM716 and tumors were harvested at 0, 4, or 72 hours followed by analysis of lysates for pHER3 and pAKT levels by MSD assay. (C) BT474 xenografts were dosed intravenously every other day with 20 mg/kg LJM716 (red) or IgG (black). (D) HBCx-13A primary human xenografts were dosed intravenously every other day with 20 mg/kg LJM716 (red) or IgG (black). (E) BxPC-3 xenografts were dosed intravenously every other day with 20 mg/kg LJM716 (red) or IgG (black). (F) FaDu xenografts were dosed intravenously every other day with 20 mg/kg LJM716 (red) or IgG (black). Two-tailed non-paired t-test was performed for A+B. Data in C–F are presented as mean tumor volume ±SEM. All delta volumes subjected to One Way ANOVA and Tukeys post hoc analysis. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.
Figure 3
Figure 3. LJM716 locks HER3 in the inactive conformation and does not block ligand binding
(A) Crystal structure of the HER3 extracellular domain in complex with MOR09825. Fab is in gold and the HER3 domains are individually colored: D1 (grey), D2 (purple), D3 (white) and D4 (blue). HER3 is in the inactive (tethered) conformation and the ligand binding site located in D1 and D3 is not occluded. (B) HER3 residues comprising the MOR09825 epitope (yellow ball-and-sticks) are clustered in D2 and D4. MOR09825 is removed from the view to aid visualization. (C) Close up of MOR09825 interactions with K267 and L268 located near the interface of the HER3 dimerization loop (D2) and auto-inhibitory domain (D4). MOR09825 light chain residues are highlighted in gold and MOR09825 heavy chain residues in orange. (D) Binding of LJM716 to HER3 mutants K267A, L268A and K267A/ L268A expressed as recombinant proteins and determined by biochemical ELISA. (E) Interaction analyses performed by capturing biotinylated NRG1 on the surface of a Bioacore™ sensor chip. Preformed HER3/ antibody complexes at the indication concentrations were injected over reference and active surfaces and their interactions with NRG1 observed. (F) MCF7 cells were incubated with the indicated concentrations of antibodies prior to the addition of 10 nM NRG1-β1 EGF domain. Cell surface bound NRG1 was quantified by flow cytometry and is plotted as mean channel fluorescence on the Y axis.
Figure 4
Figure 4. LJM716 displays in vitro and in vivo combination activity with trastuzumab and cetuximab
(A) HER2 amplified cell lines grown in full-serum were treated with 10 nM LJM716 or trastuzumab for one hour and the impact on both pHER3 (Y1197) and pAKT (S473) determined by MSD assay. % inhibition relative to control IgG treated cells is visualized in the form of a heat map colored from blue (0% inhibition) to red (100% inhibition). Cell lines harboring hotspot PI3K mutations are highlighted in red. (B) The HER2 amplified cell lines UACC812, MDA-MB-453, NCI-N87 and SKBR-3, grown in full-serum, were treated with increasing doses of LJM716 or trastuzumab for one hour and the impact on pHER3 (Y1197) was determined by MSD assay. (C) HER2 amplified cell lines were dosed with LJM716 (33nM), trastuzumab (33nM) or LJM716 plus trastuzumab. Cells were grown for 5 days and cell viability determined by CTG. % inhibition relative to untreated cells is visualized in the form of a heat map. (D) BT474 tumor xenografts were grown in NSG mice and treated with IgG (20 mg/kg, q2d), LJM716 (20 mg/kg, q2d), trastuzumab (10 mg/kg, q2w) or LJM716/ trastuzumab. Data are presented as mean tumor volume ±SEM. *p<0.05 by ANOVA post hoc Holm-Sidak test. (E) SCCHN cell lines were treated with LJM716 (11nM), Cetuximab (11nM) or LJM716/ Cetuximab. Cells were grown for 5 days in full-serum and cell viability determined by CTG. % inhibition relative to untreated cells is visualized in the form of a heat map. (F) FaDu tumor bearing mice were dosed for 14 days with IgG (20 mg/kg, q2d), LJM716 (20 mg/kg, q2d), Cetuximab (20 mg/kg, q2w) or LJM716/ Cetuximab. The re-growth of tumors was specifically monitored following cessation of dosing on day 28. *p<0.05 by Kruskal-Wallis ANOVA on ranks post hoc Dunn’s test.
Figure 5
Figure 5. The addition of LJM716 to trastuzumab enables more potent HER3/PI3K pathway signaling and growth inhibition than trastuzumab/pertuzumab combination, particularly in trastuzumab insensitive models
(A) Cells were plated at 10,000 to 50,000 cells per well in 6-well plates and treated in triplicate with DMSO, 10 µg/ml LJM716, pertuzumab, and/or trastuzumab. Media containing antibodies was replenished every 3–4 days. Cells were stained with crystal violet when control treated cells were confluent, ranging from 14–21 days. Representative images and quantification of integrated intensity (% control) are shown. *, P < 0.05, t test. (B) Cells were seeded in Matrigel and allowed to grow in the absence or presence of 10 µg/ml LJM716, pertuzumab, and/or trastuzumab as indicated. Medium was subsequently changed every 3 days. Images shown were recorded 15–19 days after cell seeding. Acini burden was quantified using the GelCount system. Each bar graph represents the mean + S.E.M. of triplicate samples. *, P < 0.05, t test. (C) BT474 and MDA453 cells were treated with 10 µg/ml LJM 716, 10 µg/ml pertuzumab and/or 10 µg/ml trastuzumab for 1 or 24 hours. Whole cell lysates were prepared and separated in a 7% SDS gel followed by immunoblot analysis with antibodies directed against the indicated proteins. (D) BT474 xenografts were treated with either IgG (20 mg/kg), trastuzumab (20 mg/kg), pertuzumab (20 mg/kg), LJM716 (20 mg/kg, q2d), trastuzumab+pertuzumab or trastuzumab+LJM716 for 35 days. Data are presented as mean tumor volume ±SEM. (E) Kaplan-Meyer survival analysis following the end of 35 days of treatment. Mice were monitored for tumor re-growth and sacrificed once tumor burden was larger than 2000 mm3.
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