Afuco™ Anti-Human ERBB3 ADCC Recombinant Antibody (LJM716), ADCC Enhanced (CAT#: AFC-232CL)

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 IgG, MAB348, or AF234 for five 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 one or 24 hours. Cell lysates were harvested and immunoblotted 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 before stimulation with NRG1 (50 ng/mL) and their impact on 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 before 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 five 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 five days using CTG. Cell proliferation values relative to unstimulated cells are plotted.

Garrett,J.T.,Sutton,C.R.,Kurupi,R.,Bialucha,C.U.,Ettenberg,S.A.,Collins,S.D.,& Arteaga, C. L.(2013).Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers. Cancer research, 73(19), 6013-6023.

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 conducted for A+B. Data in C–F are presented as mean tumor volume ±SEM. All δ volumes subjected to One Way ANOVA and Tukey post hoc analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Garrett,J.T.,Sutton,C.R.,Kurupi,R.,Bialucha,C.U.,Ettenberg,S.A.,Collins,S.D.,& Arteaga, C. L.(2013).Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers. Cancer research, 73(19), 6013-6023.

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 (gray), 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 conducted by capturing biotinylated NRG1 on the surface of a Biacore sensor chip. Preformed HER3/antibody complexes at the indicated 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 before 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.

Garrett,J.T.,Sutton,C.R.,Kurupi,R.,Bialucha,C.U.,Ettenberg,S.A.,Collins,S.D.,& Arteaga, C. L.(2013).Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers. Cancer research, 73(19), 6013-6023.

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 nmol/L LJM716 or trastuzumab for one hour and the impact on both pHER3 (Y1197) and pAKT (S473) determined by MSD assay. Percentage 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 (33 nmol/L), trastuzumab (33 nmol/L), or LJM716 plus trastuzumab. Cells were grown for five days and cell viability determined by CTG. Percentage of 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 (11 nmol/L), cetuximab (11 nmol/L), or LJM716/cetuximab. Cells were grown for five days in full serum, and cell viability determined by CTG. Percentage 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 regrowth of tumors was specifically monitored following cessation of dosing on day 28. *, P < 0.05 by Kruskal–Wallis ANOVA on ranks post hoc Dunn test. q2d, dosed every other day; q2w, dosed every second week.

Garrett,J.T.,Sutton,C.R.,Kurupi,R.,Bialucha,C.U.,Ettenberg,S.A.,Collins,S.D.,& Arteaga, C. L.(2013).Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers. Cancer research, 73(19), 6013-6023.

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 six-well plates and treated in triplicate with dimethyl sulfoxide (DMSO), 10 μg/mL LJM716, pertuzumab, and/or trastuzumab. Media containing antibodies was replenished every three to four days. Cells were stained with crystal violet when control-treated cells were confluent, ranging from 14 to 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 three days. Images shown were recorded 15 to 19 days after cell seeding. Acini burden was quantified using the GelCount system. Each bar graph represents the mean + SEM. 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 one 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–Meier survival analysis following the end of 35 days of treatment. Mice were monitored for tumor regrowth and sacrificed when tumor burden was larger than 2,000 mm3. q2d, dosed every other day; q2w, dosed every second week.

Garrett,J.T.,Sutton,C.R.,Kurupi,R.,Bialucha,C.U.,Ettenberg,S.A.,Collins,S.D.,& Arteaga, C. L.(2013).Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers. Cancer research, 73(19), 6013-6023.