Anti-HER3 domain 1 and 3 antibodies reduce tumor growth by hindering HER2/HER3 dimerization and AKT-induced MDM2, XIAP, and FoxO1 phosphorylation

Abstract

Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2(low) cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.

Figure 1

Selected anti-HER3 Abs bind to the extracellular region of human HER3 but not to the other HER receptors, with or without interference with NRG binding. (A) Cross-reactivity of anti-HER3 moAbs and huAbs toward mouse or human soluble HER3 was assessed by ELISAs. The absorbance values at 490 nm of the irrelevant control Abs Px and H3A-02 are indicated. Each value represents the mean ± SD of triplicate determinations in three independent experiments. (B) Flow cytometric analysis of the anti-HER3 Ab binding to NIH/3T3 fibroblasts transfected with membrane EGFR, HER2, HER3, HER2/HER3, or EGFR/HER4 in comparison to parental NIH/3T3 cells. Binding of the FITC-conjugated secondary Abs and of the control Abs H3A-02 and Px is also indicated. Results (geometric mean) are representative of two different experiments. (C) BiaCore determination of the binding kinetics between anti-HER3 huAbs and recombinant human HER3 (3.12–6.25 nM) and between moAbs and human HER3 (extracellular)-Fc recombinant protein (77 nM), respectively. (D) NRG interference with anti-HER3 Ab binding in HER3-positive SKBR3 cells was determined by competitive flow cytometry. The binding (%) of the moAbs 9F7-F11 (□), 16D3-C1 (○), and 12H8-B11 (◊), and of the huAbs H4B-121 (▴), H3A-122 (▪), and H4B-25 (◆) to SKBR3 cells, after incubation with various NRG concentrations, is indicated. Each value represents the mean ± SD of triplicate determinations in three independent experiments. No cell binding was observed when only the FITC-conjugated secondary Ab was added after incubation with NRG.

Figure 2

The ³⁵²DPWHKI³⁵⁸ motif recognized by the Ab H4B-121 is located in D3 of HER3. (A) Scan analysis of the 213 overlapping pentadecapeptides that covered the entire extracellular domain of HER3 (amino acids 1–645) and were frame-shifted by one or three residues (generated by the SPOT method). Binding of H4B-121 was observed only in the region between amino acids 340 and 360. No binding was observed with the secondary Ab alone. The Spot intensity was measured using ImageJ. (B) Alanine scanning (Alascan) of the pentadecapeptides ³⁴²LDFLITGLNGDPWHK³⁵⁶, ³⁴³DFLITGLNGDPWHKI³⁵⁷, and ³⁴⁴FLITGLNGDPWHKIP³⁵⁸ that cover the region between amino acids 342 and 358 of HER3. (C) Quantitative analysis of H4B-121 binding to the membrane pentadeca-peptides in B using ImageJ. Each bar represents the reactivity of H4B-121 toward a pentadecapeptide sequence inwhich the indicated amino acid was substituted by alanine. The mean spot reactivity for each residue was calculated from the results obtained in the three pentadecapeptides.

Figure 3

The Abs H4B-25 and H3A-122 recognize motifs located in D3 and D4, respectively, whereas the Abs 16D3-C1 and 9F7-F11 bind to motifs within D1 of HER3. (A) Quantitative analysis of the reactivity of H4B-25, H3A-122, 16D3-C1, and 9F7-F11 toward pentadeca-peptides generated by SPOT synthesis on cellulose membranes using ImageJ. (B) Amino acid alignment of the identified Ab-binding motifs in human, monkey, and mouse HER3 and in human EGFR, HER2, and HER4. (C) The Ab-binding motifs were localized on the crystal structure of unliganded HER3 (pdb 1M6B).

Figure 4

The anti-HER3 D1 Abs 9F7-F11 (■) and 16D3-C1 (△) and the anti-HER3 D3 Ab H4B-121 (○) inhibit tumor growth (left panel) and increase survival time (right panel) compared to vehicle (NaCl; ◆) in mice xenografted with HER2 non-amplified pancreatic carcinoma BxPC3 cells (wild-type PIK3CA and p53) (A), NRG-addicted, HER2 non-amplified epidermoid carcinoma A431 cells (wild-type PIK3CA and mutant p53) (C), or triple-negative, EGFR-amplified breast carcinoma MDA-MB-468 cells (wild-type PIK3CA and mutant p53) (D). BxPC3 tumor lysates from vehicle- or Ab-treated mice were prepared 24 days after the beginning of the treatment (tumor sizes are indicated) and HER3 phosphorylation at Tyr¹²⁸⁹ was assessed by Western blot analysis (B). Tumor growth data are presented as the mean tumor volume ± SEM for each group of nude mice (n = 8 per condition, but for the MDA-MB-468 xenograft model, n = 7). Kaplan-Meier survival curves were calculated when tumors reached a volume of 2000 mm³ and mice were sacrificed.

Figure 5

Dual treatment with the anti-HER3 D1 Ab 16D3-C1 and trastuzumab (●) improves tumor growth inhibition in nude mice xenografted with HER2^low A431 epidermoid (A) or A549 lung (B) carcinoma cells in comparison to mice treated with vehicle (NaCl; ◆), 16D3-C1 (▵) or trastuzumab (○) alone. Tumor growth data are presented as the mean tumor volume ± SEM for each group of eight nude mice.

Figure 6

The anti-HER3 Abs block NRG-stimulated BxPC3 pancreatic cancer cells in the G₁ phase of the cell cycle (A), inhibit their proliferation (B), and promote cell apoptosis (C). Cell cycle progression was analyzed using propidium iodide staining; cell proliferation was quantified on the basis of the cell incorporation of Alexa Fluor 488-conjugated 5-ethynyl-2′-deoxyuridine, and apoptosis was demonstrated by staining with Annexin V and 7-aminoactinomycin D.

Figure 7

The anti-HER3 D1 Abs 16D3-C1 and 9F7-F11 and the anti-HER3 D3 Ab H4B-121, but not the Abs 12H8-B11, H4B-25, and H3A-122, block HER2/HER3 heterodimerization in NIH/3T3 fibroblasts that express HER2 and HER3. The concentration of HER3 Abs or NRG is indicated. The blockade of HER2/HER3 heterodimerization was quantified by using Ab-based TR-FRET assays.

Figure 8

The anti-HER3 D1 Abs 16D3-C1 and 9F7-F11 and the anti-HER3 D3 Ab H4B-121 inhibit NRG-induced phosphorylation of HER3 as well as of AKT, leading to reduced phosphorylation of the downstream targets MDM2, XIAP, FoxO1, and GSK-3 (A) and to HER3 down-regulation (B). BxPC3 carcinoma cells were incubated with anti-HER3 Abs or the control Ab Px for various times and then stimulated with NRG for 10 minutes. Phosphorylation was analyzed after 15-minute Ab incubation at 37°C by Western blot analysis using the appropriate Abs. HER3 down-regulation was determined after Ab incubation at 37°C or 4°C for 15 minutes, 1 hour, and 2 hours. M, medium.