All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members. This may be overcome by a HER ligand binding molecule that sequesters multiple EGF-like ligands, preventing ligand-dependent receptor activation. Toward this end, we have combined the HER-1/EGFR and HER-3 ligand binding domains, dimerized with fusion of an Fc fragment of human IgG1. This resulted in a mixture of HER-1/Fc homodimer (HFD100), HER-3/Fc homodimer (HFD300), and HER-1/Fc:HER-3/Fc heterodimer (RB200), also termed Hermodulins. The purified first-generation RB200 bound EGF and neuregulin 1 (NRG1)-beta1 ligands, determined by cross-linking and direct binding studies. The binding affinity for both was approximately 10 nmol/L by dissociation-enhanced lanthanide fluorescence immunoassay using europium (Eu)-labeled ligands. Competition studies with RB200 using Eu-EGF or Eu-NRG1-beta1 revealed that RB200 bound HER-1 ligands, including transforming growth factor-alpha and heparin-binding EGF, and HER-3 ligands NRG1-alpha and NRG1-beta3. RB200 inhibited EGF- and NRG1-beta1-stimulated tyrosine phosphorylation of HER family proteins, proliferation of a diverse range of tumor cells in monolayer cell growth assays, tumor cell proliferation as a single agent and in synergy with tyrosine kinase inhibitors, lysophosphatidic acid-stimulated cell proliferation, and tumor growth in two human tumor xenograft nude mouse models. Taken together, the data reveal that RB200 has the potential to sequester multiple HER ligands and interfere with signaling by HER-1, HER-2, and HER-3.
AACR Centennial Conference: Translational Cancer Medicine-- July 20-23, 2008; Monterey, CA
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Resistance to single-targeted anti-HER agents has been attributed to multiple HER co-activation that occurs through receptor dimerization. We constructed and purified novel molecules comprising HER1/EGFR and HER3 ligand binding domains tethered to a human Ig Fc region. By design, these Hermodulins bind the majority of HER ligands and provide a therapeutic anti-cancer strategy similar to clinically-proven molecules developed for both TNF-alpha and VEGF-related diseases. RB200, a prototypic Hermodulin, was evaluated for activity against a spectrum of cell lines derived from human malignancies. Effects on monolayer cell growth in the presence of serum were measured. Dose-dependent inhibition of cell growth by RB200 was demonstrated in many cell lines. Synergistic growth inhibition in vitro was also demonstrated when RB200 was combined with small molecule tyrosine kinase inhibitors (TKIs) and conventional cytotoxic chemotherapies. Nude mice bearing established subcutaneous human NSCLC tumors, derived from the H1437 tumor cell line, were used to evaluate in vivo activity of RB200. Mice (N=10/group) were treated with RB200, PBS or cetuximab. Tumor sizes were measured via external calipers. These studies showed ~70% inhibition of tumor growth at 10 mg/kg vs. PBS control. Greater anti-tumor effects were observed when RB200 was combined with the TKIs. Optimized versions of Hermodulins with enhanced activity are being developed.
The human epidermal growth factor (EGF) receptor (HER) family members cooperate in malignancy. Of this family, HER2 does not bind growth factors and HER3 does not encode an active tyrosine kinase. This diversity creates difficulty in creating pan-specific therapeutic HER family inhibitors. We have identified single amino acid changes in epidermal growth factor receptor (EGFR) and HER3 which create high affinity sequestration of the cognate ligands, and may be used as receptor decoys to downregulate aberrant HER family activity. In silico modeling and high throughput mutagenesis were utilized to identify receptor mutants with very high ligand binding activity. A single mutation (T15S; EGFR subdomain I) enhanced affinity for EGF (two-fold), TGF-α (twenty-six-fold), and heparin-binding (HB)-EGF (six-fold). This indicates that T15 is an important, previously undescribed, negative regulatory amino acid for EGFR ligand binding. Another mutation (Y246A; HER 3 subdomain II) enhanced neuregulin (NRG)1-β binding eight-fold, probably by interfering with subdomain II–IV interactions. Further work revealed that the HER3 subunit of an EGFR:HER3 heterodimer suppresses EGFR ligand binding. Optimization required reversing this suppression by mutation of the EGFR tether domain (G564A; subdomain IV). This mutation resulted in enhanced ligand binding (EGF, ten-fold; TGF-α, thirty-four-fold; HB-EGF, seventeen-fold; NRG1-β, thirty-one-fold). This increased ligand binding was reflected in improved inhibition of in vitro tumor cell proliferation and tumor suppression in a human non-small cell lung cancer xenograft model. In conclusion, amino acid substitutions were identified in the EGFR and HER3 ECDs that enhance ligand affinity, potentially enabling a pan-specific therapeutic approach for downregulating the HER family in cancer.
Abstract Abstract #4127 Signalling by the human EGF (HER) receptor family can induce tumorigenicity and metastasis of cancer cells. Activation of these receptors has been associated with aggressive phenotypes and poor prognosis of breast cancer and other malignancies. Currently approved HER family targeted therapeutics are primarily directed against one member of the HER family.
 We have engineered a family of bispecific (HER1:HER3) heterodimers comprised of a human IgG1-Fc fragment fused with modified extracellular domains of HER1 and HER3. These compounds, termed Hermodulins, act as “ligand traps” and inhibit ligand-stimulated activity of all members of the HER family. In this study we have shown that Hermodulins can inhibit in vitro growth and motility in breast cancer cell lines, act synergistically with other HER-directed tyrosine kinase inhibitors and monoclonal antibody therapeutics in tumor cell antiproliferation assays, and decrease the formation of osteolytic bone metastases in animal models. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4127.
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