Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

Abstract

BackgroundOne-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α).MethodsMice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation.ResultsWe produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-α.ConclusionsNovel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-α provide evidence for a complex interplay between HER2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

Harnessing the immune system for the treatment of breast cancer

“…It was recently shown that targeting transmembrane TNF-α was effective in delaying tumor growth and inhibiting tumor metastasis (Yu M. et al, 2013). TNF-α inhibition may synergize with anti-HER2 therapy to improve treatment outcomes (Ceran et al, 2012). These studies suggest that TNF-α may play a significant role in breast tumor progression not only by directly acting on breast cancer cells, but also by recruiting and activating suppressive immune cells.…”

Design of in silico Trifunctional Antibody (hIgG1-FC: mouse-antiHER2× human-B7.1) Gene Cassettes and Expression Vectors: The Stage Prior to Production

“…Human epidermal growth factor receptor (HER) family consists of four different high affinity tyrosine kinase receptors including EGFR (HER 1 or ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4) [1,2]. The HER2 over-expression, as a part of neoplastic progression, has been implicated in tumor proliferation, division, and angiogenesis [3].…”

“…In the absence of a known ligand, hetero-dimerization of this receptor with other family members, particularly with EGFR which results in effective activation of intracellular signaling pathways, promotes cell proliferation and survival [3][4][5]. Since these cell surface receptors (HER2 and EGFR) are amplified and over-expressed in a large number of tumor types, they are considered as ideal targets for selective molecular therapy of cancer [1][2][3][4][5].…”

“…Since these cell surface receptors (HER2 and EGFR) are amplified and over-expressed in a large number of tumor types, they are considered as ideal targets for selective molecular therapy of cancer [1][2][3][4][5]. Monoclonal antibodies' (mAbs) development against the extra-cellular domain of these receptors is an approach which is, currently, being taken to prevent their signaling.…”

“…Monoclonal antibodies' (mAbs) development against the extra-cellular domain of these receptors is an approach which is, currently, being taken to prevent their signaling. In recent years, various antibodies including humanized, chimeric, and more recently single-domain mAbs have been successfully developed against these receptors [1][2][3][4][5].…”