Browsing by Author "Ceran, Ceyhan"
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Item Open Access Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α(BioMed Central, 2012) Ceran, Ceyhan; Çokol, M.; Cingöz, S.; Taşan, İpek; Öztürk, Mehmet; Yağcı, TamerBackground: One-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). Methods: Mice 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. Results: We 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. Conclusions: Novel 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.Item Open Access Novel monoclonal antibodies targeting conformational ERBB2 epitopes(Bilkent University, 2012) Ceran, CeyhanERBB2 is a tyrosine kinase receptor which can act as homodimers or heterodimers with other members of the ERBB family. Nearly 30% of breast cancers overexpress ERBB2, which can be effectively targeted by anti-ERBB2 monoclonal antibodies. Trastuzumab directed against an epitope on subdomain IV of the extracellular domain (ECD) of ERBB2 is a clinically used therapeutics but the response rate is poor and acquired resistance is frequent. Pertuzumab that binds to subdomain II and inhibits receptor dimerization is another promising therapeutics under clinical trials. Anti-ERBB2 antibodies directed to novel epitopes are potentially useful tools for replacement and combinatorial therapies. We produced five new anti-ERBB2 antibodies, all directed against epitope(s) present only on the native ECD. They performed selective growth inhibitory effects depending on the level of ERBB2 expression and cellular background. When used alone, novel anti-ERBB2 antibodies displayed modest but significant growth inhibition on SK-BR-3, BT-474 and MDA-MB-361 cells with ERBB2 overexpression; while no detectable inhibition was observed on MCF-7 and T47D cells lacking ERBB2 amplification. When the antibodies were tested in combination with TNF-α, they acted synergistically on SK-BR-3 cells, producing upto 80% growth inhibition; but performed antagonistically on BT-474 cells. Detailed investigation of a representative antibody indicated G1-arrest as the main mechanism of the anti-proliferative effects exerted on SK-BR-3 cells. Antibody treatment induced permanent inhibition of DNA synthesis, leading to accumulation of cells at G1-phase; an effect which was accelerated in the presence of TNF-α. In addition, treated SK-BR-3 cells displayed inhibition of Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest, independently from TNF-α. Novel antibodies against conformational epitopes present on the extracellular domain of ERBB2 receptor may serve as new analytical and diagnostic tools, in addition to being potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-ERBB2 antibodies and TNF-α provide evidence for a complex interplay between ERBB2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of ERBB2-directed therapeutic interventions.