Open Access Highly Accessed Research article

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

Ceyhan Ceran1, Murat Cokol2, Sultan Cingoz3, Ipek Tasan1, Mehmet Ozturk1* and Tamer Yagci14*

Author Affiliations

1 BilGen Genetics and Biotechnology Research Center, Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey

2 Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, 34956, Istanbul, Turkey

3 Department of Medical Biology, Dokuz Eylul University Medical School, Izmir, Turkey

4 Faculty of Science, Department of Molecular Biology and Genetics, Gebze Institute of Technology, 41400, Kocaeli, Turkey

For all author emails, please log on.

BMC Cancer 2012, 12:450  doi:10.1186/1471-2407-12-450

Published: 4 October 2012

Additional files

Additional file 1:

Anti-HER2 antibodies did not react with EGFR. Mice were immunized with SK-BR-3 cells, followed by a recombinant protein composed of extracellular domain of HER2 fused to human IgG1 Fc domain (HER2 ECD). ELISA plates that have been coated with either HER2 ECD or EGFR ECD were incubated with antibody-containing hybridoma supernatants, followed by alkaline phosphatase-conjugated anti-mouse IgG antibodies. The Y axis shows the absorbance (OD) reading at 405 nm following incubation with alkaline phosphatase substrate (assays in duplicate).

Format: PDF Size: 34KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 2:

Antibody binding to endogenously expressed HER2 protein as tested by immunoprecipitation-western blot assay. T47D-derived lysates were subjected to immunoprecipitation with different anti-HER2 antibodies. Antigen-antibody complexes were captured onto Protein G-conjugated beads, eluted and subjected to western blot assay using CB11 antibody. Blots were overexposed to visualize the weakly positive HER2 immunoprecipitated by BH5 antibody. Tzm: Trastuzumab used as a positive control antibody. (−): No primary antibody.

Format: PDF Size: 350KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 3:

Epitope mapping of anti-HER2 antibodies using combined immunoprecipitation-western blot assay. Huh7 cells were transfected in 6-well plates using a set of mammalian expression plasmids encoding full-length or N-terminally truncated HER2 protein. Transfected cells were cultivated for 48 h; cell lysates were subjected to immunoprecipitation with different anti-HER2 antibodies. Antigen-antibody complexes were captured onto Protein G-conjugated beads, eluted and subjected to western blot assay using CB11 antibody. Arrows: immunoreactive bands specific for full-length and truncated HER2 protein forms. IP: immunoprecipitation; *: non-specific bands originating from antibodies used for immunoprecipitation.

Format: PDF Size: 541KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 4:

Interaction between anti-HER2 antibodies and tumor necrosis factor-α. MDA-MB-361 cells were partially sensitive to anti-HER2 antibodies, but highly sensitive to TNF-α (top). MCF-7 cells were resistant to anti-HER2 antibodies, but highly sensitive to TNF-α (middle). T47D cells were resistant to both anti-HER2 antibodies and TNF-α (bottom). Growth measurements observed under 5μg/ml antibody (white columns), 1000 U/ml TNF-α (striped columns), and 5 μg/ml antibody + 1000 U/ml TNF-α (black columns) were obtained experimentally, as described in Figure 6. Growth level under a drug condition was defined as the growth under that condition normalized by growth under no drug condition. Expected growth level under no interaction (gray columns) was calculated by multiplying the growth levels under each individual drug. The observed growth level for each combination was divided by the expected growth level to find an interaction score according to Bliss Independence Model for drug interactions. Interaction scores were not calculated for these cell lines.

Format: PDF Size: 40KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 5:

In vitro molecular responses of SK-BR-3 cells to tumor necrosis factor-α. SK-BR-3 cells were treated up to 24 h with TNF-α (1000 U/ml). Cell lysates were prepared from cells harvested at indicated times (hr, hours) and the expressions of phospho-HER2 (pHER2), phospho-Akt (pAkt), total Akt (Akt), phospho-ERK1/phospho-Erk2 (pErk1/2), total Erk1/Erk2 (Erk 1/2) and Cyclin D1 were analyzed by western blotting. Calnexin was used as a loading control.

Format: PDF Size: 62KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data