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Characterization of an anti-p185HER2 monoclonal antibody that stimulates receptor function and inhibits tumor cell
J C Sarup1, R M Johnson, K L King
1Department of Developmental Biology, Genentech, Inc., South San Francisco, California 94080.
Abstract:
The HER2 protooncogene encodes a growth factor receptor-like transmembrane protein tyrosine kinase (p185HER2) whose ligand remains to be fully characterized. The overexpression of p185HER2 is implicated in aggressive forms of breast and ovarian cancers. The role of p185HER2 in aggressive malignancy, as well as its cell surface localization, makes it an attractive target for therapeutic monoclonal antibodies. In this report we have studied the modulation of p185HER2 function with 2 monoclonal antibodies, termed 4D5 and 6E9, which bind the extracellular domain of p185HER2. 4D5 inhibited proliferation of p185HER2 overexpressing SK-BR-3 human breast carcinoma cells (ED50 of approximately 1 nM) but did not inhibit proliferation of cultured human breast carcinoma MCF7 cells, low expressors of p185HER2. Monoclonal antibody 6E9 does not inhibit the growth of either cell line. Antibody binding studies revealed 2 populations of p185HER2 molecules on SK-BR-3 cells: one of high abundance (approximately 2 x 10(6) sites/cell) recognized by 4D5 (Kd approximately 6 nM) and the other of low abundance (2 x 10(4) sites/cell) recognized by 6E9 (Kd approximately 0.1 nM). 4D5, in an agonistic manner, downregulated SK-BR-3 cell surface p185HER2, was internalized, and stimulated p185HER2 phosphorylation in intact cells. Phosphoamino acid analysis of p185HER2 derived from SK-BR-3 cells incubated with the 4D5 monoclonal antibody demonstrated increased tyrosine, serine and threonine phosphorylation. 4D5, on short term (5 min) exposure to SK-BR-3 cells, stimulated inositol lipid hydrolysis as evidenced by increased intracellular levels of inositol polyphosphates (InsP) and sn-1,2-diacylglycerol (sn-1,2-DAG). On longer (24 h) exposure to the cells, the antibody appeared to downregulate this signalling pathway since the intracellular levels of InsP and sn-1,2-DAG decreased by 30 to 40%. 6E9 did not inhibit SK-BR-3 cell proliferation, downregulate surface p185HER2, stimulate receptor phosphorylation, or stimulate the second messenger pathway. Despite these agonistic properties, 4D5 was an inhibitor of SK-BR-3 cell proliferation at all concentrations tested (0.7 to 70 pM). The data suggest that 4D5 is a partial or weak agonist and thus may inhibit cell proliferation by mimicking ligand-like receptor downregulation.
Insights
Monoclonal antibody 4D5 targets HER2 (human epidermal growth factor receptor 2) in breast cancer cells. It acts as a weak agonist, inhibiting proliferation by downregulating the receptor.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- The HER2 protooncogene encodes p185HER2, a transmembrane protein tyrosine kinase implicated in aggressive breast and ovarian cancers.
- p185HER2's role in malignancy and cell surface localization makes it a target for therapeutic monoclonal antibodies.
Purpose of the Study:
- To investigate the functional modulation of p185HER2 by two monoclonal antibodies, 4D5 and 6E9.
- To characterize the binding and effects of these antibodies on HER2-overexpressing cancer cells.
Main Methods:
- Utilized monoclonal antibodies 4D5 and 6E9 targeting the extracellular domain of p185HER2.
- Assessed antibody effects on SK-BR-3 (high HER2 expressor) and MCF7 (low HER2 expressor) human breast carcinoma cell proliferation.
- Conducted antibody binding studies to determine receptor populations and affinity.
- Analyzed p185HER2 phosphorylation and inositol lipid hydrolysis following antibody treatment.
Main Results:
- Antibody 4D5 inhibited proliferation of SK-BR-3 cells (ED50 ~1 nM) but not MCF7 cells.
- Antibody 6E9 did not inhibit proliferation of either cell line.
- 4D5 demonstrated agonistic activity, downregulating cell surface p185HER2, inducing internalization, and stimulating receptor phosphorylation and inositol lipid hydrolysis.
- Despite agonistic properties, 4D5 inhibited SK-BR-3 cell proliferation, suggesting a partial or weak agonist mechanism.
Conclusions:
- Monoclonal antibody 4D5 exhibits partial agonistic activity, inhibiting cancer cell proliferation through a mechanism potentially involving ligand-like receptor downregulation.
- The differential effects of 4D5 and 6E9 highlight distinct binding populations and functional modulation of p185HER2.
- These findings support the therapeutic potential of targeting HER2 with specific monoclonal antibodies.