Reorienting the Fab domains of trastuzumab results in potent HER2 activators

PLoS One. 2012;7(12):e51817. doi: 10.1371/journal.pone.0051817. Epub 2012 Dec 20.

Abstract

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab')(2)-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

MeSH terms

  • Antibodies, Monoclonal, Humanized / immunology
  • Antibodies, Monoclonal, Humanized / pharmacology*
  • Apoptosis
  • Blotting, Western
  • Breast Neoplasms / drug therapy*
  • Breast Neoplasms / metabolism
  • Cell Proliferation
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Flow Cytometry
  • Humans
  • Immunoenzyme Techniques
  • Immunoglobulin Fab Fragments / immunology
  • Immunoglobulin Fab Fragments / pharmacology*
  • Mitogen-Activated Protein Kinases / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphorylation
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptor, ErbB-2 / metabolism*
  • Trastuzumab
  • Tumor Cells, Cultured

Substances

  • Antibodies, Monoclonal, Humanized
  • Immunoglobulin Fab Fragments
  • Phosphatidylinositol 3-Kinases
  • Receptor, ErbB-2
  • Proto-Oncogene Proteins c-akt
  • Mitogen-Activated Protein Kinases
  • Trastuzumab

Grant support

The authors have no support or funding to report.