title: Reorienting the Fab domains of trastuzumab results in potent HER2 activators.
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.
authors.affiliation: Department of Protein Chemistry, Genentech Inc., South San Francisco, CA, USA. scheer.justin@gene.com
authors.forename: Justin M
authors.initials: JM
authors.lastname: Scheer
authors.forename: Wendy
authors.initials: W
authors.lastname: Sandoval
authors.forename: J Michael
authors.initials: JM
authors.lastname: Elliott
authors.forename: Lily
authors.initials: L
authors.lastname: Shao
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Luis
authors.forename: Sock-Cheng
authors.initials: SC
authors.lastname: Lewin-Koh
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schaefer
authors.forename: Richard
authors.initials: R
authors.lastname: Vandlen
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D049109:Cell Proliferation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D000068878:Trastuzumab; D014407:Tumor Cells, Cultured
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007140:Immunoglobulin Fab Fragments; D020928:Mitogen-Activated Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab