title: Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids.
abstract: Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical "handle" by which immunoconjugates and multimers can be engineered.
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Hutchins
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Kazane
authors.forename: Karin
authors.initials: K
authors.lastname: Staflin
authors.forename: Jane S
authors.initials: JS
authors.lastname: Forsyth
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Felding-Habermann
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
authors.forename: Vaughn V
authors.initials: VV
authors.lastname: Smider
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology
chemical_list: D000596:Amino Acids; D007140:Immunoglobulin Fab Fragments; D010649:Phenylalanine; D018719:Erb-b2 Receptor Tyrosine Kinases; C476995:3-(4-acetylphenyl)-2-aminopropanoic acid; C508053:ERBB2 protein, human