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Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids. 2011

Benjamin M Hutchins, and Stephanie A Kazane, and Karin Staflin, and Jane S Forsyth, and Brunhilde Felding-Habermann, and Peter G Schultz, and Vaughn V Smider
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

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.

UI MeSH Term Description Entries
D007140 Immunoglobulin Fab Fragments Univalent antigen-binding fragments composed of one entire IMMUNOGLOBULIN LIGHT CHAIN and the amino terminal end of one of the IMMUNOGLOBULIN HEAVY CHAINS from the hinge region, linked to each other by disulfide bonds. Fab contains the IMMUNOGLOBULIN VARIABLE REGIONS, which are part of the antigen-binding site, and the first IMMUNOGLOBULIN CONSTANT REGIONS. This fragment can be obtained by digestion of immunoglobulins with the proteolytic enzyme PAPAIN. Fab Fragment,Fab Fragments,Ig Fab Fragments,Immunoglobulins, Fab Fragment,Fab Immunoglobulin Fragments,Immunoglobulin Fab Fragment,Immunoglobulins, Fab,Fab Fragment Immunoglobulins,Fab Fragment, Immunoglobulin,Fab Fragments, Immunoglobulin,Fragment Immunoglobulins, Fab,Fragment, Fab,Immunoglobulin Fragments, Fab
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008956 Models, Chemical Theoretical representations that simulate the behavior or activity of chemical processes or phenomena; includes the use of mathematical equations, computers, and other electronic equipment. Chemical Models,Chemical Model,Model, Chemical
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D010649 Phenylalanine An essential aromatic amino acid that is a precursor of MELANIN; DOPAMINE; noradrenalin (NOREPINEPHRINE), and THYROXINE. Endorphenyl,L-Phenylalanine,Phenylalanine, L-Isomer,L-Isomer Phenylalanine,Phenylalanine, L Isomer
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000596 Amino Acids Organic compounds that generally contain an amino (-NH2) and a carboxyl (-COOH) group. Twenty alpha-amino acids are the subunits which are polymerized to form proteins. Amino Acid,Acid, Amino,Acids, Amino
D055503 Protein Multimerization The assembly of the QUATERNARY PROTEIN STRUCTURE of multimeric proteins (MULTIPROTEIN COMPLEXES) from their composite PROTEIN SUBUNITS. Protein Dimerization,Protein Heteromultimerizaton,Protein Multimer Assembly,Protein Trimerization,Assembly, Protein Multimer,Dimerization, Protein,Heteromultimerizaton, Protein,Heteromultimerizatons, Protein,Multimer Assembly, Protein,Multimerization, Protein,Trimerization, Protein
D018719 Receptor, ErbB-2 A cell surface protein-tyrosine kinase receptor that is overexpressed in a variety of ADENOCARCINOMAS. It has extensive homology to and heterodimerizes with the EGF RECEPTOR, the ERBB-3 RECEPTOR, and the ERBB-4 RECEPTOR. Activation of the erbB-2 receptor occurs through heterodimer formation with a ligand-bound erbB receptor family member. HER-2 Proto-Oncogene Protein,Proto-Oncogene Protein HER-2,Proto-Oncogene Protein p185(neu),c-erbB-2 Protein,erbB-2 Proto-Oncogene Protein,erbB-2 Receptor Protein-Tyrosine Kinase,neu Proto-Oncogene Protein,Antigens, CD340,CD340 Antigen,Erb-b2 Receptor Tyrosine Kinases,Metastatic Lymph Node Gene 19 Protein,Neu Receptor,Oncogene Protein HER-2,Proto-Oncogene Proteins c-erbB-2,Proto-oncogene Protein Neu,Receptor, Neu,Receptors, erbB-2,Tyrosine Kinase-type Cell Surface Receptor HER2,p185(c-neu),p185erbB2 Protein,CD340 Antigens,Erb b2 Receptor Tyrosine Kinases,ErbB-2 Receptor,HER 2 Proto Oncogene Protein,Oncogene Protein HER 2,Proto Oncogene Protein HER 2,Proto Oncogene Proteins c erbB 2,Proto-Oncogene Protein, HER-2,Proto-Oncogene Protein, erbB-2,Proto-Oncogene Protein, neu,Tyrosine Kinase type Cell Surface Receptor HER2,c erbB 2 Protein,erbB 2 Proto Oncogene Protein,erbB 2 Receptor Protein Tyrosine Kinase,erbB-2 Receptors,neu Proto Oncogene Protein

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