Site-Specific Coupling and Sterically Controlled Formation of Multimeric Antibody Fab Fragments with Unnatural Amino Acids

JOURNAL OF MOLECULAR BIOLOGY

卷:406

Article

Hutchins, Benjamin M.^1,2  Kazane, Stephanie A.^1,2  Staflin, Karin³  Forsyth, Jane S.³  Felding-Habermann, Brunhilde³  Schultz, Peter G.^1,2  Smider, Vaughn V.⁴ 

[1] Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA

[2] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA

[3] Scripps Res Inst, Dept Mol & Expt Med, La Jolla, CA 92037 USA

[4] Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA

关键词: nonnatural amino acid;    immunoconjugate;    Fab multimer;    directed protein assembly;    controlled multimer geometry;   

DOI  :  10.1016/j.jmb.2011.01.011

来源: Elsevier

【 摘 要 】

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. (C) 2011 Published by Elsevier Ltd.

【 授权许可】

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