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.

Selective chemical protein modification

“…For example, hydroxylamines have been used to generate glycoprotein mimics 155 , to label G-protein-coupled receptors 156 , antibodies 157 and therapeutic proteins for increased pharmacokinetics 6 , and for dual protein tagging and formation of bifunctional antibodies 158,159 . However, despite its widespread use, the reactions of aldehydes and ketones suffer from a number of key drawbacks.…”

Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR

“…Thus, steric factors can influence either the binding and/or amplification. Interestingly, a biotinylated S202pAcF mutant was previously shown to more efficiently form neutravidin tetramers and inhibited Her2 phosphorylation more effectively than the K169pAcF mutant in previous studies (26).…”

“…Both mutants were either expressed in shake flasks or fermented in Escherichia coli with similar yields to wild-type Fab (>200 mg∕L with fermentation) and purified using Protein G chromatography. Mass spectrometry and SDS-PAGE gel showed >95% purity (26).…”

“…For example, nonspecific electrophilic conjugation can modify lysine residues in the antigen binding site. We have developed an approach using genetically encoded UAAs that allows conjugation to antibodies in a site-specific manner (26,27). This approach relies on the site-specific incorporation of p-acetylAuthor contributions: S.A.K., P.K., P.G.S., and V.V.S.…”

“…We mutated sites K169 and S202 in anti-Her2 Fab (Fig. 1B) to pAcF because of their surface accessibility and previously demonstrated high coupling efficiencies to aminooxy-modified Alexa Fluor 488 dyes (26). pAcF was site-specifically introduced into the anti-Her2 Fab in response to the amber nonsense codon TAG with an orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pair derived from Methanococcus jannaschii (28).…”