Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding

Abstract

Expansion of the amino‐acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti‐EGF‐receptor antibody 059–152 were systematically replaced with 3‐bromo‐ and 3‐chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17‐fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.

Natural and Synthetic Halogenated Amino Acids—Structural and Bioactive Features in Antimicrobial Peptides and Peptidomimetics

“…This can interestingly find applications from very small molecules to large complexes such as antibodies. For example, halogenated Tyr improved 10 fold the binding affinity of the Fab fragment of an anti-EGF-receptor (059-152) for its antigen [ 14 ].…”

“…Different types of halogenated Trp, prepared by employing specific halogenases (e.g., marine haloperoxidases) or haloindoles during biosynthesis by tryptophan synthase, can be introduced into peptides or proteins to tune biocompatibility and activity [ 226 ]. Moreover, the use of halogenated Tyr has been recently shown to increase the binding affinity of an antibody towards its antigen [ 14 ].…”

Chemical Diversification of Simple Synthetic Antibodies

“…Several strategies for expanding the chemical repertoire of antibodies are feasible, [29][30][31][32] including posttranslational modification of antibody structures containing canonical amino acids 31,[33][34] and the introduction of genetically encoded noncanonical amino acids (ncAAs). 32,[35][36][37][38] Numerous approaches to antibody chemical diversification have been explored extensively within the context of antibody-drug conjugates (ADCs). [39][40][41][42][43][44][45][46] However, most ADC development strategies focus on modification sites located in antibody constant regions that have little or no effect on antigen binding; [46][47][48][49][50][51][52] the result is modular addition of new chemistries that leave antibody binding function unchanged.…”

“…53 Sakamoto and coworkers recently reported improved antigen affinities in two antibodies upon substituting halogenated tyrosines in place of tyrosines. 38 Multiple groups have integrated ncAAs containing side chains known to interact with target antigens into antibodies for the purpose of phage display screening (Schultz, Smider, and coworkers) [54][55][56] and systematic biochemical characterizations (Chang Liu and coworkers) 57 to identify antibody constructs with binding properties improved by the use of one or more ncAAs. Several strategies for altering antibody binding functions with an expanded chemical repertoire have used antibodies containing only canonical amino acids as starting points.…”

Iodinated Diketopiperazines: Synthesis and Biological Evaluation of Iodinated Analogues of Cyclo(L‐Tyrosine‐L‐Tyrosine) Peptides

“…The iodinated tyrosine is also employed effectively to remedy the phase problem in the field of protein X‐ray crystallography [18] . The incorporation of halogens in the tyrosine side chain of protein at specific sites is found to impact the binding of antigen [19,20] . CYP121 converts the substrate Cyclo (L‐Tyr‐L‐Tyr) into the formation of Mycocyclosin (Scheme 1C) by the oxidative C−C bond formation [21–25] .…”