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A facile yeast-display approach for antibody mask discovery.

Nithya M Badarinath,Basudeb Mondal,C. Yellman,Kendreze L Holland,Hee Jun Lee,H. Phuengkham,Andrew P Cazier,Jaewoo Son,Jacob R Smith,J. R. Cox,Andrew J Kristof,Yusef Haikal,G. Kwong,J. Blazeck

Published 2025 in Protein engineering, design & selection : PEDS

ABSTRACT

Tuning in vivo activity of protein therapeutics can improve their safety. In this vein, it is possible to add a 'mask' moiety to a protein therapeutic such that its ability to bind its target is prevented until the mask has been proteolytically removed, for instance by a tumor-associated protease. As such, new methods to isolate functional masking sequences can aid development of protein therapies. Here, we describe a yeast display-based method to discover peptide sequences that prevent binding of antibody fragments to their antigen target. Our method includes an in situ ability to screen for restoration of binding by scFvs after proteolytic mask removal, and it takes advantage of the antigenic target itself to guide mask discovery. First, we genetically linked a yeast-displayed αPSCA scFv to overlapping 'tiles' of its target. By selecting for reduced antigen binding via flow cytometry, we discovered two peptide masks that we confirmed to be linear epitopes of the αPSCA scFv. We then expanded our method towards developing masks for three-dimensional epitopes by using a co-crystal structure of an αHer2 antibody in complex with its antigen to guide combinatorial mask design. In sum, our efforts show the feasibility of employing yeast-displayed, antigen-based libraries to find antibody masks.

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