Abstract
Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.
Highlights
Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies
In the first step of monoclonal antibodyguided peptide identification and engineering (MAGPIE), CD25-binding peptide sequences were selected from the complementarity-determining regions (CDRs) of the guide antibody (gAb), daclizumab
The resulting cDNA library was inserted into a plasmid that encoded superfolder GFP along with other elements required to express Zif-FLuctuation-regulated Affinity Proteins (FLAPs) candidates on the cell surface (Fig. 1a)
Summary
Small antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). Our recent studies have identified human epidermal growth factor receptor 2 (HER2)-binding FLAPs composed of a fibronectin type III domain (FN3) scaffold These HER2-binding FLAPs have relatively high binding affinities (dissociation constants of 24–65 nM as measured by ELISA), and we have succeeded in demonstrating the efficiency and simplicity of the methods, and the target specificity and potential of FLAPs as non-Ig-derived antibody alternatives[16,17]. The new method, which we have called monoclonal antibody-guided peptide identification and engineering (MAGPIE), allows us to identify high-affinity FLAPs from a small library of candidates in a single selection round. A humanized mAb against interleukin-2 receptor alpha chain (IL2Rα, CD25)[19], as a gAb and efficiently identified CD25-binding FLAPs called Zif-FLAPs composed of target-binding peptides immobilized in the first zinc-finger domain of Zif[268] (Zif), which acts as a small non-Ig protein scaffold. The successful identification of high affinity Zif-FLAPs using MAGPIE demonstrates its potential as a tool for efficient development of small non-Ig antibody mimetics using any mAb
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