Antibody-guided design and identification of CD25-binding small antibody mimetics using mammalian cell surface display

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.

Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing

“…As qAbs, the scFv of pertuzumab, trastuzumab, and a CD25-targeting monoclonal antibody daclizumab, designated scFv(Per), scFv(Tra), and scFv(Dac), respectively, were designed (Supplementary Fig. 2a ), and cDNAs allowing each scFv to be displayed on the cell surface as a His-tagged qAb with an intracellular green fluorescent protein sfGFP 27 were constructed (Fig. 2c ).…”

“…For surface display of scFvs on cells, the DNA fragment of scFv(Dac) in the previously constructed plasmid pCSII/scFv-sfGFP 27 was replaced with that of other scFvs. The resultant plasmids expressed sfGFP together with scFv-fused proteins.…”

“…We previously developed a screening system for identifying antigen-binding peptides, namely monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a peptide library was displayed on the surface of antigen-expressing mammalian cells 27 . In the system, library design and screening for antigen-binding peptides are guided by a validated antigen-binding antibody, called the guide antibody.…”

Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display

“…We published an article aimed only at achieving affinity maturation of antibodies using CHO cell display [ 25 ]. In addition, antibody engineering with a mammalian display by other research groups includes Robertson’s and See’s works [ 26 , 27 ]. Here, we just performed two rounds of evolution and significantly improved both stability and affinity ( Figure 6 and Figure 7 , Table 1 ).…”

Protein scaffolds: antibody alternatives for cancer diagnosis and therapy

“…184 Tetsuya's group grafted two CD25-binding residues GGGV-D of daclizumab and YGY-RS-Y of basiliximab onto a zinc-finger peptide scaffold to construct a small antibody mimetic library, three CD25-binding ligands with 30 nM affinity were successfully selected. 189 Cassie's group designed PD-1 agonist by inserting three binding motifs ''WDYKY'', ''ADYKR'', ''WDYKR'' into 34840 de novo-designed scaffolds, after Rosetta, fold-fromloopsm, flow cytometry sorting and affinity maturation processed, an agonist PD-MP1 were selected, monomeric PD-MP1 showed 6 mM affinity to mouse PD-1, and trimeric PD-MP1 strongly inhibited murine T cell activation in vitro. 190 A helixkinked-helix motif of PfEMP1 molecules which forms core binding site of endothelial protein C receptor was integrated to a three-helical bundle scaffold as a vaccine immunogen, the final binder could elicit antibodies, although not as effective as the whole CIDRalpha1 domain.…”