Design Strategy to Create Antibody Mimetics Harbouring Immobilised Complementarity Determining Region Peptides for Practical Use

Abstract

300 s. The dissociation of the interaction was subsequently measured for 300 s. Systematic baseline drift correction was done by subtracting the shift recorded for sensors loaded with ligand but incubated without analyte. Data analysis and curve fitting were done using Octet software version 11.0. Experimental data were fitted with the binding equations available for a 1:1 interaction with local fitting and the mean ± standard error of the mean (SEM) values of k on and k off were calculated from the data of five different concentrations of an analyte. The K D was calculated as the ratio of k off /k on. cell immunostaining. The human cervical cancer cell line HeLa and human gastric cancer cell line N87 or human breast cancer cells expressing firefly luciferase SK-BR-3/Luc were obtained from the RIKEN Bio-Resource Center (Tsukuba, Japan) or JCRB Cell Bank (Osaka, Japan), respectively, and maintained at 37 °C in 5% FBS-DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) (Nacalai Tesque). The cells (1.0 × 10 4 cells/well) were seeded on a slide chamber plate, cultured for 16 h and fixed by treatment with a 4% paraformaldehyde solution (Nacalai Tesque) for 10 min at 25 °C. The cells were blocked with 1% BSA/PBS for 1 h at 25 °C. After the addition of 50 nM purified His-tagged FLAPs or 5 nM trastuzumab in 1% BSA/PBS to the chamber, the cells were incubated for 16 h at 4 °C. Thousand-fold diluted Alexa Fluor 488-conjugated mouse anti-His tag secondary antibody (Medical & Biological Laboratories, Aichi, Japan) or Alexa Fluor 488-conjugated mouse anti-human IgG Fc secondary antibody in 1% BSA/PBS were used to fluorescently label the His-tagged FLAPs or trastuzumab, respectively. After washing three times with PBS, the stained cells were mounted with Fluoromount (Diagnostic ByoSystems, CA, USA) containing 1/1000 Hoechst 33342 (Nacalai Tesque). All photos were taken using a BZ-X700 microscope (Keyence, Osaka, Japan). Statistical analysis. Data are presented as means ± SEM and were statistically analysed with a two-sided Student's t-test; p values of < 0.05 were considered statistically significant.

Strategic design to create HER2-targeting proteins with target-binding peptides immobilized on a fibronectin type III domain scaffold

“…The HBP of HBP-FLAP must be exposed on the surface of the molecule because the GA site was selected because it is a solvent exposed loop in the FN3 structure. 18 Therefore, the proteolysis resistance of the HBP in HBP-FLAP is not because of reduced protease access to the target sequence but is likely due to its immobilized structure, which restricts protease activity.…”

“…22,23 In addition, previous calculations revealed that residues 51-56 of FN3 are a gra acceptor (GA) site in which the structure of graed hexapeptides can be immobilized. 18 FN3 has been used for the development of affinity proteins targeting various antigens by protein engineering, 23,24 some of which have entered clinical trials, 25 suggesting that FN3 is a promising scaffold for creating HBP-FLAP. The HBPs were rst isolated from a phage-displayed peptide library ( Fig.…”

“…17 Based on this nding, we reported a computational design strategy for developing antibody mimetics named FLAP, which is uctuation-regulated affinity protein (FLAP) having structurally immobilized mAb-derived peptide. 18 In this study, we focused on the development of a HER2targeting small protein with a structurally immobilized HBP, HBP-FLAP. The optimal HBP in an HBP-FLAP showed improved proteolysis resistance and exhibited specic HER2-binding in vitro and in vivo.…”

Reconstitution of an Anti‐HER2 Antibody Paratope by Grafting Dual CDR‐Derived Peptides onto a Small Protein Scaffold

“…This is in line with previous findings that light chain CDRs generally play a more secondary or supporting role in target binding, such as stabilization of other CDRs. [15][16][17] Finally, we calculated the RMSD values of the HER2-binding residues between the three models and the antigen-free and antigen-bound structures of trastuzumab Fab. The rank 1 model yielded RMSD values of 1.64 and 1.86 Å to the antigen-free and antigen-bound structures, respectively; rank 2, 1.73 and 2.00 Å; and rank 3, 1.74 and 2.10 Å.…”

“…[11,12] An alternative approach is to use mAb-derived CDRs. Since antibody paratopes are usually formed by a combination of dominant and ancillary CDRs [15][16][17] that bind to linear or planar epitopes, [14] optimal paratope reconstitution is difficult with a single CDR graft onto a small protein scaffold. However, grafting multiple CDR peptides onto a small protein scaffold is challenging.…”