Fig. 4: Evaluation of mirror-image monobody.

A Binding affinity and specificity analyzed by BLI. L-MCP-1 or D-MCP-1 was immobilized on a streptavidin-sensor chip, and synthetic monobody 9D or 9 L (2.5, 5.0, 10, 20 nM) was used in the kinetic analysis. B CD spectra of synthetic monobodies. 9D and 9 L (5 µM) dissolved in PBS (pH7.4) were measured. C Tryptic digestion of synthetic monobodies. 9D and 9 L (5 µM) dissolved in PBS (pH 7.4) containing 1% PEG were incubated with trypsin (300 nM) for 0.5, 1, and 2 h. The data are presented as mean ± SD, n = 3 biological replicates. D Evaluation of the immunogenicity of 9D and 9L . A 1:1,000 dilution of immunized plasma from each mouse collected on days 0, 14, 28, and 35 was added to the 9D or 9L-coated plates. Generation of antibodies against 9D or 9 L was detected using HRP-anti-mouse IgG (H + L). Bars represent mean ± SD calculated from independent experiments (4 mice for 9L: 5 mice for 9D). Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparisons test. **, p = 0.0047; ***, p < 0.0001. E MCP-1/CCR2 inhibition assay with cultured cells using the PathHunter® β-Arrestin eXpress GPCR Assay kit (DiscoverX). The cultured cells were incubated at 37 °C for 90 min with 9D or 9 L (ranging from 0.10 to 680 nM in a 3-fold serial dilution) and 7 nM L-MCP-1. Chemiluminescence was detected in a plate reader. The experiments were performed in 96-well plates with three wells for each condition (n = 3, biological replicates) and the bars represent mean ± SD. The dotted horizontal line denotes basal RLU as shown in Supplementary Fig. S26. F Cell migration assay was performed with and without MCP-1, and with or without the inhibitor (i.e., carlumab or D-Monobody, 9D), using three independent wells for each condition to obtain biological replicates (n = 3). Data are expressed as mean ± SD. Inhibitor concentrations were varied at 0, 0.1, 1, 10, and 100 nM. Cells that migrated to the empty chamber were quantified by Calcein-AM staining. Source data are provided as a Source Data file.