Kinetics-Based Structural Requirements of Human Immunoglobulin G Binding Peptides

Abstract

Currently, antibodies are widely used not only in research but also in therapy. Hence, peptides that selectively bind to the fragment crystallizable site of an antibody have been extensively utilized in various research efforts such as the preparation of antibody–drug conjugates (ADC). Consequently, appropriate peptides that bind to immunoglobulin G (IgG) with a specific Kd value and also kon and koff values will be useful in different applications, and these kinetic parameters have been perhaps overlooked but are key to development of peptide ligands with advantageous binding properties. We prepared structural derivatives of IgG-binding peptide 1 and evaluated the binding affinity and kinetic rates of the products by surface plasmon resonance assay and isothermal titration calorimetry to obtain novel peptides with beneficial antibody binding properties. In this way, 15-Lys8Leu with fast-binding and slow-release features was obtained through a shortened peptide 15-IgBP. On the other hand, we successfully obtained distinctive peptide, 15-Lys8Tle, with a similar Kd value but with kon and koff values that were as much as six-fold different from those of 15-IgBP. These new peptides are useful for the elucidation of kinetic effects on the function of IgG-binding peptides and various applications of antibody or antibody–drug interactions, such as immunoliposome, ADC, or half-life extension strategy, by using a peptide with the appropriate kinetic features.

Development of a High‐Affinity Antibody‐Binding Peptide for Site‐Specific Modification

“…The affinity was three times weaker than that of 15‐Lys8Leu with no amino group (entry 1, K d =8.19 nM). This result is consistent with a previous result that the tertiary carbon structure at γ‐position slightly decreases the affinity in both association and dissociation steps compared to the secondary carbon structure (15‐Lys8Ala( t Bu)) [13a] . 15‐Lys8Aecl exhibited, however, the best affinity among the reported peptides with the side chain amino group at the position 8, and is applicable to the CCAP method [13]…”

“…The previous study also suggests that the high affinity of 15‐Lys8Leu can be attributed to the γ‐dimethyl structure of leucine, which interacts with the hydrophobic region of the antibody [13a] . In this study, we proposed new unnatural amino acid, 4‐(2‐aminoethylcarbamoyl)leucine (Aecl), containing both a primary amino group for the crosslinking and γ‐dimethyl structure necessary for the high binding affinity (Figure 1b).…”

“…Its binding affinity was found to be slightly decreased (entry 3, K d =89.8 nM) compared to unacetylated 15‐Lys8Aecl (entry 2), mainly due to the slower association step ( k on =1.26 vs. 0.478 s −1 ⋅ μM −1 ). The amino group at the position 8 might contribute partially to the high affinity of 15‐Lys8Aecl by interacting with Glu380 of the antibody [13a] . However, 15‐Lys8Aecl(Ac) still retained a higher binding affinity than 15‐Lys8Lys(Ac) ( K d =268 nM), indicating that 15‐Lys8Aecl is a promising peptide for the CCAP method.…”

“…Acylation of side chains of 15‐Lys8Aecl and FITC‐15‐Lys8Aecl was performed in solution with the previously described conditions [13a] . After disulfide bridging, the purified peptide was dissolved in DMSO at a concentration of 2.5 μM.…”

“…b) Design concept of high‐affinity IgG binding peptide (IgBP) applicable to CCAP method. IgG‐binding peptides 15‐IgBP, [13a] 15‐Lys8Leu [13a] and 15‐Lys8Aecl developed by our group.…”