Pinpoint Insertion of a Single Amino Acid into a Framework Region Enhanced the Affinity of an Anti-cortisol Antibody to Enable Sensitive Immunoassays

Abstract

“Antibody engineering” is a promising strategy for generating high-affinity antibodies required for developing sensitive immunoassays. Therein, the variable domains (V_H and V_L) of the parental antibody are genetically randomized and combined to produce diverse single-chain Fv fragment (scFv) molecules. Subsequently, high-affinity scFv mutants are selectively isolated. In the randomization process, mutations have conventionally been targeted to the complementarity-determining regions (CDRs) in the variable domains, which often interact directly with antigens. However, we previously discovered that, pinpoint insertion of only a single amino acid (leucine, asparagine, aspartic acid, proline, glutamine, arginine, or histidine) between positions 6 and 7 in the framework region 1 (FR1) of the V_H, which is unlikely to interact with antigens, enhanced the affinity of an anti-cortisol scFv (original K_a, 3.6 × 10⁸ M⁻¹) up to 17–61-fold. These findings prompted us to conduct a comprehensive study of this affinity-enhancement phenomenon involving the remaining amino acids. Thus, we generated the necessary 13 scFv mutants and compared their K_a values. Remarkably, all mutants showed enhanced affinities, similar to those of the previous 7 mutants. Among the 20 mutants, the leucine-inserted scFv showed the largest K_a (2.2 × 10¹⁰ M⁻¹) and consequently enabled a 75-fold more sensitive enzyme-linked immunosorbent assay (midpoint, 9.86 pg/assay) compared to the assay using the parental scFv (midpoint, 744 pg/assay). In silico modeling suggested that, regardless of the amino acid inserted, elongated FR1 can alter the conformation of the CDR3 in V_H to facilitate a favorable interaction with cortisol.