Structure-Guided CDR3 Insertion Enables Affinity Maturation of Naive Single-Domain Antibodies against Small-Molecule Haptens

Abstract Single-domain antibodies (sdAbs) derived from naive phage display libraries offer a time-efficient alternative to animal immunization but often exhibit suboptimal affinity, particularly for small-molecule haptens, where restricted binding interfaces limit the efficacy of traditional saturation mutagenesis. For instance, the wild-type sdAb (B1)-targeting ethoxyquin (EQ) exhibits a moderate equilibrium dissociation constant (KD) at the submicromolar level (within the 10−7 M range), restricting its practical sensitivity. To overcome the structural and energetic barriers inherent in hapten recognition, a “structure-guided directed modification” strategy was developed that focuses on conformational tuning rather than simple side-chain replacement. Integrating AlphaFold2 modeling and AutoDock mechanistic analysis identified key interactions (e.g., the ASP19-EQ-N8 hydrogen bond). Subsequently, a random single-amino acid insertion strategy was implemented within the CDR3 loop (residues 77−90) to fine-tune local loop geometry. Top candidates were screened via MM/GBSA binding free energy calculations and experimentally validated using biolayer interferometry. This approach yielded three high-affinity mutants—77A, 79C, and 85H—with calculated ΔΔG values of −10.56, −4.35, and −6.69 kcal/mol, respectively. The mutants achieved enhanced affinities in the tens-of-nanomolar range (down to 35.1 nM), representing up to approximately 3 times overall improvement. Importantly, orthogonal surface plasmon resonance analysis using nonconjugated, free EQ successfully verified this affinity maturation trend, showing that mutant 79C bound free EQ with a KD of 1.30 × 10−5 M (a 2.3 times improvement over wild-type) through simultaneously accelerated association and slowed dissociation. Mechanistically, this substantial increase in affinity is attributed not only to reinforced noncovalent networks that significantly stabilize the complex but also to favorable CDR3 geometric outward flips that alleviate steric hindrance, leading to an approximate 3.8 times acceleration in the kon. Crucially, these mutants demonstrated notable anti-interference tolerance in complex aquatic food matrices (e.g., sea bass extracts). Ultimately, this work provides a practical and efficient computational framework for assisting the rapid evolution of low-affinity hapten sdAbs into high-performing biorecognition elements with potential for next-generation biosensing architectures.