A side-by-side biophysical comparison of five single-domain antibody scaffolds used for synthetic display libraries

The development of complex antibody therapeutics requires building blocks with high binding specificity, favorable biophysical traits, and high degree of humanness. Single-domain antibodies (sdAbs) have emerged as promising candidates due to their small size, stability, and versatility. While originally derived from camelids and cartilaginous fish, humanized and human sdAbs are now accessible via synthetic libraries and AI/ML tools. Multiple sdAb phage display libraries have been created using different scaffolds, but systematic comparison is difficult due to varying methodologies. In this study, we report a side-by-side evaluation of five sdAb scaffolds frequently used in synthetic display libraries. Using sequence analysis, biophysical assays, and AlphaFold-based structure prediction, we evaluated performance across key parameters, including humanness, expression yield, hydrophobicity, monomericity, stability, and display efficiency. Analyzed camelid sdAbs exhibited superior biophysical traits but the lowest degree of humanness. On the other hand, analyzed sdAbs based on human or humanized VHs show close sequence homology to human germline sequences, but limitations in biophysical traits. Furthermore, we observed that mutations aimed at improving stability or humanization compromised biophysical properties. Our findings underline the complexity of multi-parameter optimization of sdAbs. Simultaneously, they highlight the continued value of synthetic display libraries for application-focused discovery of sdAbs for advanced therapeutic antibody formats.