Kim, Y.; Kwon, H.; Hong, J.; Kang, C. K.; Park, W. B.; Kim, H.-R.; Lee, C.-H.

Background: Combinatorial fragment antigen-binding (Fab) libraries encode an immense heavy-light chain pairing space, often exceeding 10^14 possible combinations, which far surpasses the diversity that can be experimentally constructed and screened in display systems. As a result, direct Fab screening samples only a small fraction of the theoretical search space, creating a practical bottleneck for functional binder discovery. Results: Here, we frame Fab discovery as a staged search problem by decoupling heavy-chain (HC) and light-chain (LC) exploration. We implemented a sequential HC preselection-remating workflow in yeast surface display, in which antigen-reactive HC variants are first enriched and subsequently recombined with a diverse LC repertoire to reconstruct a focused Fab library. In a SARS-CoV-2 spike-targeted campaign, HC and LC libraries of 2.05 x 10^7 and 2.33 x 10^7 members corresponded to a theoretical pairing space of approximately 4.8 x 10^14 combinations. Sequential HC enrichment followed by LC remating allowed recovery of multiple functional Fab clones from a tractable library scale of approximately 10^8, including clones that shared a common HC scaffold but carried distinct LC partners. A representative recombinant IgG output showed broad but heterogeneous spike/RBD binding, measurable pseudovirus neutralization activity (EC50 = 11.1 nM), and compatibility with standard early biophysical characterization after full-length IgG reformatting. Conclusions: These results provide proof of principle that combinatorial Fab discovery can be approached as a staged exploration problem under realistic library-size constraints. By focusing downstream Fab reconstruction on an antigen-compatible HC subspace, sequential HC preselection followed by LC remating offers a practical strategy for exploring otherwise intractable antibody pairing landscapes in eukaryotic display systems.