Harel, N.; Sung, K.; Dumm, W.; Johnson, M. M.; Rich, D.; Fukuyama, J.; Haddox, H. K.; Matsen, F. A.

Entrenchment - epistasis that locks in amino acid differences between homologous proteins, so each disfavors substitutions toward the other's state - has been demonstrated along individual protein lineages over deep evolutionary time. Antibodies offer a unique system for studying entrenchment: multiple homologous germline V gene paralogs provide diverse starting points, and the rapid somatic evolution of affinity maturation generates dense phylogenies from which selection on germline-encoded residues can be inferred. Using DASM, a deep learning model that separates selection from mutation in antibody repertoire data, we test for entrenchment across immunoglobulin heavy chain variable (IGHV) genes. We detect entrenchment at two levels of germline divergence, driven by different sources of epistasis. Within V gene families (up to ~20% amino acid divergence), entrenched sites cluster at the borders of the complementarity-determining regions (CDRs, the antigen-binding loops) and show high germline diversity. These sites contact antigen, light chain, and the heavy chain CDR3 loop, all of which are encoded independently of the IGHV germline. This pattern is consistent with epistasis from genetically uncoupled partners. Between V gene families, at deeper levels of divergence (25-40%), entrenchment additionally includes positions in the framework scaffold distant from binding interfaces, suggesting a larger contribution from intra-heavy-chain structural constraints. Observed mutation frequencies in human repertoires corroborate these predictions where data are sufficient. Together, these results demonstrate that the rapid somatic evolution of antibodies can serve as a lens for revealing epistatic constraints acting on germline-encoded residues, including constraints imposed by genetically uncoupled partners assembled during B cell development.