Characterization of adalimumab Fab variants with the CH1 domain replaced by the Cα1 domain of IgA

Antibody constant domains (C domains) contribute to structural stability. However, studies focusing on Fab fragments with heterologous constant domains are limited. Here, we engineered an IgA-type Fab (Fab_CH1IgA) by replacing the CH1 domain of an IgG1-type adalimumab Fab with the corresponding Cα1 domain of IgA1. Fab_CH1IgA was expressed in CHO cells at approximately 50 mg L^-1 and purified to homogeneity. DSC showed comparable thermal stability, with <i>T</i> _m = 74.8 °C for Fab_CH1IgA and 75.2 °C for the IgG1-type Fab. SPR analysis showed similar antigen-binding kinetics, with <i>K</i> _D = 2.23 n M for Fab_CH1IgA and 1.77 nM for the IgG1-type Fab. Structural analysis identified Pro124 and Tyr211 in the C domain as part of the hydrophobic core-bridging variable and constant domains. Substitution of these residues with their IgG-type counterparts reduced thermal stability, underscoring the critical contribution of V-C domain interactions. Although the sequence identity between the IgG1 and IgA1 constant domains was not particularly high, the CH1 domain in adalimumab Fab could be replaced by the IgA Cα1 domain without markedly compromising stability or activity. These findings highlight cooperative packing at the V-C interface, offering important insights into Fab engineering to enhance function while preserving biophysical integrity, and supporting the design of IgG-IgA chimeric antibodies and novel chimeric Fab formats.