This observation has prompted us to replace the pair of the Fab constant domains with the paired IgG1 CH3 domains. Interestingly, in this study we found that the structure of an IgG1 CH3 domain pair shares high homology with that of a CH1–CL heterodimer. Therefore, it is possible that the stability of the Fab fragments could be improved by incorporating the IgG CH3 domains. We previously conducted in-depth studies on the constant regions of the IgG antibody, and found that the stability of these regions is maintained primarily by the extensive hydrophobic and electrostatic interactions between the two CH3 domains that form a stable homodimer. The Fab fragments maintain the N-terminal of the natural IgG antibody but lack the C-terminal constant region, resulting in loss of the effector functions and changes in the physicochemical properties, such as stability and aggregation. The major reason is that the CH1–CL interaction within Fab only assists in the pairing but not the stabilization of the variable regions. However, Fabs are weaker in protein structure stability than the intact antibodies. Therefore, the development of antibody fragments with higher stability is crucial for clinical use.įab fragments represent one of the most successful antibody fragments in clinic with several Fab products already approved by the US Food and Drug Administration (FDA) for clinical use (e.g., certolizumab pegol, ranibizumab, abciximab, idarucizumab). Protein instability affects many critical aspects of the biologic drug discovery and development process, including binding affinity, biological activity, protein expression, manufacturability, storage, handling, delivery, efficacy, and safety. However, most of these engineered antibody fragments are less stable than the natural full-size IgG antibodies. Consequently, several engineered antigen-binding formats with reduced size have been investigated for their potential of enhanced tissue penetration and facilitated production, such as antigen-binding fragment (Fab), variable fragment (Fv), single-chain variable fragment (scFv), heavy chain variable domain (VH), and light chain variable domain (VL). Although these mAbs have demonstrated significant clinical benefits in treating certain diseases, their relatively large size limits their capability of penetrating into tissues (e.g., solid tumors) and binding to epitopes on the surface of targets that are only accessible by molecules of smaller size. Since 1986, more than 100 mAbs have been approved for clinical use, with the vast majority being full-size antibodies in the IgG1 format (≈150 kDa).
Recombinant human monoclonal antibodies (mAbs) have become important protein-based therapeutics for the treatment of various diseases including cancer, immune disorders, and infectious diseases due to their high affinity and specificity.
Overall, the stabilized FabCH3 described in this report provides a versatile platform for engineering Fab-like antibody fragments with higher stability and antigen-binding affinity that can be used as a distinct class of antibody therapeutics. The high-resolution crystal structures of m912 FabCH3 and m912 Fab are determined, and the comparative analysis reveals more rigid structures in both constant domains and complementarity-determining regions of FabCH3, explaining its enhanced stability and affinity. This construct, designated as FabCH3, maintains the natural N-terminus and C-terminus of IgG antibody, can be expressed at a high level in bacterial cells and, importantly, exhibits much higher stability and affinity than the parental Fab when tested in a mesothelin-specific Fab m912, as well as a vascular endothelial growth factor A (VEGFA)-specific Fab Ranibizumab (in vivo). Herein, a novel Fab-like antibody fragment generated via an in silico-based engineering approach where the CH1 and CL domains of Fab are replaced by the IgG1 CH3 domains is described. With increasing interest in applying recombinant monoclonal antibodies (mAbs) in human medicine, engineered mAb fragments with reduced size and improved stability are in demand to overcome current limitations in clinical use.