Knowledge Bank

Understanding antibody-target interactions is essential for the efficient development of immune therapeutics. Antibody variable domains contain the Complementarity-Determining Regions (CDRs) responsible for antigen binding, and framework regions (FR) that provide structural support. Various antibody numbering schemes have been developed in hopes of better characterization of CDRs, but the binding hotspots identified are not consistent across schemes. This inconsistency can lead to delays in drug discovery screenings as possible binding epitopes may be untested or mutations of the FR destabilize potentially viable proteins. Here we studied the binding of the 3E8 single chain variable fragment (scFv), part of a family of antibodies that bind to the sialyl-Tn (STn) glycan antigen on proteins, using mutagenesis studies. There is relatively little known of how glycan-binding antibodies interact with antigens, which adds to the complexity of developing therapeutics targeting these interactions. The 3E8 scFv CDR mutants were developed based on Kabat numbering scheme and through alanine scanning, to assess biochemical properties. Mutants’ functions were evaluated via dot blots and surface-plasmon resonance (SPR) assays and structurally defined by thermostability melts and X-ray crystallography. Based on analysis of the 3E8 scFv, current numbering schemes cannot predict all crucial residues, and some identified residues were unessential for binding. Only heavy chain CDR loops were observed to be critical for binding, specifically HCDR3, as mutations in this loop lead to significant decrease in binding ability. HCDR2 was identified as a target for further optimization studies as single point mutations in this loop were well tolerated. Single point CDR mutants highlighted a precise side pocket for antibody binding that was not accurately identified by sequence databases. These findings underscore the critical role of heavy chain CDRs, in antibody binding affinity and specificity, highlighting the need for refined numbering schemes to enhance the development of immune therapeutics. Further research into glycan-binding antibodies is needed to better understand their mechanisms and improve therapeutic strategies targeting glycan antigens.