Structure of the Fab fragment of a humanized 5E5 antibody to a cancer-specific Tn-MUC1 epitope.

Abstract

The structure of the humanized Fab from murine monoclonal antibody 5E5 specific for tumor antigen Tn-MUC1 has been determined to 1.57 Å resolution. Despite undertaking thousands of crystallization trials of the humanized 5E5 (h-5E5) Fab in the presence of either the singly or doubly glycosylated peptide antigens corresponding to Tn-MUC1, the Fab is only observed unliganded in the crystal. The conformations of the complementarity-determining regions (CDRs) of the combining site on the h-5E5 Fab do not differ significantly from those reported for liganded murine scFv at 3.0 Å resolution. While the affinity of the murine 5E5 has previously been reported as KD = 1.7 nM for the 24-mer Tn-MUC1 peptide PPAHGVT*SAPDTRPAPGS*T*APPAH prepared by in vitro glycosylation of a synthetic 24-mer MUC1 peptide, the KD of the h-5E5 Fab for the shorter doubly glycosylated glycopeptide antigens PAPGS*T*AP and APGS*T*AP was measured here as only 41 and 61 µM, respectively. Interestingly, the single Fab molecule in the asymmetric unit of space group C2 is observed packed head-to-head with a symmetry-related Fab across a crystallographic twofold axis such that a polypeptide loop from the light chain of each Fab is observed to insert into the antigen-binding pocket of the symmetry-related Fab. While this might suggest that binding of the Tn-MUC1 peptides may have been inhibited by a homophilic association, none was detected. The humanization process has imposed changes in the framework regions of the Fv which may have affected the Vh-Vl interface.