9ECI

Crystal structure of a humanized 5E5 antibody

Summary for 9ECI

• Entry DOI: 10.2210/pdb9eci/pdb
• Descriptor: 5E5 Fab heavy chain, 5E5 Fab light chain, (4S)-2-METHYL-2,4-PENTANEDIOL, ... (4 entities in total)
• Functional Keywords: humanized 5e5 antibody, tn-muc1, cancer, immune system
• Biological source: synthetic construct; More
• Total number of polymer chains: 2
• Total formula weight: 47898.49

Authors

Li, W.,Evans, S.V. (deposition date: 2024-11-14, release date: 2025-04-30, Last modification date: 2025-05-14)

Primary citation

Li, W.,Mandel, U.,van Faassen, H.,Parker, M.J.,Legg, M.S.G.,Hussack, G.,Clausen, H.,Evans, S.V.
Structure of the Fab fragment of a humanized 5E5 antibody to a cancer-specific Tn-MUC1 epitope.
Acta Crystallogr D Struct Biol, 81:223-233, 2025

PubMed 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 K = 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 K 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.

PubMed: 40221891
DOI: 10.1107/S2059798325002554

Experimental method

X-RAY DIFFRACTION (1.57 Å)