Structure/activity studies of the anti‐MUC1 monoclonal antibody C595 and synthetic MUC1 mucin‐core‐related peptides and glycopeptides

Abstract

MUC1 mucin is a large complex glycoprotein expressed on normal epithelial cells in humans and overexpressed and under or aberrantly glycosylated on many malignant cancer cells which consequently allows recognition of the protein core by antibodies. In order to understand how glycosylation may modulate or regulate antibody binding of mucin protein core epitopes, we have analyzed the antibody C595 (epitope RPAP) for its structure, stability, and its binding to a series of synthetic peptides and glycopeptides by a number of spectroscopic methods. Thermal and pH denaturation studies followed by changes in the CD spectrum of the antibody indicate critical involvement of specific residues to the stability of the antibody. Fluorescence binding studies indicate that α‐N‐acetylgalactosamine (GalNAc) glycosylation of a MUC1 mucin synthetic peptide TAPPAHGVT9SAPDTRPAPGS20T21APPA at threonine residues 9 and 21 and serine residue 20 enhanced the binding of antibody. The structural effects of GalNAc glycosylation on the conformation of the MUC1 peptide were studied. CD of the peptides and glycopeptides in a cryogenic mixture cooled to approximately −97°C revealed that a left‐handed polyproline II helix (PPII) is adopted by the peptides in solution, which appears to be further stabilized by addition of the GalNAc residues. Consistent with the PPII helical structure, which has no intra‐amide hydrogen bonds, high‐field NMR spectroscopy of the glycopeptide revealed no sequential dNN, medium‐range, or long‐range nuclear Overhauser effect (NOE) connectivities. These studies indicate that stabilization of the PPII helix by GalNAc glycosylation present the epitope of C595 antibody with a favorable conformation for binding. Furthermore, they illustrate that glycosylation of the MUC1 tumor marker protein with a simple O‐linked saccharide expressed in many cancers, can enhance the binding of the clinically relevant C595 antibody. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 79–91, 1999

A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

“…19 The antibody binds specifically and preferentially to differentially glycosylated MUC1 in immunohistochemistry. Thus, although the epitope of the PH1 antibody may fall outside the amino acids that are putatively glycosylated, its binding may still be affected because of steric hindrance or carbohydrate-induced conformational changes of the epitope within the tandem repeat region, as described by Spencer and colleagues 28 for the antibody C595 that binds to the RPAP epitope.…”

Antibody recognition of a unique tumor-specific glycopeptide antigen

“…Although SM3 is specific for a glycopeptide, X-ray crystallography and NMR studies revealed that glycosylation was not required for binding, but that the GalNAc O-glycosylation induced conformational changes in the peptide that enhanced its interactions with the antibody (20,34). Similarly, mAb C595 raised against another peptide epitope in MUC1 (sequence RPAP) was found to have enhanced affinity because of conformational changes induced by Tn glycosylation, which was attributed to the stabilizing of a left-handedpolyproline II helix by di-or triglycosylation of the peptide (35).…”

Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat

“…The binding of the antibody SM3 to the PDTR region of MUC1 has been studied in detail by X-ray crystallography and NMR spectroscopy (Dokurno et al 1998;Moller et al 2002), and GalNAc O-glycosylation of the Thr residue in PDTR induces conformational changes in the peptide region, enhancing antibody interaction with the peptide core, but without siginificant interaction with the GalNAc residue. Similarly, binding of the MAb C595 to the RPAP peptide epitope is enhanced by conformational changes induced by Tn-glycosylation of Thr in VTSA as well as Ser and Thr in GSTA regions (Spencer et al 1999). In contrast, NMR studies of the binding of the antibody B27.29 suggest that the antibody epitope maps to two separate parts of the glycopeptide, with the core peptide epitope spanning the PDTRP sequence and a second carbohydrate epitope comprising Tn-glycans at Thr and Ser in the VTSA region (Grinstead et al 2002).…”