An Essential Epitope of Anti-MUC1 Monoclonal Antibody KL-6 Revealed by Focused Glycopeptide Library

Abstract

Human serum Krebs von den Lungen-6 (KL-6) antigen, a high-molecular-weight glycoprotein classified as a polymorphic epithelial mucin (MUC1), is a biomarker of diseases such as interstitial pneumonia, lung adenocarcinoma, breast cancer, colorectal adenocarcinoma, and hepatocellular carcinoma. Anti-KL-6 monoclonal antibody (anti-KL-6 MAb) is therefore a potential diagnostic and therapeutic reagent. Although glycosylation at Thr/Ser residues of the tandem-repeating MUC1 peptides appears to determine the disease-associated antigenic structures of KL-6, an essential epitope structure recognized by anti-KL-6 MAb remains unclear. In the present study, a novel compound library of synthetic MUC1 glycopeptides allowed the first rapid and precise evaluation of the specific epitope structure of anti-KL-6 MAb by combined use of a tailored glycopeptides library and common ELISA protocol. We demonstrated that the minimal antigenic structure, an essential epitope, recognized by anti-KL-6 MAb is a heptapeptide sequence Pro-Asp-Thr-Arg-Pro-Ala-Pro (PDTRPAP), in which the Thr residue is modified by Neu5Ac alpha2,3Gal beta1,3GalNAc alpha (2,3-sialyl T antigen, core 1-type O-glycan). Anti-KL-6 MAb did not bind with other tumor-relevant antigens, such as GalNAc alpha (Tn), Neu5Ac alpha2,6GalNAc alpha (STn), and Gal beta1,3GalNAc alpha (T), except for Neu5Ac alpha2,3Gal beta1,3(Neu5Ac alpha2,6)GalNAc alpha (2,3/2,6-disialyl T). However, anti-KL-6 MAb could not differentiate the above minimal antigenic glycopeptide from some core 2-based glycopeptides involving this crucial epitope structure and showed a similar binding affinity toward these compounds, indicating that branching at the O-6 position of GalNAc residue does not influence the interaction of anti-KL-6 MAb with some MUC1 glycoproteins involving an essential epitope. Actually, anti-KL-6 MAb reacts with 2,3/2,6-disialyl T having a 2,3-sialyl T component. This is why anti-KL-6 MAb often reacts with various kinds of tumor-derived MUC1 glycoproteins as well as a clinically important MUC1 glycoprotein biomarker of interstitial pneumonia, namely KL-6, originally discovered as a circulating pulmonary adenocarcinoma-associated antigen. In other words, combined use of anti-KL-6 MAb and some probes that can differentiate the sugars substituted at the O-6 position of the GalNAc residue in MUC1 glycopeptides including the PDTRPAP sequence might be a promising diagnostic protocol for individual disease-specific biomarkers. It was also revealed that glycosylation at neighboring Thr/Ser residues outside the immunodominant PDTRPAP motif strongly influences the interaction between anti-KL-6 MAb and MUC1 glycopeptides involving the identified epitope. Our novel strategy will greatly facilitate the processes for the identification of the tumor-specific and strong epitopes of various known anti-MUC1 MAbs and allow for their practical application in the generation of improved antibody immunotherapeutics, diagnostics, and MUC1-based cancer vaccines.

Levels of Surfactant Proteins A and D and KL-6 Are Elevated in the Induced Sputum of Chronic Obstructive Pulmonary Disease Patients: A Sequential Sputum Analysis

“…KL-6 is a high-molecular-weight (>200 kDa) mucin-like glycoprotein which has been classified as human MUC1 mucin [34,35]. Serum/plasma SP-A, SP-D and KL-6 have been reported to represent sensitive markers for lung diseases, to the extent that in Japan they are used in clinical practice during the identification of idiopathic pulmonary fibrosis and other types of interstitial lung diseases.…”

A High-Throughput O-Glycopeptide Discovery Platform for Seromic Profiling

“…Chemical and chemoenzymatic methods for synthesis of O-glycopeptides are available and smaller libraries of Oglycopeptides have been synthesized. [19][20][21][22] Methods for display on microarrays have also been developed, 21,23,24 but a method for robust high through-put synthesis and display of Oglycopeptide libraries on microarray has not.…”

Characterization of the Viral O -Glycopeptidome: a Novel Tool of Relevance for Vaccine Design and Serodiagnosis

“…While this approach was possible for shorter neutral O-glycan structures, such as GalNAc and Gal␤1-3GalNAc, chemical synthesis becomes costly with increased complexity and number of O-glycans and length of the peptide sequence. Although certain glycans, including sialylated structures such as STn (Neu5Ac␣2-6GalNAc) and ST (Neu5Ac␣2-3Gal␤1-3GalNAc), are extremely difficult to incorporate by Fmoc synthesis, several elegant examples exist for a limited number of peptide libraries (10,31).…”