A Tumor-Selective Monoclonal Antibody from Immunization with a Tumor-Associated Mucin Glycopeptide

Li Xia,Elijah F Edmondson,J Sebastian Temme,Kaitlyn Whalen,Jeffrey C Gildersleeve,Joseph J Barchi,Karin Abarca-Heideman,Kevin R Trabbic

doi:10.1038/s41598-019-42076-2

Abstract

We have previously studied the generation of immune responses after vaccination with tumor-associated carbohydrate antigen (TACA)-containing glycopeptides from the tandem repeat (TR) sequence of MUC4, an aberrantly expressed mucin in pancreatic adenocarcinomas. A specific lead antigen from that study containing the Thomsen-Friedenreich TACA disaccharide facilitated the pursuit of a monoclonal antibody to this synthetic hapten. Initial evaluation of polyclonal antiserum resulting from immunization with a KLH conjugate of this glycopeptide into rabbits showed high titer antibodies by ELISA assays, and selective immunoreactivity with MUC4+ cells by western blot and flow cytometry techniques. Glycan microarray analysis showed an intriguing binding pattern where the antiserum showed near complete specificity for MUC4 TR glycopeptides and peptides, relative to all components on the array. Tissue staining also showed distinct tumor specificity to pancreatic tumor tissue in relation to normal pancreatic tissue, with a preference for more aggressive tumor foci. Based on this data, we produced a monoclonal antibody whose binding and reactivity profile was similar to that of the polyclonal serum, with the added benefit of being more specific for the N-terminal glycosylated peptide domain. This epitope represents a novel immunogen to potentially develop diagnostic antibodies or immunotherapies against various MUC4-positive cancers.

Highlights

At present, there are very few biomarkers for the most common form of PC, pancreatic ductal adenocarcinomas (PDAC)
The selection of the 5TFag-TRMUC4 construct stemmed from results of our initial studies showing that vaccination of mice with a tumor-associated glycopeptide, a C3d adjuvant peptide and a doping agent coated on small gold nanoparticles can generate an immune response to the glycopeptide[38]
Immunization with the construct with the TFag in the Ser[5] position produced high titer IgG antibodies, and mice that were implanted with 4T1 breast tumors had longer survival than those vaccinated with other tandem repeat (TR)-glycopeptides where the TFag disaccharide was linked to other positions

Summary

Introduction

There are very few biomarkers for the most common form of PC, pancreatic ductal adenocarcinomas (PDAC). Notwithstanding, there are a variety of new therapeutic options that are in various stages of clinical evaluation Several of these are forms of immunotherapy, either vaccines or antibodies, that target a range of PDAC surface markers. Vaccine preparations such as GVAX4, IMM-1015 and Algenpantucel-L6 have all been evaluated in different stages of clinical trials; none have emerged as viable therapeutic options as most www.nature.com/scientificreports/. Three of the most prevalent of these so-called tumor associated carbohydrate antigens (TACAs) are, (1) the Tn antigen (GalNAcα-O-Ser/Thr), (2) the TF antigen (TFag, Galβ1-3GalNAcα-O-Ser/Thr), and (3) the sialyl-Tn (Neu5Acα2-6-GalNAcα-O-Ser/Thr) antigen[11,12] These shorter glycan chains serve to expose underlying peptide sequences that were masked by the larger “normal” oligosaccharides, and these newly presented glycopeptide epitopes are the subject of immune surveillance ( the term “antigen”). We show through various assays (Western blots, ELISA, Flow Cytometry, tissue staining and glycan microarray analysis) that this mAb is tumor selective and has preferential binding to a glycosylated peptide structure relative to the peptide sequence alone

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