Dataset Information

Isolation of a novel GPA33 monoclonal antibody that detects a redox regulated epitope

ABSTRACT: Membrane receptor targets have been identified using proteogenomics platforms in oesophageal adenocarcinoma (OAC) that are specific for tumour compared to normal adjacent oesophageal and gastric tissue. One of these receptors, GPA33, is expressed in patients including involved lymph nodes suggesting it might form a clinically relevant diagnostic and/or therapeutic target. We have used a massively diverse scFv-phage display library to identify recombinant scFv sequences enriched during biopanning against native GPA33 using both next-generation CDR3 DNA sequencing and functional ELISA. One active scFV, redox-sensitive epitope 05 (RSE-05), was converted using gene synthesis into a single cistronic full-length IgG expression plasmid containing a self-cleavage site in between the heavy and light chain. The recombinant RSE-05 MAB can bind to the outer surface of living, non-permeabilized adherent MCF7 cells transfected with the full-length GPA33 gene as defined by immunofluorescence or flow cytometry. Unexpectedly, the inclusion of DTT in a pre-incubation stage abrogated RSE-05 MAB binding using immunoblotting, suggesting that disulphide bridges form the native epitope. GPA33 is composed of two IgG-like lobes, containing three disulphide bonds. Mutation of the disulphide bridge at C22S or C96S in Lobe 1 abolishes RSE-05 MAB binding in a native immuno dotablot suggesting this lobe forms the primary epitope. Peptide-phage display of the RSE-05 MAB identified a linear epitope within Lobe 1. Cross-linking mass spectrometry (XL-MS) using DSS identified a motif in FR2/CDR2 of the IgG that forms a covalent bond with S27, which is consistent with the core epitope motif (27-STS-29) identified by peptide-phage display. XL-MS also identifed a covalent bond between Lobe 1 and Lobe 2 and suggesting that the GPA33 protein can exist in two confromational states. Accordingly, mutation of Cysteine residues in Lobe 2 abolishes the binding of RSE-05 MAB in a denaturing immunoblot, suggesting that disruption of the conformational integrity of Lobe 2 can imapct on the presumed primary epitope formed by the C22-C96 disulphide. Immunohistochemistry of an oesophageal cancer tumour microarray indicated that higher tumor grades, lymph node involvement, and venous invasion are associated with higher CD3, CD8, and GPA33 scores, indicating a potential link between immune response and tumor aggressiveness. Together, these data identify a novel monoclonal antibody that contacts a redox regulatable epitope in GPA33 protein and that can be used to study allosteric interactions in the GPA33 protein. This biologic might also form a novel diagnostic and therapeutic biologic for use in metastatic upper GI cancers.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast

DISEASE(S): Esophageal Cancer

SUBMITTER: Lenka Hernychova

LAB HEAD: prof. Ted Hupp

PROVIDER: PXD060098 | Pride | 2026-04-27

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	1165_24SP010_E_70min_2-25_trap_1ul_30ng_1_1-29-2024_19-47-42.d.zip	Other
	1167_24SP010_D_70min_2-25_trap_1ul_30ng_1_1-29-2024_21-52-02.d.zip	Other
	checksum.txt	Txt
	plots.xls	Xls

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Publications

GPA33 forms a distinct diagnostic target class to Claudin 18.2 in oesophageal adenocarcinoma enabling the development of a novel GPA33 antibody-based detection platform.

Brydon Jack J Krejcir Radovan R Zavadil-Kokas Filip F Singh Ashita A Henek Tomas T Hernychova Lenka L Coleman Skye S Al Shboul Sofian S Hrabal Vaclav V Kuncova Zuzana Z Mayordomo Marcos Yébenes MY Arcimowicz Łukasz Ł Ball Kathryn L KL Padariya Monikaben M Kalathiya Umesh U Vojtesek Borivoj B Hupp Ted T O'Neill J R JR

Cellular & molecular biology letters 20260310 1

<h4>Background</h4>Oesophageal adenocarcinoma (OAC) is a cancer of high unmet clinical need. Because of tumour heterogeneity, it is likely that OAC will be stratified into several subtypes. Claudin 18.2 antibodies form emerging novel therapeutics in patients with a subtype of OAC. A large-scale proteogenomics screen in OAC identified glycoprotein A33 (GPA33) protein as a dominating cancer-specific target. We set out to determine whether GPA33 is distinct from or overlaps with Claudin 18.2 as a t ...[more]

PMID: 41808009

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