Immunogenic regions of the GA733–2 tumour-associated antigen recognised by autoantibodies of patients with colorectal carcinoma

Abstract.

The tumour-associated antigen (TAA) GA733–2 is overexpressed by >90% of human colorectal carcinomas (CRC). The antigen has previously been shown to be recognised by B and T cells. The aim of the present study was to define B cell epitopes of GA733–2. Fifteen percent of CRC patients with no previous immunotherapy have recently been shown to elicit an anti-GA733–2 IgG antibody response. Sera of these patients (n=136) were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies against 23 partly overlapping synthetic peptides (18 amino acids: aa) derived from the extracellular domain of GA733–2. An 18-aa long sequence at the N-terminal region of the antigen (peptide 2) was found to be an immunodominant B cell epitope. Fifty percent of the patients had antibodies against peptide 2, while 8% to 9% had antibodies against peptides 1, 4, 7, 8 or 20. In healthy donors (n=30) antibodies against peptides 2 and 8 were also detected in 13% and 3% of cases respectively, while no antibodies were found against the other peptides and the complete protein. Thirteen percent of CRC patients (n=30) with no IgG antibodies against the GA733–2 antigen elicited antibodies against peptide 2. The specificity of peptide-reactive sera was verified by inhibition ELISA. The binding of sera to GA733–2 was significantly inhibited by peptides to which CRC sera bound, but not by control peptides. Binding to peptide 2 of sera showing both peptide 2 and GA733–2 reactivity was specifically inhibited by the complete GA733–2 antigen, while binding of peptide 2-reactive sera showing no GA733–2 reactivity was not inhibited. CRC sera interfered with the binding of monoclonal antibody (mAb) 17–1A and mAb C215 that recognise distinct epitopes of GA733–2. No significant correlation was found between the presence of anti-peptide antibodies in CRC patients and clinical stage or overall survival. The results provide additional evidence for immune recognition of CRC by the host.