Recombinant antigen expression on yeast surface (RAYS) for the detection of serological immune responses in cancer patients

The serological analysis of antigens by recombinant expression cloning (SEREX) has identified a multitude of new tumor antigens in many different tumor entities. These antigens can be grouped into different classes according to their specificities, with cancer/testis antigens appearing to be the most attractive candidates for vaccine development. The observation that CD8 and CD4 T-cell responses against cancer/testis antigens such as NY-ESO-1 correlate with the presence of specific antibodies demonstrates the importance of serological monitoring patients participating in vaccine trials. However, all serological assays available (Western blot, phage display and ELISA) are hampered by the fact that the protein cannot be analyzed in its natural conformation. We have thus developed a yeast display system where the antigen is expressed on the yeast surface (RAYS), allowing for a more natural folding of the protein. To validate this approach we displayed the A33 colorectal cancer antigen on the yeast cell surface and demonstrated specific binding by an A33 monoclonal antibody recognizing a conformation-dependent epitope on the A33 antigen. We then compared RAYS with the more commonly used ELISA and Western blot serological monitoring methods by analyzing 50 sera from cancer patients with known NY-ESO-1 antibody status and 10 sera from patients with unknown SSX2 antibody status in a blind fashion. RAYS appears at least equivalent to both ELISA and Western blotting for the monitoring of antibodies against NY-ESO-1 as regards specificity and sensitivity, while antibodies against SSX2 were detected more frequently by RAYS than by ELISA or phage display.