The
cancer-testis antigens are a group of unrelated
proteins aberrantly expressed in various
cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic
vaccines. However, expression levels often vary amongst patients presenting the same
cancer type, and these
antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of
antigen expression may provide correlates of specific
cancer types and
disease progression. Herein, we describe the development of a novel, readily customizable
cancer-testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti-
cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between
autoantibodies and
tumor antigens, we achieved linearity of response and an
autoantibody quantitation limit in the pg/mL range-equating to a million-fold serum dilution. By using oriented attachment of folded, recombinant
antigens and a
polyethylene glycol microarray surface coating, we attained minimal non-specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between
monoclonal antibodies and
tumor antigens for detection, the high sensitivity and specificity realized using our
autoantibody-based approach may facilitate the development of better
cancer biomarkers, as well as potentially enabling pre-symptomatic diagnosis. We illustrated the usage of our platform by monitoring the response of a
melanoma patient cohort to an experimental therapeutic NY-ESO-1-based
cancer vaccine; inter alia, we found evidence of determinant spreading in individual patients, as well as differential CT
antigen expression and
epitope usage.