title: Development of a novel, quantitative protein microarray platform for the multiplexed serological analysis of autoantibodies to cancer-testis antigens.
abstract: 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.
authors.affiliation: Institute of Infectious Disease and Molecular Medicine/Division of Medical Biochemistry, University of Cape Town, Cape Town, South Africa; Biosciences Division, Council for Scientific and Industrial Research, Pretoria, South Africa.
authors.forename: Natasha
authors.initials: N
authors.lastname: Beeton-Kempen
authors.forename: Jessica
authors.initials: J
authors.lastname: Duarte
authors.forename: Aubrey
authors.initials: A
authors.lastname: Shoko
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Serufuri
authors.forename: Thomas
authors.initials: T
authors.lastname: John
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cebon
authors.forename: Jonathan
authors.initials: J
authors.lastname: Blackburn
mesh_terms: D000912:Antibodies, Neoplasm / Q000097:blood*; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000954:Antigens, Surface / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood*; D019496:Cancer Vaccines / Q000276:immunology / Q000627:therapeutic use; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006801:Humans; D057230:Limit of Detection; D008545:Melanoma / Q000097:blood / Q000276:immunology / Q000628:therapy; D040081:Protein Array Analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
chemical_list: D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D001323:Autoantibodies; C510162:CTAG2 protein, human; D019496:Cancer Vaccines; D011993:Recombinant Fusion Proteins
keywords: antigen microarrays; cancer diagnosis; cancer-testis antigens; predictive markers; tumor immunology