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.

Expression of the cancer testis antigen IGF2BP3 in colorectal cancers; IGF2BP3 holds promise as a specific immunotherapy target

“…To further assess a prospective cancer testis protein or any other protein as a vaccine target it is useful to assess preoperative plasma or serum specimens for the presence of antibodies to that protein to evaluate the occurrence of spontaneous immune response [ 23 ]. The finding of auto antibodies in a subset of patients would suggest that this protein can elicit an immune response that may help fighting the tumor.…”

Perspective: cancer vaccines in the era of immune checkpoint blockade

“…Biomarkers might include the presence of effectors (a hot microenvironment), an IFNγ gene signature (Thorsson et al 2018) or the expansion of antigen-specific T cell clones either in the tumour or in peripheral blood (Gros et al 2016). Similarly the presence of antibodies against tumour antigens in peripheral blood clearly identifies evidence of immune engagement with tumour albeit by B cells (Beeton-Kempen et al 2014). For those patients where an immune response has been generated but immunity has failed, the characterisation of regulatory mechanisms can enable therapy to be directed against those mechanisms that are subverting the immune response.…”

Autoantibody-Based Diagnostic Biomarkers: Technological Approaches to Discovery and Validation

“…Miniaturisation allows a high overall sensitivity as analyte measurement is conducted while retaining the highest concentration per unit volume attainable for the given sample, with decreased reaction times due to short diffusion distances [71]. Furthermore, fluorescent-based signal detection in protein microarrays offers lower limits of detection (as low as 1 pg/mL; [72]) and greater dynamic range (up to 5 orders of magnitude; [73]) than colourimetric readouts in typical ELISAs. In addition to their greater sensitivity compared to ELISA, protein arrays are also superior in terms of multiplexing, as thousands of proteins can be printed onto glass slides in replicates and analysed simultaneously.…”