Quantification of multiple human immune checkpoint molecules using antibody array technology

Abstract

Abstract Immune checkpoint molecules play an important role in T cell functionality after TCR/MHC signaling. Blockade of two B7/CD28 family checkpoint molecules, CTLA-4 and PD-1 have already demonstrated superior efficacies in treatment for a variety of malignancies. Recently, the US FDA approved one anti-CTLA-4 monoclonal antibody (ipilimumab, 2011) and two anti-PD-1 monoclonal antibodies (pembrolizumab 2014, and nivolumab 2014) for the treatment of advanced melanoma. Other immune checkpoint molecules have been increasingly considered as new targets for cancer immunotherapy. To facilitate better identification of novel targets and elucidation of the checkpoint blockade, simultaneous detection of these pathway molecules in complex biological samples is essential. With antibody array technology, a novel checkpoint molecule array was developed for quantitative measurement 10 of the most important B7/CD28 family checkpoint molecules including B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2/ICOSL, B7-H3, CD28, CTLA-4, ICOS, PD-1, and PD-L2/B7-DC. Antigen-specific capture antibodies were fixed on glass slides with a non-contact arrayer, and multiplex ELISA detection of these proteins was achieved through laser fluorescence detection. All the markers could be detected at pg/ml level. The array was validated by cross reactivity and media recovery tests, and was further validated by conventional ELISA testing of normal human sera. In summary, our system provides a powerful, efficient, and low-cost tool for immunology research and biomarker discovery.