Cancer Antigen Discovery Is Enabled by RNA Sequencing of Highly Purified Malignant and Nonmalignant Cells

Abstract

Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit anti-tumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms. Experimental design: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison to patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by immunohistochemistry, and pre-existing T cell immunogenicity towards these antigens tested. Results: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancertestis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines in vitro, and the IFN- + response was markedly magnified by control of immunosuppression with cyclophosphamide in cancer patients. Conclusion: This study highlights how prior methods that sequence whole tumor fractions (i.e. inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.

Cancer stem cell-targeted chimeric antigen receptor (CAR)-T cell therapy: Challenges and prospects

“…The approaches employed to identify such antigens have evolved over several years. Overall, earlier cell-based and biochemical-based (low throughput) methods such as serological analysis of recombinant cDNA expression libraries (SEREX), phage display, protein arrays and surface receptor screening have now led to higher throughput methods such as gene expression profiling by microarray and RNA-seq 23 , 24 , 25 , 26 . These approaches might be very helpful for identification of various intracellular and extracellular (cell surface) antigens which will be targeted by different immune-based therapies.…”