Neoantigens: From Discovery to Cancer Immunotherapy

This article was originally posted on March 16, 2020.|

Neoantigen, also called tumor-specific antigen (TSA), is a repertoire of peptides that arise as the result of DNA mutations in tumor cells. Unlike the other two common types of tumor antigens, named tumor-associated antigens (TAAs) and cancer-germline antigens (CGAs), tumor neoantigen is the truly foreign protein and entirely absent from normal human organs or tissues. As a result, targeting neoantigens could allow the patient’s immune system to exclusively find and attack cancer cells without harming normal healthy cells. Decades of work has increasingly shown their potential for cancer immunotherapy. 

We will systematically introduce the history of understanding and identification of neoantigens, how to develop personalized neoantigen-based vaccines, how neoantigen vaccines work, their role on current cancer immunotherapies and the key players in the industry.

Chapter 1. What are neoantigens and how to identify them?

Genetic instability of tumor cells often leads to a large number of mutations, and expression of non-synonymous mutations can produce tumor-specific antigens, called neoantigens [1]. Some of the neoantigens can be expressed, processed and presented on the cell surface, and subsequently recognized by T cells in the context of major histocompatibility complexes (MHCs) molecules. Because neoantigens are not expressed in normal tissues, neoantigen-specific T cells are not subject to central and peripheral tolerance, and also lack the ability to induce normal tissue destruction. As a result, neoantigens appear to be the ideal targets for T cell-based cancer immunotherapy [2]. 

How are neoantigens discovered?

In the early twentieth century, many findings revealed that the immune system can recognize and eliminate tumor cells. However, the nature of antigens that could trigger antitumor immune response was unclear during that time. Until 1988 De Plaen and colleagues identified the first neoantigen that can be recognized by T cells in a mouse tumor model by using cDNA library screening [3, 4]. They found that only one nucleotide differed between the normal and tumor gene and this mutation produced an amino acid change. After that, a series of neoantigens derived from somatic mutations were identified in various human tumors including melanoma and renal cell carcinoma [3].

Fig. 1 Historical overview of tumor neoantigens. The figure is originally published by Tao Jiang et al [3] and designed by MolecularCloud.

How to identify neoantigens in tumor cells?

According to articles published during 1995-2013, most of the neoantigens were identified by cDNA library screening. In this approach, cDNA library and MHC molecules were over-expressed in cell lines, and then co-cultured with T cells to identify antigens that could induce the T cell activation [2]. However, this method is labor-intensive, expensive and cannot effectively identify all tumor antigens. The rapid development of next-generation sequencing (NGS) technology makes it possible to rapidly compare the DNA sequences of tumor cells and normal cells and identify tumor-specific mutations. In 2012, NGS technology was firstly reported to be applied in identifying neoantigens in mouse tumor models [5, 6] and soon widely used. Currently, based on the whole exome sequencing (WES) technology and constantly optimized bioinformatics algorithms, a lot of neoantigens have been identified with high efficiency, wide coverage, and low false negative rate [1]. Even though sophisticated machine learning methods are used to winnow down candidate numbers returned by prediction algorithms, scientists have to go back to wet lab experiments to validate that the potential neoantigens are active and could be able to trigger bona fide antitumor responses in patients [7].

Fig. 2 Overview of neoantigen identification using tumor WES [8].

Step 1: Identification of tumor non-synonymous mutations (NSM). WES is performed on tumor and normal DNA to identify tumor-specific NSM. When available, RNA-seq is used to select mutations that are expressed. 

Step 2: Selection of candidate neoantigens. Once NSMs are identified, three strategies can be used to select the list of candidate neoantigens that will be assessed for immunogenicity. 

Step 3: Evaluation of immunogenicity of candidate neoantigens. Finally, the immunogenicity of the selected candidate peptides is evaluated with different immunological screening assays.