What Are Neoantigens?

Neoantigens are an important feature of cancer cells and help to stimulate anti-cancer immune responses. In this article, we will define what neoantigens are, explore how they arise, the different types of neoantigens and understand how neoantigen-targeting therapies work.

Neoantigen definition

To understand neoantigens, we first need to define what an antigen is. Antigens are substances that induce the immune system to produce antibodies against that material (literally an antibody generator).¹ Antigens are commonly abbreviated as Ag. Examples of neoantigens include proteins or sugar molecules (polysaccharides) located on the outside of cells.

Neoantigens are produced by tumor or cancer cells. Cancer cells accumulate many DNA mutations that can alter the structure of proteins, and the resulting neoantigens earn their name from the Greek neo, meaning “new”.² Therefore, neoantigens are a class of tumor-specific antigens – they are absent from normal tissue. The resulting neoantigens are displayed by human leukocyte antigens (HLA) on the surface of cancer cells, helping to stimulate immune responses when immune cells – such as T cells – recognize the neoantigens as “non-self”, in a similar way to bacteria and viruses.³

How do neoantigens arise?

The development of neoantigens is caused by non-synonymous mutations in tumor cells, i.e., a mutation that changes the amino acid sequence of a protein.⁴ Mutational events include point mutations (also known as single nucleotide polymorphisms; SNPs) and insertions or deletions (indels).^5,6 Neoantigens can also be produced as a result of viral infection, alternative splicing or gene rearrangements.⁷

The steps of neoantigen development:

1. Mutations alter the sequence of a gene.
2. The gene is transcribed into RNA.
3. RNA is translated into a protein with an altered amino acid sequence.
4. Introduction of a different amino acid alters the structure of the resulting protein, which the immune system recognizes as non-self.
5. The neoantigen is presented by HLA molecules on the cell surface.
6. T cells recognize the presented non-self antigens and initiate an immune response specific to the neoantigen.

Figure 1: Graphic illustrating the differences in antigen presentation in a normal cell (left) compared to neoantigen production and presentation in a mutated cell (right). Credit: Technology Networks.

Types of neoantigens

There are two main types of neoantigens – shared and personalized.⁷

Shared neoantigens are:

• Caused by mutations that are commonly found in many different cancer patients.
• Derived from driver/hotspot mutations.
• More common in some cancer types than others.
• Not present in the normal human genome.
• Highly immunogenic.
• Promising for use as targets for “off-the-shelf” anti-cancer vaccines.

Personalized neoantigens are:

• Unique to the individual.
• Different from patient to patient.
• Arise from somatic mutations in tumor cells.
• Potential targets for personalized therapies.

Identifying neoantigens

Efficient and fast identification of neoantigens has been enabled by the advancement of next-generation sequencing technologies. These technologies have improved the identification of tumor-specific mutations in individual patients that give rise to neoantigens. This is achieved using techniques such as whole-exome or whole-genome sequencing, in which DNA or RNA is compared from paired tumor and non-tumor samples.⁸ Neoantigens are predicted using bioinformatics and computational algorithms.

The identification of neoantigens usually follows this sequence of events:⁹

1. List genomic mutations/alterations identified from next-generation sequencing of paired normal and tumor cells.
2. Convert these into “neopeptides” of the appropriate length.
3. Predict the binding affinity between neopeptides and HLA alleles specific to the patient using computational tools.
4. Assess immunogenicity (the ability to generate an immune response) and validate T-cell responses against cancer cells.

Neoantigen-targeting therapies

Cancer therapies that target neoantigens are promising therapeutic prospects due to their tumor-specific nature, which means they are highly specific and have few off-target effects. Examples of neoantigen-targeting therapies include cancer vaccines and T-cell therapies. Cancer vaccines “train” immune cells within the body, whereas T-cell therapies produce additional patient-derived cells in the laboratory ex vivo then deliver them back to the patient. These kinds of therapies are known as “autologous”, meaning the cells are taken from and administered to the same individual.¹⁰

Neoantigens are associated with tumors with a high mutational load. These tumors typically have an abundance of tumor-infiltrating immune cells and can benefit from immunotherapy such as immune checkpoint inhibitors (ICIs). Such patients usually have a more favorable prognosis. For example, colorectal cancers (CRCs) with DNA mismatch repair deficiency (dMMR) are commonly hypermutated, have better patient outcomes than MMR-proficient tumors and the use of ICIs has been approved by the FDA in some of these cases.^11,12

Cancer vaccines

Personalized neoantigen vaccines are designed to produce a tumor-specific T-cell response against the neoantigen, typically to reduce the risk of “off-target” damage to non-tumor tissues.⁵ Additionally, immunological memory from neoantigen-based vaccines can provide long-term and persistent protection against tumor recurrence. Various forms of neoantigen vaccines are in development, such as peptide, nucleic acid (DNA or RNA) and dendritic cell vaccines.⁶

Antigen-presenting cells, such as dendritic cells, recognize neoantigens administered in the vaccine and present them to T cells, initiating an immune response. Neoantigens can either be delivered as proteins, or as DNA or RNA that code for the neoantigen and is translated into protein by the patients’ cells. Dendritic cell vaccines based on neoantigens involve taking a patient’s dendritic cells, exposing them to neoantigens, then administering them back into the patient where they promote immune responses specific to the neoantigen.

Neoantigen-based therapeutic cancer vaccines have shown promising anti-tumor activity in trials of patients with melanoma, glioblastoma and other cancers.^5,13,14 However, the utility of personalized neoantigen-based vaccines is limited by the considerable time and money required for their development.

T-cell therapy

T-cell therapy involves collecting T cells from the patient, modifying them then administering them back to the patient as a kind of cell therapy, or “living drug”. Technologies include T-cell receptor (TCR) engineering/therapy and neoantigen-targeted T-cell therapy.

In TCR engineering/therapy, T cells are genetically engineered to produce a TCR that recognizes and binds to a specific neoantigen.⁶ Once a desired neoantigen is identified, T cells specific to that neoantigen are isolated and their TCRs sequenced. The patient’s T cells can then be genetically engineered by introducing these TCR sequences specific to the neoantigen. After their activity against the neoantigen is confirmed, they can then be infused back into the patient.

Another example is neoantigen-targeted T-cell therapy (also known as personalized enrichment of antigen-specific T-cells).¹⁵ This involves using cultures of a patient’s tumor-infiltrating lymphocytes (TILs), or naturally occurring neoantigen-reactive T cells.¹⁶ T cells are isolated from the patient's population of infiltrating or circulating lymphocytes. Once the desired neoantigens have been identified and validated, the isolated antigen-specific T cells are enriched by bulk culturing in vitro to reach the quantity required for reinfusion back into the patient.