Identification of neoantigens for individualized therapeutic cancer vaccines

Abstract

Somatic mutations in cancer cells can generate tumour-specific neoepitopes, which are recognized by autologous T cells in the host. As neoepitopes are not subject to central immune tolerance and are not expressed in healthy tissues, they are attractive targets for therapeutic cancer vaccines. Because the vast majority of cancer mutations are unique to the individual patient, harnessing the full potential of this rich source of targets requires individualized treatment approaches. Many computational algorithms and machine-learning tools have been developed to identify mutations in sequence data, to prioritize those that are more likely to be recognized by T cells and to design tailored vaccines for every patient. In this Review, we fill the gaps between the understanding of basic mechanisms of T cell recognition of neoantigens and the computational approaches for discovery of somatic mutations and neoantigen prediction for cancer immunotherapy. We present a new classification of neoantigens, distinguishing between guarding, restrained and ignored neoantigens, based on how they confer proficient antitumour immunity in a given clinical context. Such context-based differentiation will contribute to a framework that connects neoantigen biology to the clinical setting and medical peculiarities of cancer, and will enable future neoantigen-based therapies to provide greater clinical benefit.

Figure Box 1. Mutation classes and neoantigen and neoepitopes derived thereof.

SNVs change a single amino acid. INDELs and fusion genes may be in-frame and preserve the original open reading frame or they may cause a frameshift, creating novel open reading frames downstream of the mutation site. Alternative splicing may occur by various mechanisms including the usage of alternative splice sites, skipping of exons or intron retention events. All of these classes may generate neoepitope sequences that are foreign to the immune system. Novel sequence regions derived from mutations are indicated in red.

Figure 1. Engineering individualised neoantigen vaccines.

Next-generation sequencing of a patient’s healthy tissue (e.g., PBMC, peripheral blood mononuclear cells) and tumour biopsies is performed. The sequencing data from tumour and normal DNA is compared to identify tumour-specific mutations. Mutations are prioritised as vaccine candidates based on their likelihood to elicit a T-cell response by computational methods such as MHC binding prediction, quantification of mutated transcript expression, clonality of the mutation and other features. Using the vaccine platform of choice (e.g. mRNA, long peptides) an individualised and poly-specific neoantigen vaccine is manufactured on-demand under GMP conditions. Neoantigen vaccination aims at restoring the cancer immunity cycle by inducing de novo T-cell responses that induce tumour killing and by supporting the shift from ignorance toward anti-tumour immunity. LOH: loss of heterozygosity, GMP: good manufacturing practice

Figure 2. Mechanisms of neo-antigen-mediated tumour control.

Dying tumour cells release neoantigens that reach the draining lymph node either in a soluble form within the extracellular fluid or are transported from the tumour site by migratory antigen presenting cells (APCs). In the lymph node, highly specialised dendritic cells present the neoantigen on MHC-I or MHC-II molecules to naïve T cells for priming and activation. Activated neoantigen-specific CD4⁺ and CD8⁺ T cells leave the lymph node, enter the tumour and exert anti-tumour activities. APCs in the tumour microenvironment can activate antigen specific memory CD4⁺ and CD8⁺

Figure 3. TCR diversity and degeneracy

A neoepitope (blue, mutant amino acid red) can be recognised by different T cells with molecularly different TCR alpha and beta chains (left, middle). The TCR/neoepitope contact residues (dark blue) may differ for individual T cells that recognise the same neoepitope. This is in particular the case for neoantigens resulting from a mutation that converts a non-binding wild type peptide into a binding mutant peptide. Also, a single TCR can recognise unrelated MHC peptide epitopes (right). For example, a T cell primed against a pathogen-derived epitope (purple) may cross-recognise a neoepitope presented on a tumour cell. MHC: major histocompatibility complex; TCR: T-cell receptor, B2M: Beta-2-Microglobulin.

Figure 4. Factors affecting neoantigen recognition and evolution.

Each lesion (primary or metastasis) of an individual tumour disease consists of different subclones, each of which may contribute sets of different neoantigens to the patient’s neoantigenome. Depending on whether the neoantigen is truncal clonal (neoantigen A), truncal clonal but lost in a metastasis by deletion or gene silencing (neoantigen B), clonal in a certain metastasis (neoantigen C) or specific for a certain subclone in a single metastasis (neoantigen D), neoepitope-specific T cells would target either all tumour cells (neoantigen A), all tumour cells of the lesions harbouring the neoantigen (neoantigen B), tumour cells of a distinct lesion (neoantigen C) or merely a single tumour subclone (neoantigen D).

Figure 5. A context based classification of neoantigens

(a) The formation and evolution of neoantigen-specific T-cell responses depends on the clinical context. While ICB therapy boosts pre-existing T-cell responses, neoantigen cancer vaccines induce de novo responses or amplify preformed ones. (b) The robustness and level of a neoantigen presentation on LN-resident DCs defines the efficiency of cognate priming of T cells, while presentation on tumour-resident APCs and tumour cells activates primed cells at the tumour site. Neoantigen presentation is a function of expression level of the mutated protein, the binding ability of the mutated peptide to MHC and stability of the respective peptide/MHC complex. Memory T cell activation can be achieved with neoantigen presentation levels 50 fold lower than those required for priming of naive T cells. Dark red: supreme neoantigen; pink cross-reactive guarding neoantigen (c) Neoantigen-specific T cell responses are driven by the presentation of neoepitopes on tumour cells, on tumour-infiltrating APCs, and on DCs in the draining lymph node. Priming of naïve T cells in the lymph node requires substantially higher neoantigen presentation than is required for stimulation of memory T cells. Guarding neoantigens are either highly expressed with superior binding and stability of the respective neoepitope/MHC complex (red) or exploit cross-reactivity to heterologously primed memory T cells (purple). Restrained neoantigens exhibit robust expression and strong MHC binding affinity/stability and are able to prime and expand naive neoantigen-specific T cells in the lymph node. Ignored neoantigens require a vaccine to generate neoantigen presentation levels in the LN that allow priming. As long as neoantigen presentation is moderate (e.g., low expression/high affinity MHC binding (dark gray) or high expression/low MHC binding (light gray), T cells can be activated for effector functions in the tumour. NP: level of neoepitope presentation, LN: lymph node, APC: antigen-presenting cell, ICB: immune checkpoint blockade

Figure 5. A context based classification of neoantigens

(a) The formation and evolution of neoantigen-specific T-cell responses depends on the clinical context. While ICB therapy boosts pre-existing T-cell responses, neoantigen cancer vaccines induce de novo responses or amplify preformed ones. (b) The robustness and level of a neoantigen presentation on LN-resident DCs defines the efficiency of cognate priming of T cells, while presentation on tumour-resident APCs and tumour cells activates primed cells at the tumour site. Neoantigen presentation is a function of expression level of the mutated protein, the binding ability of the mutated peptide to MHC and stability of the respective peptide/MHC complex. Memory T cell activation can be achieved with neoantigen presentation levels 50 fold lower than those required for priming of naive T cells. Dark red: supreme neoantigen; pink cross-reactive guarding neoantigen (c) Neoantigen-specific T cell responses are driven by the presentation of neoepitopes on tumour cells, on tumour-infiltrating APCs, and on DCs in the draining lymph node. Priming of naïve T cells in the lymph node requires substantially higher neoantigen presentation than is required for stimulation of memory T cells. Guarding neoantigens are either highly expressed with superior binding and stability of the respective neoepitope/MHC complex (red) or exploit cross-reactivity to heterologously primed memory T cells (purple). Restrained neoantigens exhibit robust expression and strong MHC binding affinity/stability and are able to prime and expand naive neoantigen-specific T cells in the lymph node. Ignored neoantigens require a vaccine to generate neoantigen presentation levels in the LN that allow priming. As long as neoantigen presentation is moderate (e.g., low expression/high affinity MHC binding (dark gray) or high expression/low MHC binding (light gray), T cells can be activated for effector functions in the tumour. NP: level of neoepitope presentation, LN: lymph node, APC: antigen-presenting cell, ICB: immune checkpoint blockade

Figure 5. A context based classification of neoantigens

(a) The formation and evolution of neoantigen-specific T-cell responses depends on the clinical context. While ICB therapy boosts pre-existing T-cell responses, neoantigen cancer vaccines induce de novo responses or amplify preformed ones. (b) The robustness and level of a neoantigen presentation on LN-resident DCs defines the efficiency of cognate priming of T cells, while presentation on tumour-resident APCs and tumour cells activates primed cells at the tumour site. Neoantigen presentation is a function of expression level of the mutated protein, the binding ability of the mutated peptide to MHC and stability of the respective peptide/MHC complex. Memory T cell activation can be achieved with neoantigen presentation levels 50 fold lower than those required for priming of naive T cells. Dark red: supreme neoantigen; pink cross-reactive guarding neoantigen (c) Neoantigen-specific T cell responses are driven by the presentation of neoepitopes on tumour cells, on tumour-infiltrating APCs, and on DCs in the draining lymph node. Priming of naïve T cells in the lymph node requires substantially higher neoantigen presentation than is required for stimulation of memory T cells. Guarding neoantigens are either highly expressed with superior binding and stability of the respective neoepitope/MHC complex (red) or exploit cross-reactivity to heterologously primed memory T cells (purple). Restrained neoantigens exhibit robust expression and strong MHC binding affinity/stability and are able to prime and expand naive neoantigen-specific T cells in the lymph node. Ignored neoantigens require a vaccine to generate neoantigen presentation levels in the LN that allow priming. As long as neoantigen presentation is moderate (e.g., low expression/high affinity MHC binding (dark gray) or high expression/low MHC binding (light gray), T cells can be activated for effector functions in the tumour. NP: level of neoepitope presentation, LN: lymph node, APC: antigen-presenting cell, ICB: immune checkpoint blockade

Figure 6. Discovery of tumour rejection antigens.

As the term “tumour rejection“ and the conditions under which to assess it are not standardised, experimental mouse model setups are used that differ conceptually and provide answers to different questions. These include tumour challenge of naive mice^, as well as of tumour- or vaccine-experienced mice^, and assessment of rejection spontaneously^, or upon various modalities of immunotherapy^,,,.