Exploiting Tumor Neoantigens to Target Cancer Evolution: Current Challenges and Promising Therapeutic Approaches

Abstract

Immunotherapeutic manipulation of the antitumor immune response offers an attractive strategy to target genomic instability in cancer. A subset of tumor-specific somatic mutations can be translated into immunogenic and HLA-bound epitopes called neoantigens, which can induce the activation of helper and cytotoxic T lymphocytes. However, cancer immunoediting and immunosuppressive mechanisms often allow tumors to evade immune recognition. Recent evidence also suggests that the tumor neoantigen landscape extends beyond epitopes originating from nonsynonymous single-nucleotide variants in the coding exome. Here we review emerging approaches for identifying, prioritizing, and immunologically targeting personalized neoantigens using polyvalent cancer vaccines and T-cell receptor gene therapy. SIGNIFICANCE: Several major challenges currently impede the clinical efficacy of neoantigen-directed immunotherapy, such as the relative infrequency of immunogenic neoantigens, suboptimal potency and priming of de novo tumor-specific T cells, and tumor cell-intrinsic and -extrinsic mechanisms of immune evasion. A deeper understanding of these biological barriers could help facilitate the development of effective and durable immunotherapy for any type of cancer, including immunologically "cold" tumors that are otherwise therapeutically resistant.

Figure 1.. The human Major Histocompatibility Complex (MHC) locus on Chromosome 6 contains several polymorphic antigen-presenting genes encoding Human Leukocyte Antigen (HLA) class I and class II molecules.

A defining hallmark of HLA molecules is the presence of thousands of allelic variants within the human population encoding for multiple antigen presenting molecules within an individual. As a consequence of polymorphism, humans are generally heterozygous at HLA loci, and loss of HLA is an important mechanism of immune evasion in cancer. Since HLA genes are patient-specific, HLA haplotyping is an essential step in facilitating neoantigen prediction, and can be performed using whole genome, exome, or transcriptome sequencing of peripheral blood, skin, or tumor samples. HLA class I and class II allelic data as of October 2020 from the Immuno Polymorphism Database of the European Molecular Biology Laboratory (http://www.ebi.ac.uk/imgt/hla/).

Figure 2.. Structure of antigen-presenting molecules encoded by MHC class I and class II loci.

MHC proteins are heterotrimeric molecules that consist of alpha and beta polypeptide chains complexed with peptide. Beta-2 microglobulin (β2m) is unique to MHC class I (MHC-I) molecules, and mutations in this gene have been implicated as a mechanism of MHC-I loss and immune evasion in cancer. The closed-ended peptide-binding groove of MHC-I molecules typically accommodates diverse peptides 8 to 12 amino acids in length that bind via anchor residues at the peptide N- and C-termini for presentation to CD8+ cytotoxic T lymphocytes. MHC class II (MHC-II) molecules, by contrast, do not have closed-ended grooves and can bind longer peptides up to 13 to 28 amino acids in length for presentation to CD4+ helper T lymphocytes. Unlike MHC-1I molecules, MHC-II molecules do not use dominant binding anchor residues, making the accurate prediction of MHC-II binding affinity and stability more challenging. aa, amino acids.

Figure 3.. The landscape of immunogenic neoantigens recognized by cytotoxic T lymphocytes.

Recent studies have extended the tumor-associated neoantigen immunopeptidome far beyond epitopes arising from nonsynonymous single nucleotide variants (SNVs). These alterations include insertions/deletions (indels), gene fusions, endogenous retroviral elements (EREs), complex structural variants, alternative splicing, intron-derived peptides, defective ribosomal products, and post-translationally modified proteins. Experimental techniques such as RNA sequencing and ribosome profiling (Ribo-Seq) can be applied to capture non-canonically translated sequences that are otherwise missed by DNA exome sequencing approaches, and serve to vastly expand the breadth of potentially targetable personalized neoantigens. NeoAg, neoantigen; TCR, T-cell receptor.

Figure 4.. Identification of personalized neoantigen targets in cancer patients.

Neoantigen discovery pipelines typically begin with whole DNA exome and RNA transcriptome sequencing, which can be used to construct a customized database for mass spectrometry (MS)-based proteomic analysis of MHC-peptide complexes eluted from tumor samples. Tumor mutational analysis combined with HLA typing facilitates prediction of allele-specific MHC-I and MHC-II binders using in silico algorithms trained on binding affinity or MHC MS eluted ligand datasets. Among the vast number of exome sequence variants, only about 1 to 3% are ultimately validated as immunogenic by T-cell functional assays. Recent advances in high-dimensional proteomic techniques such as mass cytometry (e.g. cytometry by time of flight) offer a high-throughput approach for screening antigen-specific T-cells. Annotation of larger immunopeptidome databases with both canonical and non-canonical peptide candidates will be important for improving the accuracy of MS-based neoantigen discovery. IP, immunoprecipitation; PDX, patient-derived xenograft.

Figure 5.. Multiple mechanisms of immunoediting can lead to the loss of tumor cell recognition by antigen-specific T-cells.

Seminal studies established the conceptual framework of cancer immunoediting wherein tumors evade immune recognition through selection for clones with decreased immunogenicity (20). Immunoediting may act upon several substrates, including tumor neoantigens, MHC molecules, and the T-cell receptor (TCR) repertoire. Clonal selection during tumorigenesis favors an oligoclonal TCR repertoire, loss of heterozygosity at the HLA locus, discrete oncogenic signaling pathways involved in immune evasion, and loss of immunogenic neoantigens. Several other tumor-extrinsic factors further define the immunogenicity of tumors and determine the cancer-immune set point, including immune suppression in the tumor microenvironment, impaired T-cell priming, and stromal barriers that block T-cell trafficking. These processes represent key barriers that must be overcome by immunotherapy to effectuate durable antitumor immunity. TGN, Trans-Golgi Network; TAP, transporter associated with antigen processing; ER, endoplasmic reticulum; β2m, beta-2 microglobulin; LOH, loss of heterozygosity.

Figure 6.. Identification of tumor-associated neoantigens can lead directly to multiple immunotherapeutic interventions to target cancer cells.

Cancer immunotherapy can be broadly categorized by treatments that enhance T-cell priming and clonal expansion, increase tumor-specific T-cell trafficking, and overcome barriers of immune suppression in the tumor microenvironment. Preclinical and clinical studies have shown that polyvalent peptide vaccines induce T-cell priming in lymph nodes and robust T-cell activation in the peripheral blood, particularly when combined with an immune adjuvant such as poly-ICLC or imiquimod. Isolation and expansion of low-frequency neoantigen-specific endogenous T-cells (ETCs) from the peripheral blood can be applied to promote antitumor responses. ETCs may also be used to validate putative neoantigens for vaccines. While technically cumbersome in most epithelial cancers, this approach provides the advantage of rapidly implementing neoantigen-specific adoptive T-cell therapy using only clinical grade peptides and patient-derived peripheral blood. Alternatively, tumor-infiltrating lymphocytes (TILs) offer an enriched population of tumor-specific T-cells but it remains unclear whether these cells retain all neoantigen specificities after culturing and expansion ex vivo. This has recently inspired efforts to isolate and clone neoantigen-specific TCRs, which can be transduced into ETCs or allogeneic T-cells for adoptive cell therapy. Future clinical trials are needed to identify the optimal formulations, delivery platforms, and combinations of neoantigen-directed immunotherapy for different cancer types. DC, dendritic cell; TLR, Toll-like receptor.