Tumor Antigens beyond the Human Exome.

With the advent of immunotherapeutics, a new era in the combat against cancer has begun. Particularly promising are neo-epitope-targeted therapies as the expression of neo-antigens is tumor-specific. In turn, this allows the selective targeting and killing of cancer cells whilst healthy cells remain largely unaffected. So far, many advances have been made in the development of treatment options which are tailored to the individual neo-epitope repertoire. The next big step is the achievement of efficacious "off-the-shelf" immunotherapies. For this, shared neo-epitopes propose an optimal target. Given the tremendous potential, a thorough understanding of the underlying mechanisms which lead to the formation of neo-antigens is of fundamental importance. Here, we review the various processes which result in the formation of neo-epitopes. Broadly, the origin of neo-epitopes can be categorized into three groups: canonical, noncanonical, and viral neo-epitopes. For the canonical neo-antigens that arise in direct consequence of somatic mutations, we summarize past and recent findings. Beyond that, our main focus is put on the discussion of noncanonical and viral neo-epitopes as we believe that targeting those provides an encouraging perspective to shape the future of cancer immunotherapeutics.

Melanoma antigens recognized by T cells and their use for immunotherapy.

Melanoma has been a prototype for cancer immunology research, and the mechanisms of anti-tumor T-cell responses have been extensively investigated in patients treated with various immunotherapies. Individual differences in cancer-immune status are defined mainly by cancer cell characteristics such as DNA mutations generating immunogenic neo-antigens, and oncogene activation causing immunosuppression, but also by patients' genetic backgrounds such as HLA types and genetic polymorphisms of immune related molecules, and environmental and lifestyle factors such as UV rays, smoking, gut microbiota and concomitant medications; these factors have an influence on the efficacy of immunotherapy. Recent comparative studies on responders and non-responders in immune-checkpoint inhibitor therapy using various new technologies including multi-omics analyses on genomic DNA, mRNA, metabolites and microbiota and single cell analyses of various immune cells have led to the advance of human tumor immunology and the development of new immunotherapy. Based on the new findings from these investigations, personalized cancer immunotherapies along with appropriate biomarkers and therapeutic targets are being developed for patients with melanoma. Here, we will discuss one of the essential subjects in tumor immunology: identification of immunogenic tumor antigens and their effective use in various immunotherapies including cancer vaccines and adoptive T-cell therapy.

Can Immune Therapy Cure Acute Myeloid Leukemia?

OPINION STATEMENT: Although safe and effective immune therapies have been developed in several cancers, this has not been so in acute myeloid leukaemia (AML). Studies of antibodies to CD33, CD123 and CLL-1 report with unconvincing efficacy and substantial adverse events. Lacking AML-specific target antigens, these approaches using non-specific antigen targets often cause unacceptable bone marrow toxicity and off-target adverse events. Studies of AML incidence in persons with immune deficiency indicate little if any immune surveillance against AML. In contrast, data studies of recipients of haematopoietic cell transplants support an effective allogeneic anti-AML effect associated with graft-versus-host disease (GvHD) and possibly a specific graft-versus-leukaemia (GvL) effect. A special problem in the immune therapy of AML is few neo-antigens compared with solid cancers because of a relatively low mutation frequency. Studies of CAR-T-, CAR-NK-adaptor CAR-T- and allogeneic NK-cells are progressing as are approaches using synthetic biology. Presently, there are no convincing data of efficacy of immune therapy in AML.

Hydrogen peroxide-induced oxidative stress and its impact on innate immune responses in lung carcinoma A549 cells.

The immune responses, involved in recognition of cancer-specific antigens, are of particular interest as this may provide major leads towards developing new vaccines and antibody therapies against cancer. An effective treatment for cancer is still a challenge because there are many mechanisms through which the tumor cells can escape the host immune surveillance. Oxidative stress or respiratory burst which is host's mechanism to kill the foreign particles is used as defense mechanism by the tumor cells. The tumor cells uses this oxidative stress to form neo-antigens which in turn makes them undetectable and can escape the host immune surveillance. The human lung carcinoma (A549) cells were treated using 100 µM H2O2 to induce oxidative stress, and the extent oxidative modifications were detected at the level of membrane and proteins in form of lipid peroxidation and protein carbonyls respectively. Nitric oxide and iNOS levels were estimated by Griess assay and immunostaining, respectively. The oxidized tumor proteins were visualized on one-dimensional SDS-PAGE. The H2O2-treated (15 min and 24 h post-treatment) A549 cells were co-cultured with THP-1 cells to subsequently visualize the phagocytic activity by Giemsa and CFSE staining to understand the role of neo (oxidized) tumor antigens in eliciting alteration in immune responses. A significant decline in the percent engulfed cells and decrease in the levels of reactive oxygen species was observed. Immunohistostaining for p47phox, which is an important indicator of the oxygen-dependent phagocytosis, showed a decrease in its levels when cells were treated for only 15 min with 100 µM H2O2, whereas at 24-h post-treatment there was no change in the p47phox levels. The study has established oxidative stress as a new pathogenic mechanism of carcinogenesis and will open new avenues for clinical intervention, adjunct therapies for cancer, and its control at the initial stage by targeting these neo-antigens.

Personalized approaches to active immunotherapy in cancer.

Immunotherapy is emerging as a promising anti-cancer curative modality. However, in contrast to recent advances obtained employing checkpoint blockade agents and T cell therapies, clinical efficacy of therapeutic cancer vaccines is still limited. Most vaccination attempts in the clinic represent "off-the shelf" approaches since they target common "self" tumor antigens, shared among different patients. In contrast, personalized approaches of vaccination are tailor-made for each patient and in spite being laborious, hold great potential. Recent technical advancement enabled the first steps in the clinic of personalized vaccines that target patient-specific mutated neo-antigens. Such vaccines could induce enhanced tumor-specific immune response since neo-antigens are mutation-derived antigens that can be recognized by high affinity T cells, not limited by central tolerance. Alternatively, the use of personalized vaccines based on whole autologous tumor cells, overcome the need for the identification of specific tumor antigens. Whole autologous tumor cells could be administered alone, pulsed on dendritic cells as lysate, DNA, RNA or delivered to dendritic cells in-vivo through encapsulation in nanoparticle vehicles. Such vaccines may provide a source for the full repertoire of the patient-specific tumor antigens, including its private neo-antigens. Furthermore, combining next-generation personalized vaccination with other immunotherapy modalities might be the key for achieving significant therapeutic outcome.

MuPeXI: prediction of neo-epitopes from tumor sequencing data.

Personalization of immunotherapies such as cancer vaccines and adoptive T cell therapy depends on identification of patient-specific neo-epitopes that can be specifically targeted. MuPeXI, the mutant peptide extractor and informer, is a program to identify tumor-specific peptides and assess their potential to be neo-epitopes. The program input is a file with somatic mutation calls, a list of HLA types, and optionally a gene expression profile. The output is a table with all tumor-specific peptides derived from nucleotide substitutions, insertions, and deletions, along with comprehensive annotation, including HLA binding and similarity to normal peptides. The peptides are sorted according to a priority score which is intended to roughly predict immunogenicity. We applied MuPeXI to three tumors for which predicted MHC-binding peptides had been screened for T cell reactivity, and found that MuPeXI was able to prioritize immunogenic peptides with an area under the curve of 0.63. Compared to other available tools, MuPeXI provides more information and is easier to use. MuPeXI is available as stand-alone software and as a web server at http://www.cbs.dtu.dk/services/MuPeXI .

Optimized Anchor-Modified Peptides Targeting Mutated RAS Are Promising Candidates for Immunotherapy.

RAS mutations occur in approximately 20% of all cancers and given their clonality, key role as driver mutation, association with poor prognosis and undruggability, they represent attractive targets for immunotherapy. We have identified immunogenic peptides derived from codon 12 mutant RAS (G12A, G12C, G12D, G12R, G12S and G12V), which bind to HLA-A*02:01 and HLA-A*03:01 and elicit strong peptide-specific CD8+ T cell responses, indicating that there is an effective CD8+ T-cell repertoire against these mutant RAS-derived peptides that can be mobilized. Alterations in anchor residues of these peptides enhanced their binding affinity to HLA-A*02:01 molecules and allowed generation of CD8+ T cells that responded to target cells pulsed with the anchor-modified and also with the original peptide. Cytotoxic T cells generated against these peptides specifically lysed tumor cells expressing mutant RAS. Vaccination of transgenic humanized HLA-A2/DR1 mice with a long peptide encompassing an anchor-modified 9-mer G12V epitope generated CD8+ T cells reactive to the original 9-mer and to a HLA-A*02:01-positive human cancer cell line harboring the G12V mutation. Our data provide strong evidence that mutant RAS can be targeted by immunotherapy.

MHC class II molecules on pancreatic cancer cells indicate a potential for neo-antigen-based immunotherapy.

MHC class II expression is a hallmark of professional antigen-presenting cells and key to the induction of CD4+ T helper cells. We found that these molecules are ectopically expressed on tumor cells in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC) and on several PDAC cell lines. In contrast to the previous reports that tumoral expression of MHC-II in melanoma enabled tumor cells to evade immunosurveillance, the expression of MHC-II on PDAC cells neither protected cancer cells from Fas-mediated cell death nor caused T-cell suppression by engagement with its ligand LAG-3 on activated T-cells. In fact and surprisingly, the MHC-II/LAG-3 pathway contributed to CD4+ and CD8+ T-cell cytotoxicity toward MHC-II-positive PDAC cells. By combining bioinformatic tools and cell-based assays, we identified a number of immunogenic neo-antigens that can be presented by the patients' HLA class II alleles. Furthermore, CD4+ T-cells stimulated with neo-antigens were capable of recognizing and killing a human PDAC cell line expressing the mutated genes. To expand this approach to a larger number of PDAC patients, we show that co-treatment with IFN-γ and/or MEK/HDAC inhibitors induced tumoral MHC-II expression on MHC-II-negative tumors that are IFN-γ-resistant. Taken together, our data point to the possibility of harnessing MHC-II expression on PDAC cells for neo-antigen-based immunotherapy.

Cancer-specific T helper shared and neo-epitopes uncovered by expression of the MHC class II master regulator CIITA.

We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral- and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses, which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important targets for cancer immunotherapy.

Healthy Donors Harbor Memory T Cell Responses to RAS Neo-Antigens.

The RAS mutations are the most frequently occurring somatic mutations in humans, and several studies have established that T cells from patients with RAS-mutant cancer recognize and kill RAS-mutant cells. Enhancing the T cell response via therapeutic cancer vaccination against mutant RAS results in a clinical benefit to patients; thus, T cells specific to RAS mutations are effective at battling cancer. As the theory of cancer immuno-editing indicates that healthy donors may clear malignantly transformed cells via immune-mediated killing, and since T cells have been shown to recognize RAS-mutant cancer cells, we investigated whether healthy donors harbor T-cell responses specific to mutant RAS. We identified strong and frequent responses against several epitopes derived from the RAS codon 12 and codon 13 mutations. Some healthy donors demonstrated a response to several mutant epitopes, and some, but not all, exhibited cross-reactivity to the wild-type RAS epitope. In addition, several T cell responses were identified against mutant RAS epitopes in healthy donors directly ex vivo. Clones against mutant RAS epitopes were established from healthy donors, and several of these clones did not cross-react with the wild-type epitope. Finally, CD45RO+ memory T cells from healthy donors demonstrated a strong response to several mutant RAS epitopes. Taken together, these data suggest that the immune system in healthy donors spontaneously clears malignantly transformed RAS-mutant cells, and the immune system consequently generates T-cell memory against the mutations.

Cancer Immune Therapy for Philadelphia Chromosome-Negative Chronic Myeloproliferative Neoplasms.

Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPN) are neoplastic diseases of the hematopoietic stem cells in the bone marrow. MPN are characterized by chronic inflammation and immune dysregulation. Of interest, the potent immunostimulatory cytokine interferon-α has been used to treat MPN for decades. A deeper understanding of the anti-cancer immune response and of the different immune regulatory mechanisms in patients with MPN has paved the way for an increased perception of the potential of cancer immunotherapy in MPN. Therapeutic vaccination targeting the driver mutations in MPN is one recently described potential new treatment modality. Furthermore, T cells can directly react against regulatory immune cells because they recognize proteins like arginase and programmed death ligand 1 (PD-L1). Therapeutic vaccination with arginase or PD-L1 therefore offers a novel way to directly affect immune inhibitory pathways, potentially altering tolerance to tumor antigens like mutant CALR and mutant JAK2. Other therapeutic options that could be used in concert with therapeutic cancer vaccines are immune checkpoint-blocking antibodies and interferon-α. For more advanced MPN, adoptive cellular therapy is a potential option that needs more preclinical investigation. In this review, we summarize current knowledge about the immune system in MPN and discuss the many opportunities for anti-cancer immunotherapy in patients with MPN.

Personalized Dendritic Cell Vaccines-Recent Breakthroughs and Encouraging Clinical Results.

With the advent of combined immunotherapies, personalized dendritic cell (DC)-based vaccination could integrate the current standard of care for the treatment of a large variety of tumors. Due to their proficiency at antigen presentation, DC are key coordinators of the innate and adaptive immune system, and have critical roles in the induction of antitumor immunity. However, despite proven immunogenicity and favorable safety profiles, DC-based immunotherapies have not succeeded at inducing significant objective clinical responses. Emerging data suggest that the combination of DC-based vaccination with other cancer therapies may fully unleash the potential of DC-based cancer vaccines and improve patient survival. In this review, we discuss the recent efforts to develop innovative personalized DC-based vaccines and their use in combined therapies, with a particular focus on ovarian cancer and the promising results of mutanome-based personalized immunotherapies.

Cancer Exome-Based Identification of Tumor Neo-Antigens Using Mass Spectrometry.

Neo-antigens expressed on tumors are targets for development of cancer immunotherapy strategies. Use of prediction algorithms to identify neo-antigens yields a significant number of peptides that must be validated in laborious and time-consuming methods; many prove to be false-positive identifications. The use of HLA peptidomics allows the isolation of the HLA-peptide complexes directly from cells and can be done on fresh tumor, patient-derived xerographs, or cell lines when the tissue sample is limited. This method can be used to identify both HLA class I and HLA class II or any different MHC from different species. Here we describe the steps to create the immune-affinity columns used from the process, the immunoprecipitation procedure, and also the isolation of the peptides that will be analyzed by mass spectrometry.

Genomics-Driven Immunoproteomics: An Integrative Platform to Uncover Important Biomarkers for Human Diseases.

Genomics-driven immunoproteomics (GDI) is a platform that helps identify antigenic protein targets of mutations and other deoxyribonucleic acid (DNA) variations that are commonly associated with pathological states. This platform utilizes data generated from deep sequencing of exomic DNA or ribonucleic acid (RNA) as input to synthesize mutant peptides into microarrays, which then can be used to detect antigenic proteins that invoke immune response in patients. The technology has been used to detect antigenic targets of multiple sclerosis, an autoimmune disease [1], and cancer to identify mutant proteins that invoke immune response in breast cancer patients [2]. This technology has many potential applications to select genomic changes that are specifically recognized by the immune system in a rapid and efficient manner.

T-Cell-Driven Inflammation as a Mediator of the Gut-Brain Axis Involved in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder affecting mainly the dopaminergic neurons of the nigrostriatal pathway, a neuronal circuit involved in the control of movements, thereby the main manifestations correspond to motor impairments. The major molecular hallmark of this disease corresponds to the presence of pathological protein inclusions called Lewy bodies in the midbrain of patients, which have been extensively associated with neurotoxic effects. Importantly, different research groups have demonstrated that CD4+ T-cells infiltrate into the substantia nigra of PD patients and animal models. Moreover, several studies have consistently demonstrated that T-cell deficiency results in a strong attenuation of dopaminergic neurodegeneration in animal models of PD, thus indicating a key role of adaptive immunity in the neurodegenerative process. Recent evidence has shown that CD4+ T-cell response involved in PD patients is directed to oxidised forms of α-synuclein, one of the main constituents of Lewy bodies. On the other hand, most PD patients present a number of non-motor manifestations. Among non-motor manifestations, gastrointestinal dysfunctions result especially important as potential early biomarkers of PD, since they are ubiquitously found among confirmed patients and occur much earlier than motor symptoms. These gastrointestinal dysfunctions include constipation and inflammation of the gut mucosa and the most distinctive pathologic features associated are the loss of neurons of the enteric nervous system and the generation of Lewy bodies in the gut. Moreover, emerging evidence has recently shown a pivotal role of gut microbiota in triggering the development of PD in genetically predisposed individuals. Of note, PD has been positively correlated with inflammatory bowel diseases, a group of disorders involving a T-cell driven inflammation of gut mucosa, which is strongly dependent in the composition of gut microbiota. Here we raised the hypothesis that T-cell driven inflammation, which mediates dopaminergic neurodegeneration in PD, is triggered in the gut mucosa. Accordingly, we discuss how structural components of commensal bacteria or how different mediators produced by gut-microbiota, including short-chain fatty acids and dopamine, may affect the behaviour of T-cells, triggering the development of T-cell responses against Lewy bodies, initially confined to the gut mucosa but later extended to the brain.

Functional evaluation of alternative splicing in the FAM190A gene.

The human FAM190A gene undergoes frequent alteration in human cancer, most commonly involving in-frame deletions in exon 9 or exons 9 & 10. These deletions form novel peptide sequences, serving as presumptive cancer-specific neo antigens. However, it remains elusive whether these in-frame deletions of FAM190A could induce oncogenic properties in vivo. In this study, we aimed to explore the functional significance of in-frame deletions in FAM190A genes. We generated two deletion mutant forms, FAM190AΔexon9 and FAM190AΔexon9&10, and examined their gain-of-function effects in vitro and in vivo. Global transcript profiling in NIH3T3 cells revealed that the transcripts displaying altered expression following introduction of FAM190AΔexon9 and FAM190AΔexon9&10 were significantly enriched for genes assigned to cellular movement and cell-to-cell signaling, respectively. Furthermore, ectopic expression of FAM190AΔexon9 and FAM190AΔexon9&10 induced in vivo tumor formation in nu/nu mice. Taken together, our results are the first to demonstrate the in vivo oncogenic properties of in-frame deletions in the FAM190A gene and indicate that these transcript variants might be clinically applicable as therapeutic targets in patients with cancer.

Polymerase epsilon mutations and concomitant ß2-microglobulin mutations in cancer.

Mutations in the exonuclease domain of polymerase epsilon (POLE), an enzyme of DNA synthesis, are involved in a newly described syndrome of colorectal polyposis and cancer, and have been associated with a high mutation burden with or without microsatellite instability (MSI) phenotype. The exonuclease domain of POLE executes a proofreading function that decreases the mutation rate during DNA replication by an estimated of one to two orders. The high mutation burden resulting from its loss of function could create a load of neo-antigens that would put the neoplastic cells in severe disadvantage of an immune attack if properly presented to the immune system. This paper investigates the mutagenic effect of different POLE mutations in various cancers, in published genomic studies and the effect that these POLE mutations have in selecting for mutations of the ß2 microglobulin (B2M) gene involved in antigen presentation.

Cancer immunotherapy: broadening the scope of targetable tumours.

Cancer immunotherapy has experienced remarkable advances in recent years. Striking clinical responses have been achieved for several types of solid cancers (e.g. melanoma, non-small cell lung cancer, bladder cancer and mismatch repair-deficient cancers) after treatment of patients with T-cell checkpoint blockade therapies. These have been shown to be particularly effective in the treatment of cancers with high mutation burden, which places tumour-mutated antigens (neo-antigens) centre stage as targets of tumour immunity and cancer immunotherapy. With current technologies, neo-antigens can be identified in a short period of time, which may support the development of complementary, personalized approaches that increase the number of tumours amenable to immunotherapeutic intervention. In addition to reviewing the state of the art in cancer immunotherapy, we discuss potential avenues that can bring the immunotherapy revolution to a broader patient group including cancers with low mutation burden.

Mutations in DNA repair genes are associated with increased neo-antigen load and activated T cell infiltration in lung adenocarcinoma.

Mutations in DNA repair genes lead to increased genomic instability and mutation frequency. These mutations represent potential biomarkers for cancer immunotherapy efficacy, as high tumor mutational burden has been associated with increased neo-antigens and tumor infiltrating lymphocytes. While mismatch repair mutations have successfully predicted response to anti-PD-1 therapy in colorectal and other cancers, they have not yet been tested for lung cancer, and few have investigated genes from other DNA repair pathways. We utilized TCGA samples to comprehensively immunophenotype lung tumors and analyze the links between DNA repair mutations, neo-antigen and total mutational burden, and tumor immune infiltration. Overall, 73% of lung tumors contained infiltration by at least one T cell subset, with high mutational burden tumors containing significantly increased infiltration by activated CD4 and CD8 T cells. Further, mutations in mismatch repair genes, homologous recombination genes, or POLE accurately predicted increased tumor mutational burden, neo-antigen load, and T cell infiltration. Finally, neo-antigen load correlated with expression of M1-polarized macrophage genes, PD-1, PD-L1, IFNγ, GZMB, and FASLG, among other immune-related genes. Overall, after defining the immune infiltrate in lung tumors, we demonstrate the potential value of utilizing gene mutations from multiple DNA repair pathways as biomarkers for lung cancer immunotherapy.

Novel Strategies for Peptide-Based Vaccines in Hematological Malignancies.

Peptides vaccination is an interesting approach to activate T-cells toward desired antigens in hematological malignancies. In addition to classical tumor associated antigens, such as cancer testis antigens, new potential targets for peptide vaccination comprise neo-antigens including JAK2 and CALR mutations, and antigens from immune regulatory proteins in the tumor microenvironment such as programmed death 1 ligands (PD-L1 and PD-L2). Immunosuppressive defenses of tumors are an important challenge to overcome and the T cell suppressive ligands PD-L1 and PD-L2 are often present in tumor microenvironments. Thus, PD-L1 and PD-L2 are interesting targets for peptide vaccines in diseases where the tumor microenvironment is known to play an essential role such as multiple myeloma and follicular lymphoma. In myelodysplastic syndromes the drug azacitidine re-exposes tumor associated antigens, why vaccination with related peptides would be an interesting addition. In myeloproliferative neoplasms the JAK2 and CALR mutations has proven to be immunogenic neo-antigens and thus possible targets for peptide vaccination. In this mini review we summarize the basis for these novel approaches, which has led to the initiation of clinical trials with various peptide vaccines in myelodysplastic syndromes, myeloproliferative neoplasms, multiple myeloma, and follicular lymphoma.