Discovery of high-expressing lncRNA-derived sORFs as potential tumor-associated antigens in hepatocellular carcinoma.

BACKGROUND: This study is based on deep mining of Ribo-seq data for the identification of lncRNAs that have highly expressed sORFs in HCC. In this paper, dynamic prospects associated with sORFs acting as newly defined tumor-specific epitopes are discussed with possible improvement in strategies for tumor immunotherapy. OBJECTIVE: Using ribosome profiling to identify and characterize sORFs within lncRNAs in HCC, identify potential therapeutic targets and tumor-specific epitopes applicable for immunotherapy. METHODS: MetamORF performed the identification of sORFs with deep analysis of the data of ribosome profiling in lncRNAs associated with HCC. The translation efficiency in these molecules was estimated, and epitope prediction was done by pVACbind. Peptide search was done to check the presence of micropeptides translated from these identified sORFs. validated translational activity and identified potential epitopes. RESULTS: Higher translation efficiency was noted in the case of lncRNAs associated with HCC compared to normal tissues. Of particular note is ORF3418981, which results in the highest expression and has supporting experimental evidence at the protein level. Epitope prediction identified a putative epitope at the C-terminus of ORF3418981. CONCLUSIONS: This study uncovers the as-yet-unknown potential of lncRNA-derived sORFs as sources of tumor antigens, shifting the research focus from protein-coding genes to non-coding RNAs also in the HCC context. Moreover, this study highlights the contribution of a subset of lncRNAs, especially LINC00152, to the development of tumors and modulation of the immune response by its sORFs.

Neoantigen Identification and Dendritic Cell-Based Vaccines for Lung Cancer Immunotherapy.

Immunotherapies can treat many cancers, including difficult-to-treat cases such as lung cancer. Due to its tolerability, long-lasting therapeutic responses, and efficacy in a wide spectrum of patients, immunotherapy can also help to treat lung cancer, which has few treatment choices. Tumor-specific antigens (TSAs) for cancer vaccinations and T-cell therapies are difficult to discover. Neoantigens (NeoAgs) from genetic mutations, irregular RNA splicing, protein changes, or viral genetic sequences in tumor cells provide a solution. NeoAgs, unlike TSAs, are non-self and can cause an immunological response. Next-generation sequencing (NGS) and bioinformatics can swiftly detect and forecast tumor-specific NeoAgs. Highly immunogenic NeoAgs provide personalized or generalized cancer immunotherapies. Dendritic cells (DCs), which originate and regulate T-cell responses, are widely studied potential immunotherapeutic therapies for lung cancer and other cancers. DC vaccines are stable, reliable, and safe in clinical trials. The purpose of this article is to evaluate the current status, limitations, and prospective clinical applications of DC vaccines, as well as the identification and selection of major histocompatibility complex (MHC) class I and II genes for NeoAgs. Our goal is to explain DC biology and activate DC manipulation to help researchers create extremely potent cancer vaccines for patients.

Lack of shared neoantigens in prevalent mutations in cancer.

Tumors are mostly characterized by genetic instability, as result of mutations in surveillance mechanisms, such as DNA damage checkpoint, DNA repair machinery and mitotic checkpoint. Defect in one or more of these mechanisms causes additive accumulation of mutations. Some of these mutations are drivers of transformation and are positively selected during the evolution of the cancer, giving a growth advantage on the cancer cells. If such mutations would result in mutated neoantigens, these could be actionable targets for cancer vaccines and/or adoptive cell therapies. However, the results of the present analysis show, for the first time, that the most prevalent mutations identified in human cancers do not express mutated neoantigens. The hypothesis is that this is the result of the selection operated by the immune system in the very early stages of tumor development. At that stage, the tumor cells characterized by mutations giving rise to highly antigenic non-self-mutated neoantigens would be efficiently targeted and eliminated. Consequently, the outgrowing tumor cells cannot be controlled by the immune system, with an ultimate growth advantage to form large tumors embedded in an immunosuppressive tumor microenvironment (TME). The outcome of such a negative selection operated by the immune system is that the development of off-the-shelf vaccines, based on shared mutated neoantigens, does not seem to be at hand. This finding represents the first demonstration of the key role of the immune system on shaping the tumor antigen presentation and the implication in the development of antitumor immunological strategies.

Expression Pattern of Tumor-associated Antigens in Neuroblastoma: Association With Cytogenetic Features and Survival.

Background/Aim: The prognosis of high-risk and relapsed neuroblastoma (NB) patients remains poor. The identification of tumor-associated markers is important for differential diagnosis, prognosis, and the development of targeted therapies. The aim of the study was to determine the expression profile of nine most common NB antigens and assess their association with clinicopathological characteristics and patient survival. Patients and Methods: Tumor samples from 86 patients with NB were evaluated for the expression of tumor-associated antigen (TAA) using quantitative PCR. Twenty-one patients with benign tumors and 17 healthy donors were assigned as controls. Results: Overexpression of tyrosine hydroxylase (TH), PHOX2B, PRAME, GPC2, B7-H3, and Survivin is the most typical for NB. Positive expression of MAGEA3, MAGEA1, and NY-ESO-1 at low levels was detected in 54%, 48%, and 52%, respectively, and was not NB specific. Higher TH expression was observed in samples without MYCN-amplification, while higher expression of Survivin, PHOX2B, and GPC2 was significantly associated with the presence of 1p.36 deletion. Overexpression of TH, PHOX2B, and MAGEA1 was associated with better event-free (EFS) and overall survival (OS). Survivin overexpression was associated with poor EFS but had no impact on OS. Multivariate analysis confirmed Survivin as independent marker for poor survival, and PHOX2B and MAGEA1 for better survival. Conclusion: High expression of TH, PHOX2B, and MAGEA1 genes are favorable prognostic factors for OS and EFS, whereas high expression of Survivin is associated with an increased risk of relapse or progression.

Delivery of Immunostimulatory Cargos in Nanocarriers Enhances Anti-Tumoral Nanovaccine Efficacy.

Finding a long-term cure for tumor patients still represents a major challenge. Immunotherapies offer promising therapy options, since they are designed to specifically prime the immune system against the tumor and modulate the immunosuppressive tumor microenvironment. Using nucleic-acid-based vaccines or cellular vaccines often does not achieve sufficient activation of the immune system in clinical trials. Additionally, the rapid degradation of drugs and their non-specific uptake into tissues and cells as well as their severe side effects pose a challenge. The encapsulation of immunomodulatory molecules into nanocarriers provides the opportunity of protected cargo transport and targeted uptake by antigen-presenting cells. In addition, different immunomodulatory cargos can be co-delivered, which enables versatile stimulation of the immune system, enhances anti-tumor immune responses and improves the toxicity profile of conventional chemotherapeutic agents.

Rethinking Antigen Source: Cancer Vaccines Based on Whole Tumor Cell/tissue Lysate or Whole Tumor Cell.

Cancer immunotherapies have improved human health, and one among the important technologies for cancer immunotherapy is cancer vaccine. Antigens are the most important components in cancer vaccines. Generally, antigens in cancer vaccines can be divided into two categories: pre-defined antigens and unidentified antigens. Although, cancer vaccines loaded with predefined antigens are commonly used, cancer vaccine loaded with mixed unidentified antigens, especially whole cancer cells or cancer cell lysates, is a very promising approach, and such vaccine can obviate some limitations in cancer vaccines. Their advantages include, but are not limited to, the inclusion of pan-spectra (all or most kinds of) antigens, inducing pan-clones specific T cells, and overcoming the heterogeneity of cancer cells. In this review, the recent advances in cancer vaccines based on whole-tumor antigens, either based on whole cancer cells or whole cancer cell lysates, are summarized. In terms of whole cancer cell lysates, the focus is on applying whole water-soluble cell lysates as antigens. Recently, utilizing the whole cancer cell lysates as antigens in cancer vaccines has become feasible. Considering that pre-determined antigen-based cancer vaccines (mainly peptide-based or mRNA-based) have various limitations, developing cancer vaccines based on whole-tumor antigens is a promising alternative.

Antigen discovery for the development of cancer immunotherapy.

Central to successful cancer immunotherapy is effective T cell antitumor immunity. Multiple targeted immunotherapies engineered to invigorate T cell-driven antitumor immunity rely on identifying the repertoire of T cell antigens expressed on the tumor cell surface. Mass spectrometry-based survey of such antigens ("immunopeptidomics") combined with other omics platforms and computational algorithms has been instrumental in identifying and quantifying tumor-derived T cell antigens. In this review, we discuss the types of tumor antigens that have emerged for targeted cancer immunotherapy and the immunopeptidomics methods that are central in MHC peptide identification and quantification. We provide an overview of the strength and limitations of mass spectrometry-driven approaches and how they have been integrated with other technologies to discover targetable T cell antigens for cancer immunotherapy. We highlight some of the emerging cancer immunotherapies that successfully capitalized on immunopeptidomics, their challenges, and mass spectrometry-based strategies that can support their development.

Pan-Cancer Analysis Identifies Tumor Cell Surface Targets for CAR-T Cell Therapies and Antibody Drug Conjugates.

Tumor cells can be recognized through tumor surface antigens by immune cells and antibodies, which therefore can be used as drug targets for chimeric antigen receptor-T (CAR-T) therapies and antibody drug conjugates (ADCs). In this study, we aimed to identify novel tumor-specific antigens as targets for more effective and safer CAR-T cell therapies and ADCs. Here, we performed differential expression analysis of pan-cancer data obtained from the Cancer Genome Atlas (TCGA), and then performed a series of conditional screenings including Cox regression analysis, Pearson correlation analysis, and risk-score calculation to find tumor-specific cell membrane genes. A tumor tissue-specific and highly expressed gene set containing 3919 genes from 17 cancer types was obtained. Moreover, the prognostic roles of these genes and the functions of these highly expressed membrane proteins were assessed. Notably, 427, 584, 431 and 578 genes were identified as risk factors for LIHC, KIRC, UCEC, and KIRP, respectively. Functional enrichment analysis indicated that these tumor-specific surface proteins might confer tumor cells the ability to invade and metastasize. Furthermore, correlation analysis displayed that most overexpressed membrane proteins were positively correlated to each other. In addition, 371 target membrane protein-coding genes were sifted out by excluding proteins expressed in normal tissues. Apart from the identification of well-validated genes such as GPC3, MSLN and EGFR in the literature, we further confirmed the differential protein expression of 23 proteins: ADD2, DEF6, DOK3, ENO2, FMNL1, MICALL2, PARVG, PSTPIP1, FERMT1, PLEK2, CD109, GNG4, MAPT, OSBPL3, PLXNA1, ROBO1, SLC16A3, SLC26A6, SRGAP2, and TMEM65 in four types of tumors. In summary, our findings reveal novel tumor-specific antigens, which could be potentially used for next-generation CAR-T cell therapies and ADC discovery.

Plasmid DNA for Therapeutic Applications in Cancer.

Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections and cancer. In most cancers, the immune system is limited or suppressed, allowing cancer cells to grow. DNA vaccination has demonstrated its capacity to stimulate the immune system to fight against cancer cells. Furthermore, plasmids for cancer gene therapy can direct the expression of proteins with different functions, such as enzymes, toxins, and cytotoxic or proapoptotic proteins, to directly kill cancer cells. The progress and promising results reported in animal models in recent years have led to interesting clinical results. These DNA strategies are expected to be approved for cancer treatment in the near future. This review discusses the main strategies, challenges, and future perspectives of using plasmid DNA for cancer treatment.

Induced pluripotent stem cells display a distinct set of MHC I-associated peptides shared by human cancers.

Previous reports showed that mouse vaccination with pluripotent stem cells (PSCs) induces durable anti-tumor immune responses via T cell recognition of some elusive oncofetal epitopes. We characterize the MHC I-associated peptide (MAP) repertoire of human induced PSCs (iPSCs) using proteogenomics. Our analyses reveal a set of 46 pluripotency-associated MAPs (paMAPs) absent from the transcriptome of normal tissues and adult stem cells but expressed in PSCs and multiple adult cancers. These paMAPs derive from coding and allegedly non-coding (48%) transcripts involved in pluripotency maintenance, and their expression in The Cancer Genome Atlas samples correlates with source gene hypomethylation and genomic aberrations common across cancer types. We find that several of these paMAPs were immunogenic. However, paMAP expression in tumors coincides with activation of pathways instrumental in immune evasion (WNT, TGF-ß, and CDK4/6). We propose that currently available inhibitors of these pathways could synergize with immune targeting of paMAPs for the treatment of poorly differentiated cancers.

CAD v1.0: Cancer Antigens Database Platform for Cancer Antigen Algorithm Development and Information Exploration.

Cancer vaccines have gradually attracted attention for their tremendous preclinical and clinical performance. With the development of next-generation sequencing technologies and related algorithms, pipelines based on sequencing and machine learning methods have become mainstream in cancer antigen prediction; of particular focus are neoantigens, mutation peptides that only exist in tumor cells that lack central tolerance and have fewer side effects. The rapid prediction and filtering of neoantigen peptides are crucial to the development of neoantigen-based cancer vaccines. However, due to the lack of verified neoantigen datasets and insufficient research on the properties of neoantigens, neoantigen prediction algorithms still need to be improved. Here, we recruited verified cancer antigen peptides and collected as much relevant peptide information as possible. Then, we discussed the role of each dataset for algorithm improvement in cancer antigen research, especially neoantigen prediction. A platform, Cancer Antigens Database (CAD, http://cad.bio-it.cn/), was designed to facilitate users to perform a complete exploration of cancer antigens online.

Emerging role of natural products in cancer immunotherapy.

Cancer immunotherapy has become a new generation of anti-tumor treatment, but its indications still focus on several types of tumors that are sensitive to the immune system. Therefore, effective strategies that can expand its indications and enhance its efficiency become the key element for the further development of cancer immunotherapy. Natural products are reported to have this effect on cancer immunotherapy, including cancer vaccines, immune-check points inhibitors, and adoptive immune-cells therapy. And the mechanism of that is mainly attributed to the remodeling of the tumor-immunosuppressive microenvironment, which is the key factor that assists tumor to avoid the recognition and attack from immune system and cancer immunotherapy. Therefore, this review summarizes and concludes the natural products that reportedly improve cancer immunotherapy and investigates the mechanism. And we found that saponins, polysaccharides, and flavonoids are mainly three categories of natural products, which reflected significant effects combined with cancer immunotherapy through reversing the tumor-immunosuppressive microenvironment. Besides, this review also collected the studies about nano-technology used to improve the disadvantages of natural products. All of these studies showed the great potential of natural products in cancer immunotherapy.

Generation of Tumor-Specific Cytotoxic T Cells From Blood via In Vitro Expansion Using Autologous Dendritic Cells Pulsed With Neoantigen-Coupled Microbeads.

For the past decade, adoptive cell therapy including tumor-infiltrating lymphocytes, genetically modified cytotoxic lymphocytes expressing a chimeric antigen receptor, or a novel T-cell receptor has revolutionized the treatment of many cancers. Progress within exome sequencing and neoantigen prediction technologies provides opportunities for further development of personalized immunotherapies. In this study, we present a novel strategy to deliver in silico predicted neoantigens to autologous dendritic cells (DCs) using paramagnetic beads (EpiTCer beads). DCs pulsed with EpiTCer beads are superior in enriching for healthy donor and patient blood-derived tumor-specific CD8+ T cells compared to DC loaded with whole-tumor lysate or 9mer neoantigen peptides. A dose-dependent effect was observed, with higher EpiTCer bead per DC being favorable. We concluded that CD8+ T cells enriched by DC loaded with EpiTCer beads are tumor specific with limited tumor cross-reactivity and low recognition of autologous non-activated monocytes or CD8+ T cells. Furthermore, tumor specificity and recognition were improved and preserved after additional expansion using our Good Manufacturing Process (GMP)-compatible rapid expansion protocol. Phenotypic analysis of patient-derived EpiTCer DC expanded CD8+ T cells revealed efficient maturation, with high frequencies of central memory and effector memory T cells, similar to those observed in autologous expanded tumor-infiltrating lymphocytes. These results indicate that DC pulsed with EpiTCer beads enrich for a T-cell population with high capacity of tumor recognition and elimination, which are features needed for a T-cell product to be used for personalized adoptive cell therapy.

CAR-T Cells Targeting TSHR Demonstrate Safety and Potent Preclinical Activity Against Differentiated Thyroid Cancer.

BACKGROUND: Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in hematological cancers but have not yet translated in treating solid tumors. The significant hurdles limiting CAR-T therapy were from a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present TSH receptor (TSHR) as a putative target for CAR-T therapy of differentiated thyroid cancer (DTC). METHODS: We undertook a large-scale screen on thyroid cancer tissues and multiple internal organs through bioinformatical analysis and immunohistochemistry to date TSHR expression. Using 3 previously described monoclonal antibodies, we generated 3 third-generation CAR-Ts. We tested anti-TSHR CAR-T in vitro activity by T-cell function and killing assay. Then we tested preclinical therapeutical efficacy in a xenograft mouse model of DTC and analyzed mice's physical conditions and histological abnormalities to evaluate anti-TSHR CAR-T's safety. RESULTS: TSHR is highly and homogeneously expressed on 90.8% (138/152) of papillary thyroid cancer, 89.2% (33/37) of follicular thyroid cancer, 78.2% (18/23) of cervical lymph node metastases, and 86.7% of radioactive iodine resistance diseases. We developed 3 novel anti-TSHR CAR-Ts from monoclonal antibodies M22, K1-18, and K1-70; all 3 CAR-Ts mediate significant antitumor activity in vitro. Among these, we demonstrate that K1-70 CAR-T can have therapeutical efficacy in vivo, and no apparent toxicity has been observed. CONCLUSION: TSHR is a latent target antigen of CAR-T therapy for DTC. Anti-TSHR CAR-T could represent a therapeutic option for patients with locoregional relapsed or distant metastases of thyroid cancer and should be tested in carefully designed clinical trials.

Implications of Antigen Selection on T Cell-Based Immunotherapy.

Many immunotherapies rely on CD8+ effector T cells to recognize and kill cognate tumor cells. These T cell-based immunotherapies include adoptive cell therapy, such as CAR T cells or transgenic TCR T cells, and anti-cancer vaccines which expand endogenous T cell populations. Tumor mutation burden and the choice of antigen are among the most important aspects of T cell-based immunotherapies. Here, we highlight various classes of cancer antigens, including self, neojunction-derived, human endogenous retrovirus (HERV)-derived, and somatic nucleotide variant (SNV)-derived antigens, and consider their utility in T cell-based immunotherapies. We further discuss the respective anti-tumor/anti-self-properties that influence both the degree of immunotolerance and potential off-target effects associated with each antigen class.

Neoantigen: A New Breakthrough in Tumor Immunotherapy.

Cancer immunotherapy works by stimulating and strengthening the body's anti-tumor immune response to eliminate cancer cells. Over the past few decades, immunotherapy has shown remarkable efficacy in the treatment of cancer, particularly the success of immune checkpoint blockade targeting CTLA-4, PD-1 and PDL1, which has led to a breakthrough in tumor immunotherapy. Tumor neoantigens, a new approach to tumor immunotherapy, include antigens produced by tumor viruses integrated into the genome and antigens produced by mutant proteins, which are abundantly expressed only in tumor cells and have strong immunogenicity and tumor heterogeneity. A growing number of studies have highlighted the relationship between neoantigens and T cells' recognition of cancer cells. Vaccines developed against neoantigens are now being used in clinical trials in various solid tumors. In this review, we summarized the latest advances in the classification of immunotherapy and the process of classification, identification and synthesis of tumor-specific neoantigens, as well as their role in current cancer immunotherapy. Finally, the application prospects and existing problems of neoantigens were discussed.

Human Endogenous Retrovirus Reactivation: Implications for Cancer Immunotherapy.

Human endogenous retroviruses (HERVs) derive from ancestral exogenous retroviruses whose genetic material has been integrated in our germline DNA. Several lines of evidence indicate that cancer immunotherapy may benefit from HERV reactivation, which can be induced either by drugs or by cellular changes occurring in tumor cells. Indeed, several studies indicate that HERV proviral DNA can be transcribed either to double-stranded RNA (dsRNA) that is sensed as a "danger signal" by pattern recognition receptors (PRRs), leading to a viral mimicry state, or to mRNA that is translated into proteins that may contribute to the landscape of tumor-specific antigens (TSAs). Alternatively, HERV reactivation is associated with the expression of long noncoding RNAs (lncRNAs). In this review, we will highlight recent findings on HERV reactivation in cancer and its implications for cancer immunotherapy.

Anti-Cancer Auto-Antibodies: Roles, Applications and Open Issues.

Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body's defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of these antibody specificities is now possible, allowing their identification at the genetic, structural, and epitope levels. In this review, we report the evidence available on the presence of auto-antibodies in the main cancer types and discuss some of the open issues that still need to be addressed by the research community.

Cancer Vaccines: Antigen Selection Strategy.

Unlike traditional cancer therapies, cancer vaccines (CVs) harness a high specificity of the host's immunity to kill tumor cells. CVs can train and bolster the patient's immune system to recognize and eliminate malignant cells by enhancing immune cells' identification of antigens expressed on cancer cells. Various features of antigens like immunogenicity and avidity influence the efficacy of CVs. Therefore, the choice and application of antigens play a critical role in establishing and developing CVs. Tumor-associated antigens (TAAs), a group of proteins expressed at elevated levels in tumor cells but lower levels in healthy normal cells, have been well-studied and developed in CVs. However, immunological tolerance, HLA restriction, and adverse events are major obstacles that threaten TAA-based CVs' efficacy due to the "self-protein" characteristic of TAAs. As "abnormal proteins" that are completely absent from normal cells, tumor-specific antigens (TSAs) can trigger a robust immune response against tumor cells with high specificity and without going through central tolerance, contributing to cancer vaccine development feasibility. In this review, we focus on the unique features of TAAs and TSAs and their application in vaccines, summarizing their performance in preclinical and clinical trials.