Immunogenic Tumor Antigens Research Articles

Preparation of Phage Display cDNA Libraries for Identifying Immunogenic Tumor Antigens: Challenges in Functional cDNA Presentation and Approaches to Overcoming Them

Preparation of Phage Display cDNA Libraries for Identifying Immunogenic Tumor Antigens: Challenges in Functional cDNA Presentation and Approaches to Overcoming Them

Self-Tumor Antigens in Solid Tumors Turned into Vaccines by α-gal Micelle Immunotherapy.

A major reason for the failure of the immune system to detect tumor antigens (TAs) is the insufficient uptake, processing, and presentation of TAs by antigen-presenting cells (APCs). The immunogenicity of TAs in the individual patient can be markedly increased by the in situ targeting of tumor cells for robust uptake by APCs, without the need to identify and characterize the TAs. This is feasible by the intra-tumoral injection of α-gal micelles comprised of glycolipids presenting the carbohydrate-antigen "α-gal epitope" (Galα1-3Galβ1-4GlcNAc-R). Humans produce a natural antibody called "anti-Gal" (constituting ~1% of immunoglobulins), which binds to α-gal epitopes. Tumor-injected α-gal micelles spontaneously insert into tumor cell membranes, so that multiple α-gal epitopes are presented on tumor cells. Anti-Gal binding to these epitopes activates the complement system, resulting in the killing of tumor cells, and the recruitment of multiple APCs (dendritic cells and macrophages) into treated tumors by the chemotactic complement cleavage peptides C5a and C3a. In this process of converting the treated tumor into a personalized TA vaccine, the recruited APC phagocytose anti-Gal opsonized tumor cells and cell membranes, process the internalized TAs and transport them to regional lymph-nodes. TA peptides presented on APCs activate TA-specific T cells to proliferate and destroy the metastatic tumor cells presenting the TAs. Studies in anti-Gal-producing mice demonstrated the induction of effective protection against distant metastases of the highly tumorigenic B16 melanoma following injection of natural and synthetic α-gal micelles into primary tumors. This treatment was further found to synergize with checkpoint inhibitor therapy by the anti-PD1 antibody. Phase-1 clinical trials indicated that α-gal micelle immunotherapy is safe and can induce the infiltration of CD4+ and CD8+ T cells into untreated distant metastases. It is suggested that, in addition to converting treated metastases into an autologous TA vaccine, this treatment should be considered as a neoadjuvant therapy, administering α-gal micelles into primary tumors immediately following their detection. Such an immunotherapy will convert tumors into a personalized anti-TA vaccine for the period prior to their resection.

Nanovaccines: Immunogenic tumor antigens, targeted delivery, and combination therapy to enhance cancer immunotherapy.

Nanovaccines have been designed to overcome the limitations associated with conventional vaccines. Effective delivery methods such as engineered carriers or smart nanoparticles (NPs) are critical requisites for inducing self-tolerance and optimizing vaccine immunogenicity with minimum side effects. NPs can be used as adjuvants, immunogens, or nanocarriers to develop nanovaccines for efficient antigen delivery. Multiloaded nanovaccines carrying multiple tumor antigens along with immunostimulants can effectively increase immunity against tumor cells. They can be biologically engineered to boost interactions with dendritic cells and to allow a gradual and constant antigen release. Modifying NPs surface properties, using high-density lipoprotein-mimicking nanodiscs, and developing nano-based artificial antigen-presenting cells such as dendritic cell-derived-exosomes are amongst the new developed technologies to enhance antigen-presentation and immune reactions against tumor cells. The present review provides an overview on the different perspectives, improvements, and barriers of successful clinical application of current cancer therapeutic and vaccination options. The immunomodulatory effects of different types of nanovaccines and the nanoparticles incorporated into their structure are described. The advantages of using nanovaccines to prevent and treat common illnesses such as AIDS, malaria, cancer and tuberculosis are discussed. Further, potential paths to develop optimal cancer vaccines are described. Given the immunosuppressive characteristics of both cancer cells and the tumor microenvironment, applying immunomodulators and immune checkpoint inhibitors in combination with other conventional anticancer therapies are necessary to boost the effectiveness of the immune response.

Immune profiling of dedifferentiated liposarcoma and identification of novel antigens for targeted immunotherapy

Dedifferentiated liposarcoma (DDLS) is an aggressive, recurring sarcoma with limited treatments. T-cell immunotherapies selectively target malignant cells, holding promise against DDLS. The development of successful immunotherapy for DDLS requires a thorough evaluation of the tumor immune microenvironment and the identification and characterization of targetable immunogenic tumor antigens. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens, we used the nCounter NanoString platform, analyzing gene expression profiles across 29 DDLS and 10 healthy adipose tissue samples. Hierarchical clustering of tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumors and 14 non-inflamed tumors, demonstrating tumor heterogeneity within this sarcoma subtype. Among the identified antigens, PBK and TTK exhibited substantial upregulation in mRNA expression compared to healthy adipose tissue controls, further corroborated by positive protein expression by IHC. This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS, and provides a novel targetable antigen in DDLS. The results of this study lay the groundwork for the development of a novel immunotherapy for this highly aggressive sarcoma.

Application of Machine Learning Algorithms for Prediction of Tumor T-Cell Immunogens

The identification and characterization of immunogenic tumor antigens are essential for cancer vaccine development. In light of the impracticality of isolating and evaluating each putative antigen individually, in silico prediction algorithms, particularly those utilizing machine learning (ML) approaches, play a pivotal role. These algorithms significantly reduce the experimental workload necessary for discovering vaccine candidates. In this study, we employed six supervised ML methods on a dataset comprising 212 experimentally validated human tumor peptide antigens and an equal number of non-antigenic human peptides to develop models for immunogenicity prediction. These methods encompassed k-nearest neighbor (kNN), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGBoost). The models underwent validation through internal cross-validation within 10 groups from the training set and were further assessed using an external test set. Remarkably, the kNN model demonstrated superior performance, recognizing 90% of the known immunogens in the test set. The RF model excelled in the identification of non-immunogens, accurately classifying 93% of them in the test set. The three top-performing ML models according to multiple evaluation metrics (SVM, RF, and XGBoost) are to be subsequently integrated into the new version of the VaxiJen server, facilitating tumor antigen prediction through a majority voting mechanism.

Engineered Bacteriophage-Based In Situ Vaccine Remodels a Tumor Microenvironment and Elicits Potent Antitumor Immunity.

In situ vaccines (ISVs) utilize the localized delivery of chemotherapeutic agents or radiotherapy to stimulate the release of endogenous antigens from tumors, thereby eliciting systemic and persistent immune activation. Recently, a bioinspired ISV strategy has attracted tremendous attention due to its features such as an immune adjuvant effect and genetic plasticity. M13 bacteriophages are natural nanomaterials with intrinsic immunogenicity, genetic flexibility, and cost-effectiveness for large-scale production, demonstrating the potential for application in cancer vaccines. In this study, we propose an ISV based on the engineered M13 bacteriophage targeting CD40 (M13CD40) for dendritic cell (DC)-targeted immune stimulation, named H-GM-M13CD40. We induce immunogenic cell death and release tumor antigens through local delivery of (S)-10-hydroxycamptothecin (HCPT), followed by intratumoral injection of granulocyte-macrophage colony stimulating factor (GM-CSF) and M13CD40 to enhance DC recruitment and activation. We demonstrate that this ISV strategy can result in significant accumulation and activation of DCs at the tumor site, reversing the immunosuppressive tumor microenvironment. In addition, H-GM-M13CD40 can synergize with the PD-1 blockade and induce abscopal effects in cold tumor models. Overall, our study verifies the immunogenicity of the engineered M13CD40 bacteriophage and provides a proof of concept that the engineered M13CD40 phage can function as an adjuvant for ISVs.

Individualized Multimodal Immunotherapy (IMI): Scientific Rationale and Clinical Experience from a Single Institution.

Oncolytic viruses and combinatorial immunotherapy for cancer (this Special Issue) are both part of cancer treatment at IOZK. This review focusses on an individual multimodal cancer immunotherapy concept developed by IOZK, Cologne, Germany. The scientific rationale for employing three main components is explained: (i) oncolytic Newcastle disease virus, (ii) modulated electrohyperthermia and (iii) individual tumor antigen and oncolytic virus modified dendritic cell vaccine (IO-VACR). The strategy involves repeated cancer-immunity cycles evoked in cancer patients by systemic oncolytic virus exposure plus hyperthermia pretreatment to induce immunogenic cell death followed by intradermal IO-VACR vaccination. As an example of the experience at IOZK, we present the latest results from combining the immunotherapy with standard treatment of patients suffering from glioblastoma multiforme. The promising clinical results in terms of overall survival benefit of additional individualized multimodal immunotherapy are presented. The cancer-immunity cycle, as introduced 10 years ago, describes key important steps occurring locally at the sites of both tumor and draining lymph nodes. This view is extended here towards systemic events occuring in blood where immunogenic cell death-induced tumor antigens are transported into the bone marrow. For 20 years it has been known that bone marrow is an antigen-responsive organ in which dendritic cells present tumor antigens to T cells leading to immunological synapse formation, tumor antigen-specific T cell activation and memory T cell formation. Bone marrow is known to be the most prominent source of de novo cellular generation in the body and to play an important role for the storage and maintenance of immunological memory. Its systemic activation is recommended to augment cancer-immunity cycles.

Immunoprecipitation methods impact the peptide repertoire in immunopeptidomics.

Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data. In this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples. Although, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it. Together, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens.

Immunohistochemical detection of the chondroitin sulfate proteoglycan 4 protein in primary and metastatic melanoma.

Treatment of malignant melanoma, the most aggressive form of skin cancer, continues to be a major challenge for clinicians. New targeted therapies with kinase inhibitors or drugs which modify the immune response are often accompanied by the development of resistance or severe side effects. In this context, chondroitin sulfate proteoglycan 4 (CSPG4), a highly immunogenic melanoma tumor antigen, could be a potential target for alternative therapeutic approaches. The aim of the present study was to identify differences in the levels of CSPG4 protein expression in primary and metastatic melanomas as well as to analyze correlations between CSPG4 expression and histopathological data and patient characteristics. A total of 189 melanoma tissue samples from Lower Austria, including primary melanomas and melanoma metastases, were immunohistochemically stained for the expression of CSPG4 and statistical analyses were performed. A total of 65.6% of melanoma tissue samples stained positive for the expression of CSPG4. Primary nodular and primary superficial spreading melanomas demonstrated a significantly higher number of positively stained tissue samples for CSPG4 compared with primary lentigo maligna melanomas. No significant differences in the expression of CSPG4 were demonstrated between primary melanomas and melanoma metastases. The present study supports the advancement of the understanding of CSPG4 tissue expression patterns in melanoma patients and provides additional information for further investigation of CSPG4 as a potential therapeutic target.

Combined expression of WT1/PRAME genes and BIRC5/PRAME genes and their clinical significance in adult patients with acute myeloid leukemia

Abstract Background Several tumor-associated antigens have been recently recognized that qualify to monitor tumor burden of acute myeloid leukemia (AML) or are used as antigen-specific approach for anticancer immunotherapy. Aim The current study was designed to assess the intracellular expression levels of the immunogenic tumor antigens ( WT1 , BIRC5, and PRAME genes) and clarify the prognostic effect of their combined overexpression, that is, WT1 /PRAME and BIRC5 / PRAME , on the clinical course, the outcome, and the response to treatment in patients with AML. Settings and design A total of 61 patients with AML were included in the study. All patients received induction chemotherapy. Detection of intracellular WT1, BIRC5, and PRAME genes in myeloid blast cells was done by real-time PCR on bone marrow samples. Their levels were correlated with the demographic data, the clinical and pathological data, the defined subgroups, the overall survival, and the response to treatment in patients with AML. Results WT1, BIRC5, and PRAME overexpression was detected in 73.8, 28, and 38.6% of patients, respectively. Nearly half of the patients (51.7%) were WT1+/PRAME− and significantly associated with absence of both favorable cytogenetic prognosis and complete remission. In addition, it expression was correlated with female sex, higher count of blasts in peripheral blood, positive CD34 expression, and higher FAB classification (M4) but with no statistically significant difference. Most patients were double negative BIRC5−/PRAME− (45.6%). A significant relation was found between BIRC5+/PRAME− and older age patients (P=0.003) and a lower overall survival (P=0.005) and also associated with higher count of blast cells but not statistically significant. Most BIRC5−/PRAME+ (72.7%) achieved complete remission at 28 days, followed by double negative BIRC5−/PRAME− (56.6%) at 28 days, most of BIRC5+/PRAME− patients died (77.8%). Conclusion In patients with AML, the expression levels of genes WT1, BIRC5, and PRAME were high, and these genes had a substantial correlation with poor prognostic factors, including a poor cytogenetic prognosis, incomplete remission, and a reduced survival rate. Therefore, they could be considered as targets for anticancer immunotherapy as well as possible markers for monitoring AML.

Identification of antigenic epitopes recognized by tumor infiltrating lymphocytes in high grade serous ovarian cancer by multi-omics profiling of the auto-antigen repertoire

Immunotherapeutic strategies aimed at enhancing tumor cell killing by tumor-specific T cells hold great potential for reducing tumor burden and prolonging survival of cancer patients. Although many potential tumor antigens have been described, identifying relevant targets when designing anti-cancer vaccines or targeted cell therapies remains a challenge. To identify novel, potentially immunogenic candidate tumor antigens, we performed integrated tumor transcriptomic, seromic, and proteomic analyses of high grade serous ovarian cancer (HGSC) patient tumor samples. We identified tumor neo-antigens and over-expressed antigens using whole exome and RNA sequencing and examined these in relation to patient-matched auto-antibody repertoires. Focusing on MHC class I epitopes recognized by CD8+ T cells, HLA-binding epitopes were identified or predicted from the highly expressed, mutated, or auto-antibody target antigen, or MHC-associated peptides (MAPs). Recognition of candidate antigenic peptides was assessed within the tumor-infiltrating T lymphocyte (TIL) population expanded from each patient. Known tumor-associated antigens (TAA) and cancer/testis antigens (CTA) were commonly found in the auto-antibody and MAP repertoires and CD8+ TILs recognizing epitopes from these antigens were detected, although neither expression level nor the presence of auto-antibodies correlated with TIL recognition. Auto-antibodies against tumor-mutated antigens were found in most patients, however, no TIL recognition of the highest predicted affinity neo-epitopes was detected. Using high expression level, auto-antibody recognition, and epitope prediction algorithms, we identified epitopes in 5 novel antigens (MOB1A, SOCS3, TUBB, PRKAR1A, CCDC6) recognized by HGSC patient TILs. Furthermore, selection of epitopes from the MAP repertoire identified 5 additional targets commonly recognized by multiple patient TILs. We find that the repertoire of TIL specificities includes recognition of highly expressed and immunogenic self-antigens that are processed and presented by tumors. These results indicate an ongoing autoimmune response against a range of self-antigens targeted by HGSC TILs.

Senescent cancer cell-derived nanovesicle as a personalized therapeutic cancer vaccine

The development of therapeutic cancer vaccines (TCVs) that provide clinical benefits is challenging mainly due to difficulties in identifying immunogenic tumor antigens and effectively inducing antitumor immunity. Furthermore, there is an urgent need for personalized TCVs because only a limited number of tumor antigens are shared among cancer patients. Several autologous nanovaccines that do not require the identification of immunogenic tumor antigens have been proposed as personalized TCVs. However, these nanovaccines generally require exogenous adjuvants (e.g., Toll-like receptor agonists) to improve vaccine immunogenicity, which raises safety concerns. Here, we present senescent cancer cell-derived nanovesicle (SCCNV) as a personalized TCV that provides patient-specific tumor antigens and improved vaccine immunogenicity without the use of exogenous adjuvants. SCCNVs are prepared by inducing senescence in cancer cells ex vivo and subsequently extruding the senescent cancer cells through nanoporous membranes. In the clinical setting, SCCNVs can be prepared from autologous cancer cells from the blood of liquid tumor patients or from tumors surgically removed from solid cancer patients. SCCNVs also contain interferon-γ and tumor necrosis factor-α, which are expressed during senescence. These endogenous cytokines act as adjuvants and enhance vaccine immunogenicity, avoiding the need for exogenous adjuvants. Intradermally injected SCCNVs effectively activate dendritic cells and tumor-specific T cells and inhibit primary and metastatic tumor growth and tumor recurrence. SCCNV therapy showed an efficacy similar to that of immune checkpoint blockade (ICB) therapy and synergized with ICB. SCCNVs, which can be prepared using a simple and facile procedure, show potential as personalized TCVs.

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.

Exposure of Immunogenic Tumor Antigens in Surrendered Immunity and the Significance of Autologous Tumor Cell-Based Vaccination in Precision Medicine.

The mechanisms by which immune systems identify and destroy tumors, known as immunosurveillance, have been discussed for decades. However, several factors that lead to tumor persistence and escape from the attack of immune cells in a normal immune system have been found. In the process known as immunoediting, tumors decrease their immunogenicity and evade immunosurveillance. Furthermore, tumors exploit factors such as regulatory T cells, myeloid-derived suppressive cells, and inhibitory cytokines that avoid cytotoxic T cell (CTL) recognition. Current immunotherapies targeting tumors and their surroundings have been proposed. One such immunotherapy is autologous cancer vaccines (ACVs), which are characterized by enriched tumor antigens that can escalate specific CTL responses. Unfortunately, ACVs usually fail to activate desirable therapeutic effects, and the low immunogenicity of ACVs still needs to be elucidated. This difficulty highlights the significance of immunogenic antigens in antitumor therapies. Previous studies have shown that defective host immunity triggers tumor development by reprogramming tumor antigenic expressions. This phenomenon sheds new light on ACVs and provides a potential cue to improve the effectiveness of ACVs. Furthermore, synergistically with the ACV treatment, combinational therapy, which can reverse the suppressive tumor microenvironments, has also been widely proposed. Thus, in this review, we focus on tumor immunogenicity sculpted by the immune systems and discuss the significance and application of restructuring tumor antigens in precision medicine.

Insights into the Role of Sialylation in Cancer Metastasis, Immunity, and Therapeutic Opportunity.

Sialylation is an enzymatic process that covalently attaches sialic acids to glycoproteins and glycolipids and terminates them by creating sialic acid-containing glycans (sialoglycans). Sialoglycans, usually located in the outmost layers of cells, play crucial biological roles, notably in tumor transformation, growth, metastasis, and immune evasion. Thus, a deeper comprehension of sialylation in cancer will help to facilitate the development of innovative cancer therapies. Cancer sialylation-related articles have consistently increased over the last four years. The primary subjects of these studies are sialylation, cancer, immunotherapy, and metastasis. Tumor cells activate endothelial cells and metastasize to distant organs in part by the interactions of abnormally sialylated integrins with selectins. Furthermore, cancer sialylation masks tumor antigenic epitopes and induces an immunosuppressive environment, allowing cancer cells to escape immune monitoring. Cytotoxic T lymphocytes develop different recognition epitopes for glycosylated and nonglycosylated peptides. Therefore, targeting tumor-derived sialoglycans is a promising approach to cancer treatments for limiting the dissemination of tumor cells, revealing immunogenic tumor antigens, and boosting anti-cancer immunity. Exploring the exact tumor sialoglycans may facilitate the identification of new glycan targets, paving the way for the development of customized cancer treatments.

Bacterial outer membrane vesicle-based cancer nanovaccines.

Tumor vaccines, a type of personalized tumor immunotherapy, have developed rapidly in recent decades. These vaccines evoke tumor antigen-specific T cells to achieve immune recognition and killing of tumor cells. Because the immunogenicity of tumor antigens alone is insufficient, immune adjuvants and nanocarriers are often required to enhance anti-tumor immune responses. At present, vaccine carrier development often integrates nanocarriers and immune adjuvants. Among them, outer membrane vesicles (OMVs) are receiving increasing attention as a delivery platform for tumor vaccines. OMVs are natural nanovesicles derived from Gram-negative bacteria, which have adjuvant function because they contain pathogen associated molecular patterns. Importantly, OMVs can be functionally modified by genetic engineering of bacteria, thus laying a foundation for applications as a delivery platform for tumor nanovaccines. This review summarizes 5 aspects of recent progress in, and future development of, OMV-based tumor nanovaccines: strain selection, heterogeneity, tumor antigen loading, immunogenicity and safety, and mass production of OMVs.

An unexplored angle: T cell antigen discoveries reveal a marginal contribution of proteasome splicing to the immunogenic MHC class I antigen pool

In the current era of T cell–based immunotherapies, it is crucial to understand which types of MHC-presented T cell antigens are produced by tumor cells. In addition to linear peptide antigens, chimeric peptides are generated through proteasome-catalyzed peptide splicing (PCPS). Whether such spliced peptides are abundantly presented by MHC is highly disputed because of disagreement in computational analyses of mass spectrometry data of MHC-eluted peptides. Moreover, such mass spectrometric analyses cannot elucidate how much spliced peptides contribute to the pool of immunogenic antigens. In this Perspective, we explain the significance of knowing the contribution of spliced peptides for accurate analyses of peptidomes on one hand, and to serve as a potential source of targetable tumor antigens on the other hand. Toward a strategy for mass spectrometry independent estimation of the contribution of PCPS to the immunopeptidome, we first reviewed methodologies to identify MHC-presented spliced peptide antigens expressed by tumors. Data from these identifications allowed us to compile three independent datasets containing 103, 74, and 83 confirmed T cell antigens from cancer patients. Only 3.9%, 1.4%, and between 0% and 7.2% of these truly immunogenic antigens are produced by PCPS, therefore providing a marginal contribution to the pool of immunogenic tumor antigens. We conclude that spliced peptides will not serve as a comprehensive source to expand the number of targetable antigens for immunotherapies.

Controllable regulation of CD3ζ and MyD88 signaling in T cells expands and enhances the neoantigen-reactive repertoire resulting in effective antitumor activity

Abstract Background Adoptive cell therapy (ACT) of tumor-reactive T cells including tumor infiltrating lymphocytes (TIL) represents one of the most promising forms of therapies for treating patients with solid tumors. However, durable responses are observed in a small subset of patients and in certain cancer types. Major hurdles to achieving durable responses include: 1) suboptimal TCR responses due to sparse and/or weakly immunogenic tumor antigen presentation, 2) development of dysfunctional or exhausted T cells, 3) low TIL persistence, and 4) tumor infiltration. We and others have demonstrated that activating MyD88 signaling in T cells strongly amplifies weak TCR responses and imparts T cells novel anti-tumor properties. Hypothesis TILs engineered with a novel fusion protein consisting of CD3ζ:MyD88 will activate MyD88 signals in a TCR-dependent manner resulting in enhanced activity and against a broader panel of neoantigens. Method & Results CD4 and CD8 TILs from a variety of human tumors engineered CD3ζ:MyD88 demonstrated: a) dramatically improved ability to expand in vitro resulting (generating 10–100X TILs than conventional TILs within a few weeks), b) persist for months in tissue culture, c) more polyfunctional cells, d) destruction of established melanoma tumors in vivo, e) enhanced responses against a panel of antigens with no overt autoimmunity. Conclusions CD3ζ:MyD88 represents a unique and universal approach to safely enhance T cell response to tumor antigens.

Upstream open reading frames regulate translation of cancer-associated transcripts and encode HLA-presented immunogenic tumor antigens

BackgroundUpstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood.MethodsWe examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs.ResultsWe provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses.ConclusionOur data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides.

1427P Cancer/testis antigen expression landscape in gastroesophageal adenocarcinoma

Cancer/testis antigens (CTA) are cancer-specific, immunogenic tumor antigens that are potential targets for cancer immunotherapy. There is limited literature regarding their co-expression and prognostic role in gastroesophageal adenocarcinoma (GEAC). This study evaluated the CTA landscape of GEAC and their correlation with different clinicopathological parameters and survival.