Tumor Antigens For Cancer Immunotherapy Research Articles

The landscape of MHC-presented phosphopeptides yields actionable shared tumor antigens for cancer immunotherapy across multiple HLA alleles

BackgroundCertain phosphorylated peptides are differentially presented by major histocompatibility complex (MHC) molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide...

Identification of tumor antigens for cancer immunotherapy

Identification of tumor antigens for cancer immunotherapy

Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.

A synergistic cancer immunotherapy nano-system for preventing tumor growth

Immunotherapy has become a promising therapy in cancer treatment. Both photothermal therapy (PTT) and genotoxic chemotherapy can trigger immune responses. On this subject, a synergistic cancer immunotherapy nano-system was developed for treating primary and distant tumors. This long-time, synergistic therapeutic system consisted of two parts: one was indocyanine green (ICG) and sepantronium bromide (YM155) co-loaded mesoporous silica nanoparticles (MSNs) for PTT and genotoxic chemotherapy, which could effectively destroy primary tumors and thus cause exposure of tumor antigens for cancer immunotherapy. The other was silica nanoparticles (nSiO2) coated with magnetic nanoparticles (MNP) and conjugated with anti-CD47 antibody (anti-CD47), which demonstrated a strong antitumor immune effect of distant tumors after PTT and genotoxic chemotherapy. In vitro and vivo results proved that this synergistic system could efficiently inhibit primary and distant tumor growth. We expected that this paper may develop a promising strategy to prevent tumor growth for basic and clinical research.

Application of radially grown ZnO nanowires on poly-l-lactide microfibers complexed with a tumor antigen for cancer immunotherapy.

Zinc oxide (ZnO)-based nanocomposites have shown promising potential for various biomedical applications, including vaccine development, owing to their multifunctionality and biocompatibility. Here, we synthesized radially grown ZnO nanowires (NWs) on poly-l-lactic acid (PLLA) microfibers with unique 3-dimensional structure and applied them as therapeutic cancer vaccines. This inorganic-organic hybrid nanocomposite has mild cellular toxicity but efficiently delivers a tumor antigen into dendritic cells, cellular bridges between innate and adaptive immunity, to stimulate them to express inflammatory cytokines and activation surface markers. We also demonstrated that the hybrid nanocomposites successfully induce tumor antigen-specific cellular immunity and significantly inhibit tumor growth in vivo. ZnO NWs on PLLA fibers systemically reduced immune suppressive TReg cells and enhanced the infiltration of T cells into tumor tissues, compared to mice immunized with PLLA fibers coated with the antigen. Our current findings open a new avenue in extending the biomedical application of inorganic metal oxide-inert organic hybrid nanocomposites as a novel vaccine platform.

Vaccination of Acute Leukemia Patients with WT1 Peptide-Pulsed Dendritic Cells Induces Immunological and Clinical Responses: A Pilot Study

Background and Purpose: Long term survival rate for acute leukemia has improved significantly with the development of monoclonal antibodies and molecular targeting agents. However, new therapeutic approaches are needed for patients with refractory or relapsed disease. Active immunizations using dendritic cells (DCs) loaded with tumor lysates or peptides have shown safety and efficacy. Wilms’ tumor 1 (WT1) gene is frequently overexpressed in leukemia and is considered to be one of the ideal tumor antigens for cancer immunotherapy. In the present pilot study, we treated acute leukemia patients with WT1 peptide-pulsed DC and examined safety, clinical and immunological responses to the vaccination.Patients and Methods: 5 AML and 2 ALL patients (4 males, 3 female; aged 16-77 years) were enrolled in the present study. 2 patients relapsed following allogeneic stem cell transplantation and 3 relapsed following standard and salvage chemotherapies. 2 patients were in CR with residual disease. 6 patients were HLA-A*24:02(A24) positive and 1 was positive for HLA-A*:02:01(A2). Autologous DCs were generated from patients’ peripheral blood monocytes which were separated by an adherence technique from mononuclear cells collected by apheresis. Monocytes were cultured with GM-CSF and IL-4 followed by maturation with OK432 and PGE2. HLA-A2 or HLA-A24-restricted 9-mer WT1 peptide-loaded mature DCs were administered intradermally every 2 weeks 5-7 times in combination with OK432. Induction of vaccine-induced T cell responses was monitored by a HLA-tetramer assay and a flow cytometry analysis. Cytotoxic activity was evaluated by an LDH release assayResults: The treatment was well tolerated and none of the patients experienced more than grade 2 adverse events during the treatment period. The most common adverse events were mild, transient erythema at injection sites and low grade fever. Of 7 patients, 3 had SD and 4 had PD following one course of the treatment. Decrease in WT1 mRNA was observed in 2 patients with SD. Survival of patients achieving SD (responder) after one course of DC vaccination was longer than those who did not respond to the treatment (median overall survival 37 vs 5.5 months). Duration of survival in responders was 46, 37 and 10 months after initiation of therapy. Two responders are still alive in remission. Increase in positivity of WT1-specific CD8+ T cells was observed in all the responders after one course of treatment. The percentage of WT1 tetramer positive cells was 0.10±0.09% before vaccination; it increased to 2.49±1.58% after the first course (approximately 25 times increase). Cytotoxic T cells (CTLs) generated by in vitro stimulation with WT1 peptide showed cytotoxic activity in an LDH release assay. CTL did not lyse irrelevant HIV peptide-pulsed HLA-A24+ target and WT1 peptide-pulsed HLA-A24- target, demonstrating antigen specificity and HLA restriction. Although absolute number of CD3+ T cells increased by 1.03±0.51 and 1.27±0.36 fold, respectively both in responders and non-responders, CD4/CD8 ratio increased by 1.45±0.97 fold in responders but decreased by 0.77±0.47 in non-responders, following the first course of DC vaccination. On the other hand, while the absolute number of CD4+CD25+Foxp3+ regulatory T cells decreased by 29.0% in responders, it increased by 43.0% in non-responders.Conclusions: The present study demonstrated that a vaccination with WT1 peptide-pulsed DC was well tolerated and no serious adverse event was observed. DC vaccination elicits both innate and acquired cellular immune responses correlated with clinical effects. These results suggest WT1 peptide-pulsed DC vaccination might be a promising novel strategy for the treatment of acute leukemia patients with relapsed or refractory disease. DisclosuresNo relevant conflicts of interest to declare.

Cancer-testis antigen expression in digestive tract carcinomas: frequent expression in esophageal squamous cell carcinoma and its precursor lesions.

Cancer-testis (CT) antigens are attractive tumor antigens for cancer immunotherapy. They comprise a group of proteins normally expressed in germ cells and aberrantly activated in a variety of human cancers. The protein expression of eight cancer-testis antigens [MAGEA, NY-ESO-1, GAGE, MAGEC1 (CT7), MAGEC2 (CT10), CT45, SAGE1, and NXF2] was evaluated by immunohistochemistry in 61 esophageal carcinomas (40 adenocarcinoma and 21 squamous cell carcinoma), 50 gastric carcinomas (34 diffuse and 16 intestinal type), and 141 colorectal carcinomas. The highest frequency of expression was found in esophageal squamous cell carcinomas: Positive staining for MAGEA, CT45, CT7, SAGE1, GAGE, NXF2, NY-ESO-1, and CT10 was observed in 57%, 38%, 33%, 33%, 29%, 29%, 19%, and 14% of squamous cell carcinomas, respectively. Similar staining patterns were observed in squamous dysplasias. Expression frequencies of cancer-testis antigens were seen in 2% to 24% of gastroesophageal adenocarcinomas and were not significantly different between adenocarcinomas of the stomach versus the esophagus, or between diffuse and intestinal types of gastric adenocarcinomas. Colorectal cancers did not express NY-ESO-1, CT7, CT10, or GAGE, and only infrequently expressed SAGE1 (0.7%) MAGEA (1.4%), CT45 (3.5%), and NXF2 (8.5%). We conclude that cancer-testis antigens are frequently expressed in esophageal squamous neoplasms. Although cancer-testis antigens are generally considered to be expressed later in tumor progression, they are found in squamous dysplasias, suggesting a potential diagnostic role for cancer-testis antigens in the evaluation of premalignant squamous lesions.

Vaccination Of Malignant Lymphoma Patients With WT1 Peptide-Pulsed Dendritic Cells Induces Immunological and Clinical Responses: A Pilot Study

BackgroundWith the advent of monoclonal antibodies such as rituximab, the duration of remission and overall survival in B-cell non-Hodgkin's lymphoma has improved significantly. However, new therapeutic approaches are needed for patients with refractory disease. Active immunizations using dendritic cells (DCs) loaded with ideotype proteins or tumor lysates have shown safety with limited efficacy. Wilms' tumor 1 (WT1) gene is frequently overexpressed in lymphoma and is considered to be one of the ideal tumor antigens for cancer immunotherapy. In the present pilot study, we treated lymphoma patients with WT1 peptide-pulsed DC and examined safety, clinical and immunological responses to the vaccination. Methods5 HLA-A*24:02(A24)+patients (4 males, 1 female; aged 51-90 years; 2 with diffuse large B cell lymphoma, 1 with mantle cell lymphoma, 1 with follicular lymphoma and 1 with peripheral T cell lymphoma) with relapsed or refractory disease were enrolled in the present study. Autologous DCs were generated from patients' peripheral blood monocytes which were separated by an adherence technique from mononuclear cells collected by apheresis. Monocytes were cultured with GM-CSF and IL-4 followed by maturation with OK432 and PGE2. HLA-A24-restricted, modified 9-mer WT1 peptide-loaded mature DCs were administered intradermally every 2 weeks 5-7 times in combination with OK432. Induction of vaccine-induced T cell responses was monitored by a HLA-tetramer assay and a flow cytometry analysis. Cytotoxicity against WT1 peptide pulsed target cells was tested in a LDH release assay. ResultsThe treatment was well tolerated and none of the patients experienced more than grade 2 adverse events during the treatment period. The most common adverse events were mild, transient erythema at injection sites and low grade fever. Of 5 patients, 1 had CR, 1 had PR, 2 had SD and 1 had PD following one course of the treatment. Survival of patients achieving CR, PR or SD (responder) after one course of DC vaccination was longer than those who did not respond to the treatment. Duration of survival in responders was 56, 32, 24 and 19 months after initiation of therapy. Three responders are still alive in remission. Increase in positivity of WT1-specific CD8+ T cells was observed in responders after one course of treatment; the percentage of HLA-A24 restricted WT1 tetramer positive cells was 0.12±0.12% before vaccination, it increased to 6.82±10.2% after the first course. WT1 tetramer positivity in one of the responding patients who received 3 courses of vaccination increased from 0.06% to 24.5% following one course of vaccination. High level of positivity persisted thereafter (14.6% and 14.6% after second and third course, respectively). Cytotoxic T cells (CTLs) generated by in vitro stimulation with WT1 peptide showed cytotoxic activity in an LDH release assay; percent specific lysis against WT1 peptide-pulsed HLA-A24+ target was 64.8% at effector/target ratio 30 to1. CTL did not lyse irrelevant HIV peptide-pulsed HLA-A24+ target and WT1 peptide-pulsed HLA-A24- target, demonstrating antigen specificity and HLA restriction. Absolute number of lymphocytes, CD3+ T cells, CD4/CD8 ratio and NK cells increased by 1.10±0.38, 2.03±1.78, 1.27±0.36, 1.31±0.74 fold, respectively, following the first course of DC vaccination in responders. On the other hand, absolute number of CD4+CD25+Foxp3+ regulatory T cells (Treg) decreased by 50.7%. Lin-CD33+HLA-DR- myeloid-derived suppressor cells (MDSC) decreased from 17.8%±10.1 to 9.0±2.5% (38.3% reduction). Similarly, monocytic MDSC (CD14+HLA-DRlow/-) and granurocytic MDSC (CD15+CD33+) decreased by 34.4% and 34.0%, respectively, indicating that DC vaccination may contribute to the reversal of immunosuppression by Treg and MDSC. ConclusionsThe present study demonstrated that a vaccination with WT1 peptide-pulsed DC was well tolerated and no serious adverse event was observed. DC vaccination elicits both innate and acquired cellular immune responses correlated with clinical effects. These results suggest WT1 peptide-pulsed DC vaccination might be a promising novel strategy for the treatment of malignant lymphoma patients with relapsed or refractory disease. Disclosures:No relevant conflicts of interest to declare.

Insights into the processing of MHC class I ligands gained from the study of human tumor epitopes.

The molecular definition of tumor antigens recognized by cytolytic T lymphocytes (CTL) started in the late 1980s, at a time when the MHC class I antigen processing field was in its infancy. Born together, these two fields of science evolved together and provided each other with critical insights. Over the years, stimulated by the potential interest of tumor antigens for cancer immunotherapy, scientists have identified and characterized numerous antigens recognized by CTL on human tumors. These studies have provided a wealth of information relevant to the mode of production of antigenic peptides presented by MHC class I molecules. A number of tumor antigenic peptides were found to result from unusual mechanisms occurring at the level of transcription, translation or processing. Although many of these mechanisms occur in the cell at very low level, they are relevant to the immune system as they determine the killing of tumor cells by CTL, which are sensitive to low levels of peptide/MHC complexes. Moreover, these unusual mechanisms were found to occur not only in tumor cells but also in normal cells. Thereby, the study of tumor antigens has illuminated many aspects of MHC class I processing. We review here those insights into the MHC I antigen processing pathway that result from the characterization of human tumor antigens recognized by CTL.

Topoisomerase II alpha as a universal tumor antigen: antitumor immunity in murine tumor models and H-2K(b)-restricted T cell epitope.

Topoisomerase II alpha (Top2alpha) is an attractive candidate to be used as a tumor antigen for cancer immunotherapy, because it is abundantly expressed in various tumors and serves as a target for a number of chemotherapeutic agents. In this study, we demonstrated the immunogenicity of Top2alpha, using dendritic cells (DC) electroporated with RNA encoding the Top2alpha C-terminus (Top2alphaCRNA/DC). Top2alphaCRNA/DC were able to demonstrate in vitro stimulation of T cells from mice that were previously vaccinated with Top2alpha-expressing tumor lysate-pulsed DC. Vaccination with Top2alphaCRNA/DC induced Top2alpha-specific T cell responses in vivo as well as antitumor effects in various murine tumor models including MC-38, B16F10, and GL26. DC pulsed with p1327 (DSDEDFSGL), defined as an epitope presented by H-2K(b), also induced Top2alpha-specific immune responses and antitumor effects. Based on these data, Top2alpha is suggested to be a universal target for cancer immunotherapy.

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Heat labile enterotoxin of E. coli: a potential adjuvant for transcutaneous cancer immunotherapy

Escherichia coli heat labile enterotoxin (LT) has been shown to penetrate intact skin and to activate adaptive immunity. A nontoxic mutant, nLT, and its B subunit (LTB), have been evaluated separately for their potential use as a tool for transcutaneous delivery of antigens for cancer immunotherapy. We have shown that FITC-labeled nLT is taken up by human dendritic cells (hDC) in vitro and in mouse skin, and induces maturation and activation of hDC in vitro. hDC matured with nLT enhanced nonspecific melanoma antigen uptake and presentation to autologous CD8+ T cells. In mouse in vivo studies, nLT or LTB were applied on the skin either mixed with recombinant gp100 or genetically fused with a multiepitope polypeptide (MEP). Fused LTB-MEP induced antibody production that was dependent on LTB cell binding. We conclude that LT derivatives may be useful for the transcutaneous delivery of tumor antigens for cancer immunotherapy.

Tumor antigens for cancer immunotherapy: therapeutic potential of xenogeneic DNA vaccines

Preclinical animal studies have convincingly demonstrated that tumor immunity to self antigens can be actively induced and can translate into an effective anti-tumor response. Several of these observations are being tested in clinical trials. Immunization with xenogeneic DNA is an attractive approach to treat cancer since it generates T cell and antibody responses. When working in concert, these mechanisms may improve the efficacy of vaccines. The use of xenogeneic DNA in overcoming immune tolerance has been promising not only in inbred mice with transplanted tumors but also in outbred canines, which present with spontaneous tumors, as in the case of human. Use of this strategy also overcomes limitations seen in other types of cancer vaccines. Immunization against defined tumor antigens using a xenogeneic DNA vaccine is currently being tested in early phase clinical trials for the treatment of melanoma and prostate cancers, with proposed trials for breast cancer and Non-Hodgkin's Lymphoma.

Cancer immunotherapy targeting WT1 protein.

The Wilms tumor gene WT1 is expressed in leukemias and various kinds of solid tumors, including lung and breast cancer, and exerts an oncogenic function in these malignancies, suggesting that WT1 protein is a novel, overexpressed tumor antigen. The WT1 protein, in fact, is an attractive tumor rejection antigen in animal models. Stimulation in vitro of peripheral blood mononuclear cells with HLA-A*2402--and HLA-A*0201--restricted 9-mer WT1 peptides elicits WT1-specific cytotoxic T-lymphocytes (CTLs), and the CTLs kill endogenously WT1-expressing leukemia or solid tumor cells. Furthermore, WT1 immunoglobulin M (IgM) and IgG antibodies are detected in patients with hematopoietic malignancies such as acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndromes, indicating that WT1 protein overexpressed by leukemia cells is indeed immunogenic. Taken together, these results demonstrate that WT1 protein is a promising tumor antigen for cancer immunotherapy against leukemias and various kinds of solid tumors, including lung and breast cancer.

A herpesvirus saimiri-based gene therapy vector with potential for use in cancer immunotherapy.

The herpesvirus saimiri (HVS) genome has the capacity to incorporate large amounts of heterologous DNA and can be maintained episomally in many different human cell types. To evaluate the efficacy of HVS-mediated gene transfer into human hemopoietic cells, we investigated the ability of an HVS-based construct, carrying the enhanced green fluorescent protein (EGFP) and neomycin resistance genes, to transduce a variety of human hemopoietic cell lines and primary CD34+ cells. As measured by flow cytometry, the numbers of EGFP+ cells at 2 days postinfection differed between various cell types ranging, from 1.3% for KG1 cells to 56.8% for THP-1 cells. In addition, the expression of EGFP in Jurkat cells was retained at >95% per round of cell division over a period of 6 weeks (comparable with Epstein-Barr virus-derived gene therapy systems). Although the virus was not specifically disabled, no lytic viral mRNAs could be detected in transduced Jurkat cells, and infectious virus could not be detected by sensitive virus recovery assay. We also describe a simple centrifugation method that increases the efficiency of transduction by >100% in some cases and may be generally applicable to other herpesvirus-based vectors for ex vivo gene delivery. Using this technique, we were able to demonstrate a tropism for CD34+/CD14+ cells, transducing 30% of the population. These cells are known to give rise to dendritic cells (the most potent of the antigen-presenting cells), suggesting that the vector could be used to deliver DNA sequences encoding tumor antigens for cancer immunotherapy.

Identification of Lung Tumor Antigens for Cancer Immunotherapy: Immunological and Molecular Approaches

(2000). Identification of Lung Tumor Antigens for Cancer Immunotherapy: Immunological and Molecular Approaches. Immunological Investigations: Vol. 29, No. 2, pp. 87-91.