Differential effects of PD-1 and CTLA-4 blockade on the melanoma-reactive CD8 T cell response.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.

Immunogenicity in humans of a transdermal multipeptide melanoma vaccine administered with or without a TLR7 agonist.

BACKGROUND: Experimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine. METHODS: Twelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund's adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot. RESULTS: CD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%. CONCLUSIONS: These data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.

Antibody-Conjugated Gel-Coated Single-Walled Carbon Nanotubes as Photothermal Agents.

Photothermal therapy (PTT) using near-infrared (NIR) light is an attractive treatment modality for cancer, in which photothermal agents absorb energy from photons and convert it into thermal energy to lead to cancer cell death. Among the various organic and inorganic materials, single-walled carbon nanotubes (SWCNTs) are promising candidates for NIR photothermal agents due to their strong absorption in this region as well as their high photothermal conversion efficiency. In the development of the SWCNT-based PTT materials, modifications of SWCNTs to offer a stable dispersion for biocompatibility as well as to target the tumor of choice while maintaining their NIR absorption have been required. While modification of SWCNTs through noncovalent methods can be achieved, these modifications can be easily reversed in the body. Contrarily, modifications through covalent attachments, while more desirable, may compromise the NIR absorption characteristics of the SWCNTs. Previously, we reported the development of a synthetic strategy to coat SWCNTs with a cross-linked polymer (i.e., a gel) through a process called CNT Micelle Polymerization and successfully introduced maleimide groups that allowed for postmodification through the ene-thiol reaction without deteriorating the NIR absorption. In this report, we postmodify thiol-containing antibodies (anti-TRP-1, a melanoma specific protein) using maleimide chemistry and find that the SWCNTs conjugated with anti-TRP-1 maintain the characteristic NIR absorption as SWCNTs with dispersion stability. It is estimated that 50 maleimide groups are incorporated in one SWCNT (ca. 280 nm long) and they are modified with 32 TRP-1 fragments. Finally, we successfully use these targeted SWCNTs for the PTT of the melanoma cell line using NIR light (1064 nm; 2 W, 5 min). Our method can be extended to a vast array of specific antibodies as well as other targeting agents.

Preparation of a new combination nanoemulsion-encapsulated MAGE1-MAGE3-MAGEn/HSP70 vaccine and study of its immunotherapeutic effect.

BACKGROUND: MAGE family genes have been studied as targets for tumor immunotherapy for a long time. Here, we combined MAGE1-, MAGE3- and MAGEn-derived peptides as a cancer vaccine and tested whether a new combination nanoemulsion-encapsulated vaccine could be used to inhibit the growth of tumor cells in humanized SCID mice. METHODS: The nanoemulsion-encapsulated complex protein vaccine (MAGE1, MAGE3, and MAGEn/HSP70 fusion protein; M1M3MnH) was prepared using a magnetic ultrasonic technique. After screening, human PBMCs were injected into SCID mice to mimic the human immune system. Then, the humanized SCID mice were challenged with M3-HHCC cells and immunized with nanoemulsion-encapsulated MAGE1-MAGE3-MAGEn/HSP70 [NE(M1M3MnH)] or M1M3MnH. The cellular immune responses were detected by IFN-γ ELISPOT and cytotoxicity assays. Therapeutic and tumor challenge experiments were also performed. RESULTS: The results showed that the immune responses elicited by NE(M1M3MnH) were apparently stronger than those elicited by M1M3MnH, NE(-) or PBS, suggesting that this novel nanoemulsion carrier induces potent antitumor immunity against the encapsulated antigens. The results of the therapeutic and tumor challenge experiments also indicated that the new vaccine had a definite effect on SCID mice bearing human hepatic cancer. CONCLUSION: Our study indicated that the combination of several tumor antigen-derived peptides may be a relatively good strategy for peptide-based cancer immunotherapy. These results suggest that the complex nanoemulsion vaccine could have broader applications for both therapy and prevention mediated by antitumor effects in the future.

Synthetic tumor-specific antigenic peptides with a strong affinity to HLA-A2 elicit anti-breast cancer immune response through activating CD8+ T cells.

Researches on tumor-associated antigen have become a hot target in immunotherapy, but it stagnated in the pre-clinical/clinical stages. Here, we developed a series of MAGE-A1-restricted antigenic peptides, which exhibited prominent inhibiting effect on specific breast cancer. Peptides were synthesized by Fmoc solid phase method and analyzed by online servers. The stability and affinity to HLA-A2 was assessed by inverted fluorescence and flow cytometry qualitatively and quantitatively. In vitro effect on dendritic cells (DCs) maturation was observed by morphology and surface markers. The secretion of IFN-γ in the supernatant was detected by co-incubation of DCs loaded with as-synthesized peptides and CD8+ T lymphocytes. The specific immune response was evaluated against 4 cell lines, and the response in MCF-7 xenografted BALB/c nude mice were further assessed. Most of the derived peptides, especially I-6, showed great HLA-A2 binding ability. Compared with cytokines, I-6 significantly induced DCs maturation and promoted CD8+ T lymphocytes activation. Additionally, it is more specific for the lethality of MAGE & HLA-A2 double positive cells compared with others. We successfully developed I-6 with a high affinity to HLA-A2 which could induce strong specific immune response. It could be a potential candidate for breast cancer immunotherapy, which deserves further studies.

Altering Antigen Charge to Control Self-Assembly and Processing of Immune Signals During Cancer Vaccination.

Biomaterial delivery systems offer unique potential to improve cancer vaccines by offering targeted delivery and modularity to address disease heterogeneity. Here, we develop a simple platform using a conserved human melanoma peptide antigen (Trp2) modified with cationic arginine residues that condenses an anionic toll-like receptor agonist (TLRa), CpG, into polyplex-like nanoparticles. We reasoned that these structures could offer several useful features for immunotherapy - such as tunable loading, co-delivery of immune cues, and cargo protection - while eliminating the need for synthetic polymers or other complicating delivery systems. We demonstrate that Trp2/CpG polyplexes can readily form over a range of Trp2:CpG ratios and improve antigen uptake by primary antigen presenting cells. We show antigen loading can be tuned by interchanging Trp2 peptides with defined charges and numbers of arginine residues. Notably, these polyplexes with greater antigen loading enhance the functionality of Trp-2 specific T cells and in a mouse melanoma model, decrease tumor burden and improve survival. This work highlights opportunities to control the biophysical properties of nanostructured materials built from immune signals to enhance immunotherapy, without the added complexity or background immune effects often associated with synthetic carriers.

Antibody Responses to Cancer Antigens Identify Patients with a Poor Prognosis among HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma Patients.

PURPOSE: The identification of high-risk patients within human papillomavirus (HPV)-positive and -negative head and neck squamous cell carcinoma (HNSCC) is needed for improved treatment and surveillance strategies. In this study, we set out to discover antibody responses (AR) with prognostic impact in HNSCC stratified by HPV status. EXPERIMENTAL DESIGN: A fluorescent bead-based multiplex serology assay on 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, and 8 oncogenes) and 29 HPV antigens was performed in samples of 362 patients with HNSCC from five independent cohorts (153 HPV positive, 209 HPV negative). A multivariable Cox proportional hazard model with bootstrapping (M = 1000) was used for validation of prognostic antibody responses. RESULTS: Antibody response to any of the cancer antigens was found in 257 of 362 patients (71%). In HPV-negative patients, antibody responses to c-myc, MAGE-A1, -A4, and Rhodopsin E2 (combined as ARhigh risk) were significantly associated with shorter overall survival. In HPV-positive patients, antibody responses to IMP-1 were discovered as a negative prognostic factor. ARhigh risk (HR = 1.76) and antibody responses to IMP-1 (HR = 3.28) were confirmed as independent markers for a poor prognosis in a multivariable Cox proportional hazard model with bootstrapping (M = 1000). CONCLUSIONS: We identified antibody responses to cancer antigens that associate with a dismal prognosis in patients with HNSCC beyond HPV-positive status. ARhigh risk may be used to detect HPV-negative patients with an extraordinarily bad prognosis. Most importantly, antibody response to IMP-1 may serve as a marker for a subgroup of HPV-positive patients who present with a poor prognosis similar to that in HPV-negative patients.

Cancer Neoepitopes for Immunotherapy: Discordance Between Tumor-Infiltrating T Cell Reactivity and Tumor MHC Peptidome Display.

Tumor-infiltrating lymphocytes (TIL) are considered enriched for T cells recognizing shared tumor antigens or mutation-derived neoepitopes. We performed exome sequencing and HLA-A*02:01 epitope prediction from tumor cell lines from two HLA-A2-positive melanoma patients whose TIL displayed strong tumor reactivity. The potential neoepitopes were screened for recognition using autologous TIL by immunological assays and presentation on tumor major histocompatibility complex class I (MHC-I) molecules by Poisson detection mass spectrometry (MS). TIL from the patients recognized 5/181 and 3/49 of the predicted neoepitopes, respectively. MS screening detected 3/181 neoepitopes on tumor MHC-I from the first patient but only one was also among those recognized by TIL. Consequently, TIL enriched for neoepitope specificity failed to recognize tumor cells, despite being activated by peptides. For the second patient, only after IFN-γ treatment of the tumor cells was one of 49 predicted neoepitopes detected by MS, and this coincided with recognition by TIL sorted for the same specificity. Importantly, specific T cells could be expanded from patient and donor peripheral blood mononuclear cells (PBMC) for all neoepitopes recognized by TIL and/or detected on tumor MHC-I. In summary, stimulating the appropriate inflammatory environment within tumors may promote neoepitope MHC presentation while expanding T cells in blood may circumvent lack of specific TIL. The discordance in detection between physical and functional methods revealed here can be rationalized and used to improve neoantigen-targeted T cell immunotherapy.

Design of Artificial Immunogens Containing Melanoma-associated T-cell Epitopes.

OBJECTIVE: Immunotherapy based on induction of T-cell responses is a promising approach to cancer treatment. The study aims to design artificial epitope-based immunogens, DNA vaccine candidates against melanoma and evaluate their ability to stimulate tumor cytotoxicity of ex vivo generated T-cells. METHODS: The original computational methods were used for predicting T-cell epitopes and designing polyepitope melanoma antigens. Artificial genes encoding the target antigens were cloned into DNA vaccine plasmid vector. Target gene expression was confirmed both at transcriptional and translational level in HEK-293T cells transfected with DNA-vaccine constructs. Dendritic cells were generated from adherent peripheral blood mononuclear cells of HLA-A*02:01+ donors. Cytotoxic activity of effector lymphocytes stimulated in co-culture with autologous antigen-presenting dendritic cells towards melanoma Mel Is cells was assessed with lactate dehydrogenase release assay. The proportion of granzyme B producing CD8+ T-cells was estimated using intracellular cytokine staining and flow cytometry. RESULTS: Two DNA vaccine constructions were created - pMEL-TCI and pMEL-A0201 - encoding polypeptides containing T-cell epitopes of six immunodominant melanoma antigens (NY-ESO-1, MART1, MAGE-A1, MAGE-A11, MAGE-A3, and MAGE-C1). Dendritic cells transfected with DNA vaccine constructs were found to stimulate both tumor cytotoxicity mediated by autologous lymphocytes and granzyme B production by CD8+ T-cells, and pMEL-A0201 was found to be the most efficient. CONCLUSION: The described approach may become a common platform for designing immunotherapeutic vaccines against oncological diseases.

A Nobel Prize-worthy pursuit: cancer immunology and harnessing immunity to tumour neoantigens.

The field of cancer immunology stepped into the limelight this year when James P. Allison and Tasuku Honjo received the Nobel Prize in Physiology or Medicine for their discovery of cancer therapy by inhibition of negative immune regulation. Among many exciting advances contributing to the coming of age of tumour immunology as a viable clinical specialty has been the ability to progress from the initial elucidation of tumour antigens, such as the melanoma antigen, MAGE-1, to high-throughput sequencing facilitating identification of T cell epitopes from diverse tumour neoantigens. This has resulted from the convergence of expertise in tumour biology, next-generation sequencing, T cell and structural immunology, and predictive algorithms. Among many examples, immunotherapy for ovarian cancer has been one of the beneficiaries of these advances, leading to a number of recent and ongoing clinical trials.

Immunization with tumor neoantigens displayed on T7 phage nanoparticles elicits plasma antibody and vaccine-draining lymph node B cell responses.

The aim of this preclinical study was to evaluate T7 bacteriophage as a nanoparticle platform for expression of neoantigens that could allow rapid generation of vaccines for potential studies in human cancer patients. We have generated recombinant T7 phage vaccines carrying neoepitopes derived from mutated proteins of B16-F10 melanoma tumor cells. With the single mutated amino acid (AA) centered, peptides were expressed on the outer coat of T7 phage. All peptides with 11 and 34 AAs were successfully expressed. Trimers of the 11-AA peptides were successfully expressed in only 3 of 8 peptides. The 11-AA peptide was better in stimulating antibodies selective for the mutated region than the longer 34-AA peptide. We observed a dose response for vaccines which provides an initial framework of the minimum phage required for vaccination. A single injection with phage-peptide vaccines in both monomer and trimer formats produced significant immune responses in mice on day 21, as assessed by lymph node cell counts, next generation sequencing (NGS), and plasma titers against T7 phage and vaccine peptides. A trimer provided no additional serum response to the monomer format. Immunization of mice with a mixture of 8 different peptide vaccines resulted in antibodies to most of the peptides. It was encouraging that induced antibodies had higher binding to the mutated peptides compared to the corresponding normal peptides. The NGS of lymph node cells demonstrated a low B cell receptor diversity and clonal hyperpolarization in vaccine-draining lymph nodes in comparison to those in unvaccinated mice nodes. The NGS data also revealed phenomenal increase in IgG and other class-switched antibodies following vaccination. These results agree with the higher plasma titers of IgG antibodies against T7 phage and vaccine peptides. Antibodies bound whole B16-F10 cells, lysates and multiple bands on Western blot. This indicates that these vaccine peptides successfully induced antibodies that bind full proteins from which the vaccine peptides were derived. We demonstrate a preclinical platform for rapid production of vaccines that can deliver mutated peptides and stimulate an appropriate B cell response. We anticipate further research in utilizing the cells from a tumor or vaccine draining lymph node as a resource for therapeutic anticancer reagents.

MAGE-A antigens as targets for cancer immunotherapy.

Targeted anti-cancer therapies aim at reducing side effects while retaining their anti-cancer efficacy. Immunotherapies e.g. monoclonal antibodies, adoptive T cell therapy and cancer vaccines are used to combat cancer, but the number of available cancer specific targets is limited and new approaches are needed to generate more effective and patient tailored treatments. Unique cancer intracellular epitopes can be presented on the cell surface by MHC class I molecules, which can function as epitopes for targeted therapies. The intracellular MAGE proteins belong to a sub-class of Cancer Testis (CT) antigens which are expressed in germline cells and a wide variety of tumors of different histological origin. Evidence has emerged that their expression is linked to pro-tumorigenic activities like increased cell motility, resisting cell death, and tumor promoting inflammation. Intracellular MAGE proteins are processed by the proteasome and their peptides are presented by MHC class I molecules on the cell surface of cancer cells thereby making them ideal cancer specific antigens. Here we review the previous and ongoing (pre-) clinical studies on the use of surface expressed MAGE antigens for their employment in targeted anti-cancer therapies. We present and analyze study outcomes and discuss possible future directions and improvements for MAGE directed anti-cancer immunotherapies.

Cancer-Germline Antigen Expression Discriminates Clinical Outcome to CTLA-4 Blockade.

CTLA-4 immune checkpoint blockade is clinically effective in a subset of patients with metastatic melanoma. We identify a subcluster of MAGE-A cancer-germline antigens, located within a narrow 75 kb region of chromosome Xq28, that predicts resistance uniquely to blockade of CTLA-4, but not PD-1. We validate this gene expression signature in an independent anti-CTLA-4-treated cohort and show its specificity to the CTLA-4 pathway with two independent anti-PD-1-treated cohorts. Autophagy, a process critical for optimal anti-cancer immunity, has previously been shown to be suppressed by the MAGE-TRIM28 ubiquitin ligase in vitro. We now show that the expression of the key autophagosome component LC3B and other activators of autophagy are negatively associated with MAGE-A protein levels in human melanomas, including samples from patients with resistance to CTLA-4 blockade. Our findings implicate autophagy suppression in resistance to CTLA-4 blockade in melanoma, suggesting exploitation of autophagy induction for potential therapeutic synergy with CTLA-4 inhibitors.

Melanoma: a prototype of cancer-testis antigen-expressing malignancies.

Melanoma is the first malignancy in which expression and immunogenicity of cancer-testis antigens (CTAs) have been documented. Several CTAs have been shown to be expressed in melanoma samples especially those with metastatic potential. Many of them have been shown to exert oncogenic effects through modulation of essential pathways involved in melanoma. The crucial role of CTAs in the pathogenesis of melanoma, the high prevalence of expression of CTA panels in melanoma and the presence of spontaneous as well as inducible immune responses against CTAs in melanoma patients potentiate CTAs as immunotherapeutic targets. Numerous clinical trials are now ongoing to evaluate CTA-based immunotherapeutic effects in melanoma patient's survival. NY-ESO-1 and MAGE antigens have the most promising results up to now.

Personalized ex vivo multiple peptide enrichment and detection of T cells reactive to multiple tumor-associated antigens in prostate cancer patients.

Personalized peptide vaccination is a promising immunotherapeutic approach in prostate cancer (PCa). We therefore examined whether an approach, utilizing personalized multiple peptide-mediated ex vivo enrichment with effector T cells reactive to multiple tumor-associated antigens (TAAs), could be employed as a basis for the development of T cell immunotherapy of PCa. In this study, we used the non-adherent fraction (lymphocytes) of cryopreserved peripheral blood mononuclear cells from a leukapheretic product of biochemically recurrent (BR, n = 14) and metastatic hormone-refractory (HR, n = 12) PCa patients. The lymphocytes were primed with a pool of mixed overlapping peptides derived from 6 PCa TAAs-PSA, PAP, NY-ESO-1, MAGE-A1, MAGE-A3 and MAGE-A4. After 2 weeks of culture, the cells were stimulated with the peptides and T cell reactivity determined by externalization of CD107a. No TAAs-reactive effector T cells were detected in the patient's lymphocytes after their reconstitution. However, following their priming with the TAAs-derived peptides and 2-week culturing, the lymphocytes became enriched with polyclonal TAAs-reactive effector CD8+ T cells in 8 out of 14 BR and 5 out of 12 HR patients. No such reactive CD8+ T cells were detected in cultured lymphocytes without the peptide priming. Stimulation of the responding cultures with peptides derived from individual TAAs revealed a unique repertoire of the reactive CD8+ T cells. Our strategy revealed that the personalized multiple peptide-mediated ex vivo enrichment with multiple TAAs-reactive T cells in the PCa patient's lymphocytes is a viable approach for development of T cell immunotherapy of PCa.

A novel electrochemical immunosensor based on ITO modified by carboxyl-ended silane agent for ultrasensitive detection of MAGE-1 in human serum.

A new, low-cost electrochemical immunosensor was developed for rapid detection of Melanoma-associated antigen 1 (MAGE-1), a cancer biomarker. The fabrication procedure of immunosensor was based on the covalent immobilization of anti-MAGE-1, biorecognition molecule, on ITO electrode by carboxyethylsilanetriol (CTES) monolayer. The biosensing MAGE-1 antigen was monitored by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) technique. Apart from these techniques, single frequency impedance (SFI) was used for investigation of antibody-antigen interactions. Scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) were utilized for characterization of the proposed biosensor. To fabricate highly sensitive, good stability immunosensor, some parameters were optimized. Under optimal conditions, the developed electrochemical immunosensor for MAGE-1 exhibited a dynamic range of 4 fg/mL and 200 fg/mL with a low detection limit of 1.30 fg/mL. It had acceptable repeatability (5.05%, n = 20) and good storage stability (3.58% loss after 10 weeks). Moreover, this electrochemical immunosensor has been successfully applied to the determination of MAGE-1 in human serum samples.

Expansion of cancer germline antigen-specific cytotoxic T lymphocytes for immunotherapy.

The cancer germline antigens MAGE-A1, MAGE-A3, and NY-ESO-1 can be used to target relapsed or therapy-resistant malignant solid tumors, and previous studies have demonstrated that these antigens can be epigenetically upregulated on the surface of tumor cells following exposure to low-dose demethylating chemotherapy agents, such as decitabine. The extent to which cancer germline antigen cytotoxic T lymphocytes can be reliably expanded from healthy donors has not been well characterized, specifically in terms of whether these T cells consistently kill antigen-bearing targets or simply produce interferon-γ in the presence of the antigen. Cancer germline antigen cytotoxic T lymphocytes were generated using conventional method and high-density lymphocyte culture method. We demonstrate that there is no difference in the extent of antigen-specific killing with or without CD25 depletion when interleukin-21 is added to the cultures. Cancer germline antigen-specific killer cells could be expanded from 8/12 healthy donors using overlapping peptide mixes derived from MAGE-A1, MAGE-A3, and NY-ESO-1 and from 7/9 healthy donors using HLA-restricted epitopes. Furthermore, cytotoxic T lymphocyte derived from 4/5 patients displayed specific cytotoxicity of target cells expressing respective cancer germline antigen and HLA partially matched tumor lines. High-density lymphocyte culture prior to stimulation with cancer germline antigen peptides resulted in antigen-specific cytotoxic T lymphocyte from healthy donors and patients from whom cancer germline antigen cytotoxic T lymphocyte culture with conventional methods was not feasible. These data demonstrate that MAGE-A1-, MAGE-A3-, and NY-ESO-1-specific T cells with antigen-specific cytotoxicity can be cultured from healthy donors and patient-derived cells making adoptive immunotherapy with these cytotoxic T lymphocyte feasible.

Development of an epitope panel for consistent identification of antigen-specific T-cells in humans.

We aimed to establish a panel of MHC-peptide multimers suitable as a positive control in the detection of HLA A*0201 restricted antigen specific T cells (ASTC) by flow cytometry. MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melanoma targets identified from a literature search and in silico prediction. Peripheral blood mononuclear cells (PBMC) from healthy donors were analysed with the MHC Dextramers using flow cytometry. The best performing epitopes were tested on PBMC from patients undergoing testing for Mycobacterium tuberculosis infection to assess the coverage of this epitope panel. Of 21 candidate epitopes, ASTC could be detected against 12 (57·1%) in at least one of 18 healthy blood donors. Reactivity to two or more epitopes was seen in 17 of the 18 donors (94·4%). We selected the six best-performing epitopes and demonstrated a positive response in 42 (97·7%) of 43 patient samples (healthy, latent and active M. tuberculosis infection). The selected panel of six antigenic epitopes sufficed as a positive control in the detection of ASTC in HLA A*0201. Performance was robust in different stages of latent and active M. tuberculosis infection, indicating reliability also during infection.

Melanoma antigens and related immunological markers.

Melanoma is a highly lethal cancer deriving from transformed dermal melanocytes. Early diagnosed primary melanoma may be curable, but the cure-rate of more advanced stages is limited, with high mortality rate. With the progression of the tumor, the melanocytes overexpress intracellular or cell-surface molecules, including ectopic normal and tumor-specific proteins. Some of these induce a specific immune response by T and B lymphocytes. Antibodies raised against melanoma antigens were proposed for differential disease diagnosis, staging, prognosis and evaluation of treatment efficiency. Nevertheless, treatments based on stimulation of specific anti-melanoma immune responses have had only limited success. It seems that efficient immunotherapy should become more feasible pending on finding new adequate antigens to target. New insights into immune regulation of the tumor microenvironment and its progression may help the development of more successful treatments. We present here up-to-date information on known major melanoma-associated antigens, which could serve as tools for diagnosis as well as for clinical immunotherapy. This approach with promising results for treating some other selected malignancies is still experimental with a very limited success in melanoma. The development of new immune modulators of the tumor microenvironment and neo-antigens may be additional promising directions and may open new opportunities for the immunotherapy of melanoma.