MAGE-A3 Peptide Research Articles

Abstract B123: Local treatment with PeptiCRAd-1, a novel cancer immunotherapy approach, mediates a systemic antitumour CD8+ T-cell response and infiltration of CD8+ and CD4+ T-cells into distant untreated tumors in a clinically relevant humanized mouse melanoma model

Abstract Multiple oncolytic viruses (OV) are currently in clinical development as cancer immunotherapy candidates, due to their high immunogenicity and immune activation capacity. However, only anecdotal clinical evidence for OV-mediated tumor-specific immune responses exists. Peptide vaccines are highly tumor-specific, but the low immunogenicity and lack of CD8+ T-cell responses limit the clinical utility of this class of cancer immunotherapy. We have developed a unique cancer immunotherapy platform, PeptiCRAd™, which utilizes a highly immunogenic genetically engineered oncolytic adenovirus to deliver tumor-specific peptides to the immune system. This creates robust Th1 biased cellular immune responses against multiple tumor-specific targets simultaneously. This study compares the immune activation characteristics of intratumorally administered PeptiCRAd-1 to intratumoral OV treatment and standard intradermal peptide vaccination in a humanized mouse melanoma model. PeptiCRAd™ is an immunotherapy platform where multiple tumor-specific peptides are adsorbed onto the negatively charged capsid of genetically engineered oncolytic adenovirus via electrostatic interactions. Our lead clinical candidate PeptiCRAd-1 is based on a state-of-the-art oncolytic adenovirus coding for human CD40L and OX40L transgenes, coated with poly-lysine extended NY-ESO-1 and MAGE-A3 peptides. NOD/Shi-scid/IL-2Rγnull immunodeficient mice were humanized using hematopoietic stem cells (CD34+, HLA-B35+) isolated from human cord blood. A375 melanoma tumors were implanted subcutaneously and treated either with PeptiCRAd-1 or the naked virus. Peptide vaccines were given intradermally with Poly-IC as an adjuvant. Secondary tumors were implanted into the contralateral flank two days after the treatments were stopped. No treatments for secondary tumors were given. Peripheral blood mononuclear cells (PBMCs) and tumor infiltrating CD8+ lymphocytes (TILs) were analyzed for NY-ESO-1 and MAGE-A3 specific CD8+ T-cells by flow cytometry with dextramer analysis. Different immune cell subsets among PBMCs and TILs were assessed. All active treatments increased the number of immune cells in primary tumors in comparison to mock treated animals. Both OV and PeptiCRAd-1 treated animals showed more T-cells (CD3, CD4, CD8) in primary tumors in comparison to peptide vaccine or mock treated animals post treatment. Furthermore, the number of T regulatory cells (CD3+/CD4+/FoxP3+) was smaller in OV and PeptiCRAd-1 treated primary tumors in comparison to primary tumors from peptide vaccine or mock treated animals. This suggests that intratumorally administered immunogenic adenovirus (either naked virus or PeptiCRAd-1) modulates the tumor microenvironment by reducing local immune-suppression. Unlike OV treated animals, PeptiCRAd-1 treated animals showed more CD4+ and CD8+ T-cells in untreated secondary tumors than in treated primary tumors, suggesting that tumor-targeting via peptide-coating of the virus was critically important for the induction of an effect in distant untreated tumors. Furthermore, PeptiCRAd-1 treated animals had more NYESO-specific CD8+ T-cells in blood post priming (mean= 4.3% of total CD8+ cells) in comparison to OV treated (mean= 0.6%) or peptide vaccine treated (mean= 0.6%) animals. PeptiCRAd™ is superior to naked oncolytic adenovirus or standard peptide vaccination in triggering systemic tumor-targeted CD8+ T-cell responses and infiltration of CD8+ TILs into untreated distant tumors. The data suggest that PeptiCRAd improves the tumor targeting specificity of a standard OV. This easily adaptable technology could potentially be used to improve the tumor-focused immune responses generated by any oncolytic adenovirus currently in development. Importantly, PeptiCRAd™ could potentially be used for personalized tumor vaccination by delivering patient-specific neoantigens. A phase I clinical trial with PeptiCRAd-1 in several solid tumor indications is under preparation. Citation Format: Erkko Ylosmaki, Tuuli Ranki, Petri Priha, Charlotta Backman, Matthew Vaughn, Vincenzo Cerullo, Sari Pesonen. Local treatment with PeptiCRAd-1, a novel cancer immunotherapy approach, mediates a systemic antitumour CD8+ T-cell response and infiltration of CD8+ and CD4+ T-cells into distant untreated tumors in a clinically relevant humanized mouse melanoma model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B123.

Abstract CT003: A phase I study of an HLA-DPB1*0401-restricted T-cell receptor targeting MAGE-A3 for patients with metastatic cancer

Abstract Background: Adoptive transfer of genetically-modified T cells is being explored as a salvage treatment for patients with selected metastatic cancers. Most of the current strategies utilize MHC class I-restricted T cell receptor (TCR) or chimeric antigen receptor (CAR) technologies to genetically modify CD8+ T cells or bulk T cells for patient treatment. Evidence indicates that CD4+ T cells can induce tumor regression, similar to CD8+ T cells. To test this hypothesis, an HLA-DPB1*0401-restricted TCR recognizing MAGE-A3 was isolated from a patient's peripheral blood after MAGE-A3 peptide vaccination. Because HLA-DPB1*0401 is present in ∼60% of the Caucasian population and MAGE-A3 is expressed in up to one third of tumor specimens from a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients. Trial Design: Eligible patients were HLA-DPB1*0401 positive with MAGE-A3 positive tumor specimens, and had not responded or had recurred following at least one standard first line therapy for their disease. Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4+ T cells transduced with the HLA-DPB1*0401-restricted MAGE-A3 TCR plus systemic high-dose interleukin-2 (IL-2). A cell dose-escalation was conducted, treating 1 patient at each cohort (0.01, 0.03, 0.1, up to 30 billion cells), followed by 6 patients at the highest dose level (up to 100 billion cells). Clinical trial information: NCT02111850. Results: Fourteen patients were treated in this phase I study, including the last 6 patients at the highest dose level (78∼100 billion cells). Objective partial responses (RECIST) were observed in a patient with metastatic cervical cancer (ongoing at 11+ months), esophageal cancer (duration 3 months) and urothelial cancer (ongoing at 4+ months). High levels of IL-6 were detected in all patients’ serum samples after adoptive cell transfer. One month after the treatment, TCR-transduced T cells persisted at high levels in the peripheral blood of 6 patients who received the highest dose level, compared to patients who receive lower dose levels (0.01 ∼ 30 billion cells) (p = 0.0082). These results demonstrate the safety of administering autologous CD4+ T cells genetically-engineered to express an MHC class II-restricted anti-tumor TCR targeting MAGE-A3 and presents evidence for efficacy. We have started the phase II clinical trial to study the efficacy of this TCR therapy in different types of metastatic cancer. This clinical trial extends the reach of TCR gene therapy to patients with metastatic cancer. To our knowledge, this clinical trial represents the first genetically-modified CD4+ T cell immunotherapy against metastatic cancer. Citation Format: Yong-Chen Lu, Linda L. Parker, Tangying Lu, Zhili Zheng, Xin Yao, Paul F. Robbins, Steven A. Feldman, Pierre van der Bruggen, Christopher A. Klebanoff, Christian S. Hinrichs, Stephanie L. Goff, Richard M. Sherry, Udai S. Kammula, James C. Yang, Steven A. Rosenberg. A phase I study of an HLA-DPB1*0401-restricted T-cell receptor targeting MAGE-A3 for patients with metastatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT003.

A randomized pilot trial testing the safety and immunologic effects of a MAGE-A3 protein plus AS15 immunostimulant administered into muscle or into dermal/subcutaneous sites.

Methods to induce T cell responses to protein vaccines have not been optimized. The immunostimulant AS15 has been administered with the recombinant MAGE-A3 protein (recMAGE-A3) i.m. but not i.d. or s.c. This study tests hypotheses that the i.d./s.c. route is safe and will increase CD4(+) and CD8(+) T cell responses to MAGE-A3. Twenty-five patients with resected stage IIB-IV MAGE-A3(+) melanoma were randomized to immunization with recMAGE-A3 combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) either i.m. (group A, n=13) or i.d./s.c. (group B, n=12). Adverse events were recorded. Ab responses to MAGE-A3 were measured by ELISA. T cell responses to overlapping MAGE-A3 peptides were assessed in PBMC and a sentinel immunized node (SIN) after 1 in vitro stimulation with recMAGE-A3, by IFN-γ ELISPOT assay and by flow cytometry for multifunctional (TNF-α/IFN-γ) responses. Both routes of immunization were well tolerated without treatment-related grade 3 adverse events. All patients had durable Ab responses. For all 25 patients, the T cell response rate by ELISPOT assay was 30% in SIN (7/23) but only 4% (1/25) in PBMC. By flow cytometry, multifunctional CD8(+) T cell responses were identified in one patient in each group; multifunctional CD4(+) T cell response rates for groups A and B, respectively, were 31 and 64% in SIN and 31 and 50% in PBMC. The MAGE-A3 immunotherapeutic was well tolerated after i.d./s.c. administration, with trends to higher CD4(+) T cell response rates than with i.m. administration. This study supports further study of AS15 by i.d./s.c. administration.

An aptamer targeting shared tumor-specific peptide antigen of MAGE-A3 in multiple cancers.

A DNA aptamer was identified against the shared tumor-specific MAGE-A3111-125 peptide antigen. The dissociation constant between the aptamer and the peptide was measured at 57 nM. Binding of the aptamer to seven types of cancer cells, melanoma, breast, colorectal, liver, lung, pancreas and oral cancer, was confirmed with flow cytometry and fluorescence imaging. Cy3-conjugated aptamers signals were specifically localized to the surface of those cancer cells. The results indicate that the DNA aptamer against the shared tumor-specific MAGE-A3 peptide can be used in cancer cell targeting and has the potential for developing into new modalities for the diagnosis of multiple cancers.

A phase I dose escalation trial of MAGE-A3 and HPV-16 specific peptide immunomodulatory vaccines in patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN).

e17014 Background: New therapeutic modalities are needed for RM-SCCHN. We conducted a phase I dose escalation study to evaluate the safety and immunologic response to peptide immunomodulatory vacci...

Combination Immunotherapy after ASCT for Multiple Myeloma Using MAGE-A3/Poly-ICLC Immunizations Followed by Adoptive Transfer of Vaccine-Primed and Costimulated Autologous T Cells

Myeloma-directed cellular immune responses after autologous stem cell transplantation (ASCT) may reduce relapse rates. We studied whether coinjecting the TLR-3 agonist and vaccine adjuvant Poly-ICLC with a MAGE-A3 peptide vaccine was safe and would elicit a high frequency of vaccine-directed immune responses when combined with vaccine-primed and costimulated autologous T cells. In a phase II clinical trial (NCT01245673), we evaluated the safety and activity of ex vivo expanded autologous T cells primed in vivo using a MAGE-A3 multipeptide vaccine (compound GL-0817) combined with Poly-ICLC (Hiltonol), granulocyte macrophage colony-stimulating factor (GM-CSF) ± montanide. Twenty-seven patients with active and/or high-risk myeloma received autografts followed by anti-CD3/anti-CD28-costimulated autologous T cells, accompanied by MAGE-A3 peptide immunizations before T-cell collection and five times after ASCT. Immune responses to the vaccine were evaluated by cytokine production (all patients), dextramer binding to CD8(+) T cells, and ELISA performed serially after transplant. T-cell infusions were well tolerated, whereas vaccine injection site reactions occurred in >90% of patients. Two of nine patients who received montanide developed sterile abscesses; however, this did not occur in the 18 patients who did not receive montanide. Dextramer staining demonstrated MAGE-A3-specific CD8 T cells in 7 of 8 evaluable HLA-A2(+) patients (88%), whereas vaccine-specific cytokine-producing T cells were generated in 19 of 25 patients (76%). Antibody responses developed in 7 of 9 patients (78%) who received montanide and only weakly in 2 of 18 patients (11%) who did not. The 2-year overall survival was 74% [95% confidence interval (CI), 54%-100%] and 2-year event-free survival was 56% (95% CI, 37%-85%). A high frequency of vaccine-specific T-cell responses were generated after transplant by combining costimulated autologous T cells with a Poly-ICLC/GM-CSF-primed MAGE-A3 vaccine.

Identification of a Titin-Derived HLA-A1–Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3–Directed T Cells

MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

Profound tumor-specific Th2 bias in patients with malignant glioma

BackgroundVaccination against tumor-associated antigens is one promising approach to immunotherapy against malignant gliomas. While previous vaccine efforts have focused exclusively on HLA class I-restricted peptides, class II-restricted peptides are necessary to induce CD4+ helper T cells and sustain effective anti-tumor immunity. In this report we investigated the ability of five candidate peptide epitopes derived from glioma-associated antigens MAGE and IL-13 receptor α2 to detect and characterize CD4+ helper T cell responses in the peripheral blood of patients with malignant gliomas.MethodsPrimary T cell responses were determined by stimulating freshly isolated PBMCs from patients with primary glioblastoma (GBM) (n = 8), recurrent GBM (n = 5), meningioma (n = 7), and healthy controls (n = 6) with each candidate peptide, as well as anti-CD3 monoclonal antibody (mAb) and an immunodominant peptide epitope derived from myelin basic protein (MBP) serving as positive and negative controls, respectively. ELISA was used to measure IFN-γ and IL-5 levels, and the ratio of IFN-γ/IL-5 was used to determine whether the response had a predominant Th1 or Th2 bias.ResultsWe demonstrate that novel HLA Class-II restricted MAGE-A3 and IL-13Rα2 peptides can detect T cell responses in patients with GBMs as well as in healthy subjects. Stimulation with a variety of peptide antigens over-expressed by gliomas is associated with a profound reduction in the IFN-γ/IL-5 ratio in GBM patients relative to healthy subjects. This bias is more pronounced in patients with recurrent GBMs.ConclusionsTherapeutic vaccine strategies to shift tumor antigen-specific T cell response to a more immunostimulatory Th1 bias may be needed for immunotherapeutic trials to be more successful clinically.

MAGE-A3 Specific T-Cell Receptor Adoptive Cell Transfer of Multiple Myeloma

Abstract 1843 Background:Multiple myeloma (MM) is a cancer of plasma cells and the second most common blood cancer. Current treatment strategies such as high dose chemotherapy, autologous stem cell rescue, and allogeneic transplantation have improved response rates and increased survival. However, these treatments often include high procedure-related morbidity and mortality and can only be applied to a small minority of myeloma patients. Therefore, safe broadly applicable immunologic strategies for myeloma, such as Adoptive Cell Therapy (ACT) are urgently needed. Methods:In this study we focused on aHLA-A*0201-restricted cancer testis antigen MAGE-A3:112–120, which is widely expressed in many forms of cancers such as metastatic melanoma, non-small cell lung cancer and MM, but not expressed in most normal tissues. To develop a system of effective strategies for T-cell therapy of multiple myeloma, we employed T-cell engineering technology using a MAGE-A3specific T-cell receptor (TCR)obtained from Dr. Steven Rosenberg at the National Cancer Institute. MAGE-A3 specific TCR was sub-cloned into a lentiviral vector and tranduced into purified CD8+ T-cells from human peripheral blood mononucleocytes (hPBMCs). To test the effector functionality of the MAGE-A3 specific TCR, the MAGE-A3 TCR-transduced CD8+ T-cells were subjected to cytokine release and chromium release assays after being co-cultured with MAGE-A3 peptide-loaded T2 cells, and U266 (MAGE-A3+/HLA-A*0201+), MM1.r (MAGE-A3+/HLA-A*0201-), KAS6 (MAGE-A3-/HLA-A*0201+), and KMS11(MAGE-A3-/HLA-A*0201-) MM tumor cell lines. Results:We observedcytokine production of INF-g and IL-2 in the MAGE-A3 TCR-transduced CD8+ T-cells generally in a dose-dependent manner to the MAGE-A3 peptide-loaded T2 cells. For example, the difference of INF-g secretion bythe MAGE-A3 TCR-transduced CD8+ T-cells wasa 10-fold increase from 0.001 uM to 0.02 uM of the loaded MAGE-A3 peptide. IL-2 secretion was also increasedby 7-fold from 0.001 uM to 0.1 uM of the MAGE-A3 peptide concentration. At 10uM of the peptide concentration, there was a 29-fold increase of the IL-2 production as compared to the 0.001 uM peptide concentration. Between 10uM and 100 uMof the peptide concentration, there was a decrease in IL-2 secretion by 2-fold, which is commonly observed at high peptide concentrations presumably due to cytotoxicity.Specific lysis of tumor cells by the MAGE-A3 TCR-transduced CD8+ T-cellswas observed in all four MM tumor cell lines, and we detected higher percentage of cell lysisin U266 (38%) and MM1.r (51%) cell lines as compared to the KAS6 (11%) and KMS11(21%) cell lines. Conclusions:Our findings suggest that the MAGE-A3 TCR-engineered CD 8+ T-cells are able to specifically recognize MAGE-A3 antigen, produce IL-2 and IFN-g, and destroy MM tumor cells loaded with the MAGE-A3 antigen. This potentially could further translate into effective MAGE-A3 specific targeted tumor rejection in vivo. We also plan to transduce the MAGE-A3 TCR into hematopoietic stem cells to and test the effector function of those cells against MM tumor cells and eventually against MM patient samples. Disclosures:No relevant conflicts of interest to declare.

Dendritic Cells Loaded With mRNA Encoding Full-length Tumor Antigens Prime CD4+ and CD8+ T Cells in Melanoma Patients

It is generally thought that dendritic cells (DCs) loaded with full-length tumor antigen could improve immunotherapy by stimulating broad T-cell responses and by allowing treatment irrespective of the patient's human leukocyte antigen (HLA) type. To investigate this, we determined the specificity of T cells from melanoma patients treated with DCs loaded with mRNA encoding a full-length tumor antigen fused to a signal peptide and an HLA class II sorting signal, allowing presentation in HLA class I and II. In delayed-type hypersensitive (DTH)-biopsies and blood, we found functional CD8(+) and CD4(+) T cells recognizing novel treatment-antigen-derived epitopes, presented by several HLA types. Additionally, we identified a CD8(+) response specific for the signal peptide incorporated to elicit presentation by HLA class II and a CD4(+) response specific for the fusion region of the signal peptide and one of the antigens. This demonstrates that the fusion proteins contain newly created immunogenic sequences and provides evidence that ex vivo-generated mRNA-modified DCs can induce effector CD8(+) and CD4(+) T cells from the naive T-cell repertoire of melanoma patients. Thus, this work provides definitive proof that DCs presenting the full antigenic spectrum of tumor antigens can induce T cells specific for novel epitopes and can be administered to patients irrespective of their HLA type.

Adjuvante systemische Therapie des Melanoms

Despite intensive clinical and research efforts during the last decades the prognosis for patients with stage IV melanoma still remains fatal. An efficient adjuvant treatment for patients with a high risk of relapse and metastases is one of the most urgent fields in clinical research. Systemic adjuvant chemotherapy was not beneficial in terms of relapse-free or overall survival improvement in several clinical trials. Treatment with IFN-α-2a and -2b treatment was the first and as yet only adjuvant therapy which has been proven to show a benefit in controlled studies and to gain approval in Germany in the indications for adjuvant therapy. Current clinical research focuses on improved treatment schedules with conventional interferon compared to pegylated interferon and on the other hand on testing new compounds, such as the CTLA4 inhibitor ipilimumab or a vaccination against the MAGE-A3 peptide.

Aktuelle Aspekte der adjuvanten Therapie des malignen Melanoms

Despite intensive clinical and research efforts during recent decades, the prognosis of patients with stage IV melanoma still remains poor. Finding effective adjuvant treatment for patients with a high risk of relapse and metastasis is one of the most urgent needs in clinical research. Systemic adjuvant chemotherapy administered in several clinical trials offered no benefit in terms of improved relapse-free or overall survival. Interferon alpha-2a and -2b treatment was the first treatment in the adjuvant setting which has shown a treatment benefit and received approval in Germany. Today clinical research focuses on improved treatment schedules with conventional interferon compared to pegylated interferon as well as on new compounds such as CTLA4 inhibitors like ipilimumab or a vaccination against the MAGE-A3 peptide.

Effect of tumor-associated antigens on specific T-cell responses in patients with metastatic lung cancer and potential use as immunogenic targets for a (polyvalent) vaccination strategy.

e13064 Background: In spite of many attempts to improve overall survival and to prolong the relapse-free survival in advanced and metastatic lung cancer, therapeutic approaches including chemotherapy and radiation have reached a plateau of efficacy. Therefore, new strategies of tumor therapy have been investigated like immunotherapeutic approaches. Tumor cells can be efficiently killed by specific T-cells of the immune system. Methods: We investigated the frequency of specific T-cell responses of CD8-positive T cells against several known but also newly characterized HLA-A2-restricted epitopes derived from the tumor-associated antigens MAGE-A3, RHAMM, hTERT, Survivin, WT-1, PRAME, HER2/neu, and G250 in patients with metastatic lung cancer. For new epitopes HLA-A2-binding motifs were predicted using computer analysis based on known binding affinities. More than 10 peptides with most favorable characteristics were evaluated in ELISPOT assays for interferon gamma and granzyme B secretion by CD8 positive T cells in patients with metastatic lung cancer. Results: Specific T-cell responses could be detected against at least one peptide in all patients with non-small cell lung cancer before treatment with chemotherapy. Most frequent responses could be detected (50% or more of patients) against a peptide derived from hTERT and PRAME. Lower frequency of specific T-cell responses was found for Survivin- (40%), G250- (33%), and MageA3-derived peptides (20%), no responses against RHAMM-, HER2neu-, and WT1-derived peptides. Conclusions: Taken together, specific T- cell responses against several TAAs could be detected for the antigens hTERT and PRAME in 50% or more of patients with lung cancer, but also in a lower frequency against MAGEA3, G250 and Survivin peptides. Therefore, immunotherapeutic approaches targeting these antigen structures is a new therapeutic option for these patients in advanced lung cancer. No significant financial relationships to disclose.

In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Abstract 4079Poster Board III-1014MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures:No relevant conflicts of interest to declare.

The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti-MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 x 10(-6) to 2 x 10(-3) among the CD4(+) blood T lymphocytes of the patients. The CD4(+) blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4(+) T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25(+) phenotype in the resting state. These CD25(+) clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-beta but none of cytokines IFN-gamma, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25(-) clones had a significant but lower suppressive activity. Most of the CD25(-) clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4(+) T cells that may exert regulatory T-cell function in vivo.

Functions of Anti-MAGE T-Cells Induced in Melanoma Patients under Different Vaccination Modalities

Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti-MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least in some patients, surprisingly few anti-MAGE-3.A1 T-cells could initiate a tumor regression process. To understand the role of these T cells, we carried out a functional analysis of anti-MAGE-3.A1 CTL clones derived from vaccinated patients who displayed tumor regression. The functional avidities of these CTL clones, evaluated in lysis assays, were surprisingly low, suggesting that high avidity was not part of the putative capability of these CTL to trigger tumor rejection. Most anti-MAGE-3.A1 CTL clones obtained after DC vaccination, but not after peptide or ALVAC vaccination, produced interleukin 10. Transcript profiling confirmed these results and indicated that approximately 20 genes, including CD40L, prostaglandin D2 synthase, granzyme K, and granzyme H, were highly differentially expressed between the anti-MAGE-3.A1 CTL clones derived from patients vaccinated with either peptide-ALVAC or peptide-pulsed DC. These results indicate that the modality of vaccination with a tumor-specific antigen influences the differentiation pathway of the antivaccine CD8 T-cells, which may have an effect on their capacity to trigger a tumor rejection response.

Marked clinical and immunological response of MAGE-A3 peptides vaccination in a patient with adenocarcinoma of the lung case report: A case report

A 56-year patient was diagnosed adenocarcinoma of the right lung and underwent lower lobectomy. Pleural dissemination was observed, and the pathological stage was T4N0M0. Systemic chemotherapy was performed with paclitaxel and vinorelbine after the surgery. Because multiple pulmonary and liver metastases were appeared 1 year after the surgery, the second line chemotherapy (CDDP and 5FU) was performed. The response of the chemotherapy was not observed, and thus the patient was enrolled in the clinical trial of MAGE-A3 peptide vaccine with OK-432. The peptide (300 mg) was injected subcutaneously at the upper thigh or upper arm a total of 6 times over 2 months (days 1, 8, 15, 22, 36, and 50) with OK432. During two courses of the vaccinations, the tumor markers (CA19-9 and CEA) decreased markedly. Furthermore, the tumor size of the lung and liver metastases decreased to 30% of their pre-treatment size. The delayed type hypersensitive reaction became positive after the first course of vaccinations. The precursor CTL for the peptide in the peripheral blood had increased after the vaccination. MAGE-A3 peptide vaccination with OK-432 has a potential to induce immunological response and clinical response in advanced lung cancer patients.

Identification of a naturally presented MAGE-A3 epitope on the surface of HLE cell line by mass spectrometry

To identify a naturally presented HLA-A2-restricted epitope of MAGE-A3 antigen, FLWGPRALV (MAGE-A3(271 - 279)), on the surface of a human hepatocellular carcinoma (HCC) cell line HLE. Synthetic peptide FLWGPRALV, served as positive control target, was analyzed by HPLC and HPLC-ESI-TOF-MSMS, in order to determine its HPLC elution time, mass-spectrometric characteristics and the lowest detection limitation by the two approaches. 3 x 10(9) HLE cells were collected, peptides naturally presented by major histocompatibility complex (MHC) molecules on the cell surface were isolated by mild acid elution, and concentrated by lyophilization, then the mixtures of peptides were fractioned by HPLC. The ingredient ranged from 2 min before the elution time determined by the synthetic peptide to 2 min after that was collected, concentrated by lyophilization, and analyzed by HPLC-ESI-TOF-MSMS, to identify the existence of the MAGE-A3(271 - 279) peptide. The HPLC-ESI-TOF-MSMS detection provided an evidence for the existence of a doubly charged ion of (m/z)(2) 529.9, which was further analyzed by collision induced dissociation. The doubly charged ion was ultimately identified as the MAGE-A3(271 - 279) peptide, its amino sequence was FLWGPRALV and its molecular weight was 1058.4 Da. MAGE-A3(271 - 279) epitope could be naturally presented by HLA-A2 molecules to the surface of HCC cell line and MAGE-A3(271 - 279) peptide may have potential immunotherapeutic value in HCC patients.

Lysis of human chondrosarcoma cells by cytolytic T lymphocytes recognizing a MAGE‐A3 antigen presented by HLA‐A1 molecules

Treatment of chondrosarcomas is limited to resection because these tumors are unresponsive to standard adjuvant treatments, such as chemotherapy and radiation. We have previously shown that high-grade chondrosarcomas express unspecified members of the Melanoma Antigen (MAGE) gene family. We show here that FS human chondrosarcoma (FS) cells express MAGE-A3 gene and HLA-A1 molecules. In vitro assays show that a cytolytic T-lymphocyte clone (CTL) specific for a MAGE-A3 peptide presented by HLA-A1 specifically lysed FS chondrosarcoma cells. Addition of antigenic peptide did not increase the susceptibility of FS cells to CTL mediated lysis, suggesting that HLA-A1 expression by the chondrosarcoma cells limited their susceptibility to lysis by the anti-MAGE-A3 CTL clone. Incubation of FS cells with 50 U/mL interferon-gamma increased surface expression of HLA class-I molecules, increased their susceptibility to lysis, and had no effect on MAGE-A3 gene expression. These results suggest that immunotherapy targeted against chondrosarcoma cells is possible.

Cancer/testis antigen expression and specific cytotoxic T lymphocyte responses in non small cell lung cancer

Non small cell lung cancers (NSCLC) express cancer/testis antigens (CTA) genes and MAGE-A expression correlates with poor prognosis in squamous cell carcinomas. We addressed cytotoxic T lymphocytes (CTL) responses to HLA class I restricted CTA epitopes in TIL from NSCLC in an unselected group of 33 patients consecutively undergoing surgery. Expression of MAGE-A1, -A2, -A3, -A4, -A10, -A12 and NY-ESO-1 CTA genes was tested by quantitative RT-PCR. Monoclonal antibodies (MAb) recognizing MAGE-A and NY-ESO-1 CTA were used to detect CTA by immunohistochemistry. CD8(+) TIL obtained from tumors upon culture with anti CD3 and anti CD28 mAb and IL-2 were stimulated with autologous mature DC (mDC) and HLA-A*0101 restricted MAGE-A1(161-169) or MAGE-A3(168-176) peptides or HLA-A*0201 restricted MAGE-A4(230-239), MAGE-A10(254-262), NY-ESO-1(157-165) or multi-MAGE-A (YLEYRQVPV) peptides or a recombinant vaccinia virus (rVV) encoding MAGE-A and NY-ESO-1 HLA-A*0201 restricted epitopes and CD80 co-stimulatory molecule. Specificity was assessed by (51)Cr release and multimer staining. At least one CTA gene was expressed in tumors from 15/33 patients. In 10 specimens, at least 4 CTA genes were concomitantly expressed. These data were largely confirmed by immunohistochemistry. TIL were expanded from 26/33 specimens and CTA-specific CTL activity was detectable in 7/26 TIL. In 6, however, specific cytotoxicity was weak, (<40% lysis at a 50:1 E:T ratio) and multimer staining was undetectable. In one case, high (>60% lysis at 50:1 E:T ratio) MAGE-A10(254-262) specific, HLA-A*0201 restricted response was observed. Supportive evidence was provided by corresponding multimer staining. Although CTA genes are frequently expressed in NSCLC, detection of CTL reactivity against CTA epitopes in TIL from nonimmunized NSCLC patients represents a rare event.