Immunotherapy of Stage III/IV Melanoma Patients

Study Overview
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Brief Summary
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The purpose of this study is to determine whether vaccination with melanoma antigen peptides [Melan-A/Mart-1 (both EAA and ELA), NY-ESO-1b analog, Long NY-ESO-1 LP and MAGE-A10] and Montanide, CpG adjuvants and low dose rIL-2 can induce an immune response in melanoma patients and to assess the safety of this vaccination.

Detailed Description
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Current peptide vaccines suffer from low efficiency, since they induce only weak immune activation. We have recently confirmed that in humans the immune response was readily detectable in local lymph nodes while no or only weak activation could be identified in circulating lymphocytes. Increased doses of antigen and adjuvant allow a better extension from local to systemic immune responses. Group 1 : vaccination with Melan-A analog (ELA) peptide + Montanide Group 2 : vaccination with Melan-A analog (ELA), NY-ESO-1b analog and MAGE-A10 peptides + Montanide Group 3: vaccination with Melan-A analog (both EAA and ELA), Mage-A10, NY-ESO-1 peptides+ Montanide + CpG adjuvant Group 4: vaccination with Melan-A (ELA), Mage-A10,long NY-ESO-1LP peptides + Montanide + CpG Group 5: vaccination with Melan-A (both EAA and ELA), Mage-A10, long NY-ESO-1 LP peptides + Montanide + CpG + low dose rIL-2

Official Title
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Vaccination of Patients With Stage III or IV Malignant Melanoma With Melanoma Antigen Peptides [Melan-A/Mart-1 Analog (ELA), NY-ESO-1b(A) Analog and MAGE-A10] and Montanide Adjuvant

Conditions
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Melanoma

Intervention / Treatment
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* Biological: Melan-A ELA + Montanide
* Biological: Melan-A ELA + NY-ESO-1b + MAGE-A10 + Montanide
* Biological: Melan-A -ELA + NY-ESO-1b + MAGE-A10 peptide + Montanide + CpG
* Biological: Melan-A-EAA/ELA + NY-ESO-1 lp + MAGE-A10 + Montanide + CpG
* Biological: Melan-A-EAA/ELA + NY-ESO-1 lp + MAGE-A10 + Montanide + CpG+ IL-2


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Histologically confirmed stage III or stage IV melanoma with at least one metastatic lymph node and/or at least one in-transit metastasis. According to the AJCC rules, this includes all patients with stage IV and stage III. Patients with or without measurable disease may be included. Tumor expression of Melan-A by reverse transcriptase and polymerase chain reaction (RT-PCR) analysis for patients of group I. Tumor expression of Melan-A and at least one of the tumor antigens MAGE-A10, NY-ESO-1, or LAGE-1 by rt-PCR analysis for patients of group II and III and for HLA-A2+ patients of groups IV and V. HLA-A2 negative patients of groups IV and V must only have NY-ESO-1 positive tumors to be eligible, while expression of Melan-A and MAGE-A10 is unimportant. If no frozen tissue is available, immunohistochemistry may be performed to detect tumor expression of Melan-A and NY-ESO-1. HLA-A2 positive (serological or molecular typing of Peripheral Blood Lymphocytes (PBL) for patients of groups 1 to 3. Patients of groups 4 and 5 may either be HLA-A2+ or HLA-A2-. Expected survival of at least five months. Full recovery from surgery. Karnofsky scale performance status of 70% or more. The following laboratory results: Neutrophil count sup or equal 2.0 x 10^9/L Lymphocyte count sup or equal 0.5 x 10^9/L Platelet count sup or equal 100 x 10^9/L Creatinine ≤ 2 mg/dL (180 micromol/L) Bilirubin ≤ 2mg/dL (34 micromol/L) Granulocyte count > 2.5x10^9/L AST < 2x upper limit of normal aPTT: within the normal ranges of the laboratory ± 25 % Age > 18 years. Able to give written informed consent. Exclusion Criteria: Clinically significant heart disease (NYHA Class III or IV). Other serious illnesses, e.g., serious infections requiring antibiotics, uncontrolled peptic ulcer, or central nervous system disorders with major dysfunction. History of immunodeficiency disease or autoimmune disease. Known HIV positivity. Known seropositivity for hepatitis B surface antigen. Chemotherapy, radiation therapy, or immunotherapy within 4 weeks before study entry (6 weeks for nitrosoureas). Concomitant treatment with steroids, antihistamine drugs. Topical or inhalational steroids are permitted. Participation in any other clinical trial involving another investigational agent within 4 weeks prior to enrollment. Pregnancy or lactation. Women of childbearing potential not using a medically acceptable means of contraception. Psychiatric or addictive disorders that may compromise the ability to give informed consent. Lack of availability of the patient for immunological and clinical follow-up assessment. Coagulation or bleeding disorders. Metastatic disease to the central nervous system, unless treated and stable.

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: 1. Melan-A ELA<br>500 mcg Melan-A ELA analog peptide + 1 ml Montanide ISA-51 | Biological: Melan-A ELA + Montanide<br>* A maximum of 3 vaccination cycles (cycles 1-3) has been given, each cycle consisting of 4 vaccines in 4 week intervals. The intervals between cycles were 8 weeks. After 3 cycles, patients without major tumor progression requiring other treatment who showed an immunological response received booster vaccinations every 3 months.<br>|
| Experimental: 2. Melan-A ELA + NY-ESO-1b + MAGE-A10<br>500 mcg Melan-A ELA analog peptide + 500 mcg NY-ESO-1b(A) analog peptide + 500 mcg MAGE-A10 peptide + 1 ml Montanide ISA-51 | Biological: Melan-A ELA + NY-ESO-1b + MAGE-A10 + Montanide<br>* A maximum of 3 vaccination cycles (cycles 1-3) has been given, each cycle consisting of 4 vaccines in 4 week intervals. The intervals between cycles were 8 weeks. After 3 cycles, patients without major tumor progression requiring other treatment who showed an immunological response received booster vaccinations every 3 months.<br>|
| Experimental: 3. Melan-A ELA + NY-ESO-1b + MAGE-A10 + CpG<br>500 mcg Melan-A ELA analog peptide + 500 mcg NY-ESO-1b(A) analog peptide + 500 mcg MAGE-A10 peptide + 1 ml Montanide ISA-51 + 2.5 mg CpG-7909/PF-3512676 | Biological: Melan-A -ELA + NY-ESO-1b + MAGE-A10 peptide + Montanide + CpG<br>* A maximum of 3 vaccination cycles (cycles 1-3) has been given, each cycle consisting of 4 vaccines in 4 week intervals. The intervals between cycles were 8 weeks. After 3 cycles, patients without major tumor progression requiring other treatment who showed an immunological response received booster vaccinations every 3 months.<br>|
| Experimental: 4. Melan-A EAA/ELA + NY-ESO-1lp + MAGE-A10+ CpG<br>If patient is HLA-A2 positive: 100 mcg Melan-A EAA native peptide (during first cycle) or 100 mcg ELA analog peptide (during other cycles) + 500 mcg NY-ESO-1lp long peptide + 100 mcg MAGE-A10 peptide + 1 ml Montanide ISA-51 (no Montanide during cycle 3) + 2.5 mg CpG-7909/PF-3512676 If patient is HLA-A2 negative: 500 mcg NY-ESO-1lp long peptide+ 1 ml Montanide ISA-51 (no Montanide during cycle 3) + 2.5 mg CpG-7909/PF-3512676 | Biological: Melan-A-EAA/ELA + NY-ESO-1 lp + MAGE-A10 + Montanide + CpG<br>* A maximum of 3 vaccination cycles (cycles 1-3) has been given, each cycle consisting of 4 vaccines in 4 week intervals. The intervals between cycles were 8 weeks. After 3 cycles, patients without major tumor progression requiring other treatment who showed an immunological response received booster vaccinations every 3 months.<br>|
| Experimental: 5. Melan-A EAA/ELA + NY-ESO-1lp + MAGE-A10+ CpG+ IL-2<br>If patient is HLA-A2 positive: 100 mcg Melan-A EAA native peptide (during first cycle) or 100 mcg ELA analog peptide (during other cycles) + 500 mcg NY-ESO-1lp long peptide + 100 mcg MAGE-A10 peptide + 1 ml Montanide ISA-51 (no Montanide during cycle 3) + 2.5 mg CpG-7909/PF-3512676 + low dose IL-2 If patient is HLA-A2 negative: 500 mcg NY-ESO-1lp long peptide+ 1 ml Montanide ISA-51 (no Montanide during cycle 3) + 2.5 mg CpG-7909/PF-3512676 + low dose IL-2 | Biological: Melan-A-EAA/ELA + NY-ESO-1 lp + MAGE-A10 + Montanide + CpG+ IL-2<br>* A maximum of 3 vaccination cycles (cycles 1-3) has been given, each cycle consisting of 4 vaccines in 4 week intervals. The intervals between cycles were 8 weeks. After 3 cycles, patients without major tumor progression requiring other treatment who showed an immunological response received booster vaccinations every 3 months.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change From Baseline in Mean Number of Adverse Events (Serious and Non Serious Events) | Safety of the vaccination was assessed according to the National Cancer Institute Common Toxicity Criteria (NCI CTC) scale. The adverse events (AE) and serious adverse events (SAE) were registered at each study visit during the 3 vaccination cycles and boost cycles. | Change from baseline to end of Cycle 1 (3 months), end of Cycle 2 (8 months), end of Cycle 3 (13 months) and end of Boost Cycles (18 months to 23 months). | 
| Fold Change From Baseline in ex Vivo Melan-A-specific CD8+ T Cells Frequency During the Vaccination Period | Ex vivo frequency of Melan-A-specific CD8+ T cells was measured by multimer technique (tetramer assay) in a multicolor flow cytometry analysis. The fold change for each time point compared to baseline was calculated as: Melan-A-specific CD8+ T cell frequency at the time point/ Melan-A-specific CD8+ T cell frequency at baseline. Significant T cell response is defined by at least 2-fold change of Melan-A-specific CD8+ T cell frequency as compared to pre-immunotherapy. | Fold change from baseline in Melan-A-specific CD8+T-cells at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months). | 
| Fold Change From Baseline in ex Vivo Frequency of Melan-A-specific IFN-γ-secreting CD8+ T Cells During the Vaccination Period | Ex vivo frequency of Melan-A-specific CD8+ T cells producing IFN-γ (Interferon-gamma) was measured through the Enzyme-Linked Immunosorbent Spot (ELISpot) assay. The fold change for each time point compared to baseline was calculated as: Melan-A-specific IFN-γ-secreting CD8+ T cell frequency at the time point/ Melan-A-specific IFN-γ-secreting CD8+ T cell frequency at baseline. | Fold change from baseline in Melan-A-specific IFN-γ-secreting CD8+T-cells frequency at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months) | 
| Fold Change From Baseline in ex Vivo Frequency of NY-ESO-1-specific CD8+ T Cells During the Vaccination Period | Ex vivo frequency of NY-ESO-1-specific CD8+ T cells was measured by multimer technique (tetramer assay) in a multicolor flow cytometry analysis. The fold change for each time point compared to baseline was calculated as: NY-ESO-1-specific CD8+ T cell frequency at the time point/ NY-ESO-1-specific CD8+ T cell frequency at baseline. | Fold change from baseline in NY-ESO-1-specific CD8+T-cells at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months) | 
| Fold Change From Baseline in ex Vivo Frequency of NY-ESO-1-specific IFN-γ-secreting CD8+ T Cells During the Vaccination Period | Ex vivo frequency of NY-ESO-1-specific CD8+ T cells producing IFN-γ (Interferon-gamma) was measured through the Enzyme-Linked Immunosorbent Spot (ELISpot) assay. The fold change for each time point compared to baseline was calculated as: NY-ESO-1-specific IFN-γ-secreting CD8+ T cell frequency at the time point/ NY-ESO-1-specific IFN-γ-secreting CD8+ T cell frequency at baseline. | Fold change from baseline in NY-ESO-1-specific IFN-γ-secreting CD8+T-cells frequency at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months) | 
| Fold Change From Baseline in ex Vivo Frequency of MAGE-A10-specific CD8+ T Cells During the Vaccination Period | Ex vivo frequency of MAGE-A10-specific CD8+ T cells was measured by multimer technique (tetramer assay) in a multicolor flow cytometry analysis. The fold change for each time point compared to baseline was calculated as: MAGE-A10-specific CD8+ T cell frequency at the time point/ MAGE-A10-specific CD8+ T cell frequency at baseline. | Fold change from baseline in MAGE-A10-specific CD8+T-cells at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months) | 
| Fold Change From Baseline in ex Vivo Frequency of MAGE-A10-specific IFN-γ-secreting CD8+ T Cells During the Vaccination Period | Ex vivo frequency of MAGE-A10-specific CD8+ T cells producing IFN-γ (Interferon-gamma) was measured through the Enzyme-Linked Immunosorbent Spot (ELISpot) assay. The fold change for each time point compared to baseline was calculated as: MAGE-A10-specific IFN-γ-secreting CD8+ T cell frequency at the time point/ MAGE-A10-specific IFN-γ-secreting CD8+ T cell frequency at baseline. | Fold change from baseline in MAGE-A10-specific IFN-γ-secreting CD8+T-cells frequency at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months) | 
| Percentage of in Vitro Stimulated NY-ESO-1 Lp-specific IFN-γ/TNF-α -Secreting CD4+ T Cells During the Vaccination Period | For each patient, total CD4+ T-cells were stimulated in the presence of peptide NY-ESO-1 long peptide (lp). After 10 days, cell cultures were challenged for 4h with the peptide or left unchallenged. The activation of NY-ESO-1 long peptide (lp)-specific CD4+ T cells were analyzed in vitro by Intracellular Cytokine Staining (ICS) via detection of IFN-γ (Interferon-gamma) and TNF-α (Tumor Necrosis Factor-alpha) producing cells. | Percentage of NY-ESO-1 lp-specific IFN-γ/TNF-α -secreting CD4+ T-cells at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months) | 
| Percentage of in Vitro Stimulated NY-ESO-1 Lp-specific IFN-γ/TNFα -Secreting CD8+ T Cells During the Vaccination Period | For each patient, total CD8+ T cells were stimulated in the presence of peptide NY-ESO-1 long peptide (lp). After 10 days, cell cultures were challenged for 4h with the peptide or left unchallenged. The activation of NY-ESO-1 long peptide (lp)-specific CD8+ T cells were analyzed in vitro by Intracellular Cytokine Staining (ICS) via detection of IFN-γ (Interferon-gamma) and TNF-α (Tumor Necrosis Factor-alpha) producing cells. | Percentage of NY-ESO-1 lp-specific IFN-γ/TNF-α -secreting CD8+ T cells at the end of Cycle 1 (3 months), at the end of Cycle 2 (8 months), at the end of Cycle 3 (13 months) and if applicable at the end of Boost cycles (18 to 24 months) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Disease Status Assessment During the Vaccination Period | The disease status was assessed by computed tomography (CT) or positron emission tomography (PET)/CT at baseline and after the fourth vaccination of each cycle. During the booster vaccines period, imagery examinations were performed every 3 months for patients with measurable disease and every 6 months for patients with non measurable disease. The tumor response was assessed according to the classification World Health Organization (WHO) 1979 and defined as: No Evidence of Disease (NED) Stable disease (SD): Change in size of all measurable lesions (the sum of the products of the greatest and perpendicular parameters), of less than a 25% increase or 25% decrease from baseline for at least 4 weeks, without appearance of new lesions or progression of any lesion. Progressing disease (PD): Appearance of new tumors, or increase in size of any measurable tumor by at least 25% of the sum of the product of the greatest and perpendicular diameter. | Disease status at baseline, after cycle 1 (3 months), after cycle 2 (8 months), after cycle 3 (13 months) and if applicable after boost cycles (16 months, 19 months or 22 months) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Immunotherapy, Vaccination, Melanoma, Melan-A/Mart-1 peptide, MAGE-A10 peptide, NY-ESO-1 peptide, Montanide, CpG