Immunotherapy of HLA-A2 Positive Stage II-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 tumor antigenic peptides and both IMP321/LAG-3 and Montanide adjuvants can induce an immune response in melanoma patients and to assess the safety and tolerability of this vaccination. Tumor responses following this vaccination will also be documented.

Detailed Description
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The primary objective of this study is: to evaluate melanoma antigen specific immune response induced by this vaccination with tumor antigenic peptides derived from MAGE-A3 (Melanoma Antigen family A3) (MHCI: MAGE-A3.A2 and MHCII: MAGE-A3.DP4), NY-ESO-1 (New York Esophageal squamous cell carcinoma antigen-1), Melan A (analog ELA and native EAA) and NA-17A with IMP321 (ImmuFact)/ LAG-3Ig (Lymphocyte activation gene-3 immunoglobulin-like domains) as adjuvant/immunostimulant, formulated with Montanide ISA-51. to assess the safety and tolerability of this vaccination The secondary objective is to document tumor responses in patients following this vaccination.

Official Title
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Vaccination of Melanoma Patients (Stage II-IV) With ImmuFact IMP321, Tumor Antigenic Peptides and Montanide

Conditions
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Melanoma

Intervention / Treatment
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* Biological: 2 vaccine injections in 1 limb
* Biological: 2 vaccine injections in distinct limbs
* Biological: 2 vaccine injections in distinct limbs


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Histologically confirmed stage II, III or IV melanoma patients. Tumor expression of Melan-A. Human leukocyte antigen-A2 (HLA-A2) positive. Expected survival of at list 3 months. Karnofsky scale performance status of 70 % or more. Age ≥ 18 years. Able to give a written informed consent. The following laboratory results: Hemoglobin ≥ 100g/L, Neutrophil count ≥ 1.5 x 109/L, Lymphocyte count ≥ 0.5 x 109/L, Platelet count ≥ 100 x 109/L, Serum creatinine ≤ 2 mg/dL (0.18mmol/L), Serum bilirubin ≤ 2mg/dL (0.034mmol/L), Granulocyte count > 2.5x109/L, Aspartate Amino Transférase (ASAT), Alanine Amino Transferase (ALAT) < 2.5 x upper limit of normal, Activated Partial Thromboplastin Time (aPTT) within the normal ranges ±25%, Thromplastin (TP) ≥ 80% Exclusion Criteria: Clinically significant heart disease. Serious illness, eg. serious infections requiring antibiotics, uncontrolled peptic ulcer, or central nervous system disorders. History of immunodeficiency disease or autoimmune disease. Metastatic disease to the central nervous system, unless treated and stable. Known HIV positivity. Known seropositivity for hepatitis B surface antigen. Concomitant treatment with steroids, antihistamine drugs. Topical or inhalation 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. Kidney dysfunction with creatinine > 2 X the upper limit of the normal value. Reported strong (allergic) reactions to previous vaccination.

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
Interventional Model Description: open label, non comparative study in patients suffering from stage II, III or IV melanoma
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: 2 vaccine injections in 1 limb<br>9 patients initially planned: patients received peptides with IMP321/LAG-3Ig and Montanide in 2 injections sites at 5 cm distance from each other 2 vaccine injections in same limb (vaccine 1 : NY-ESO-1, MAGE-3.A2, NA-17 peptides + IMP321 + Montanide) (vaccine 2 : Melan-A, MAGE-A3-DP4 peptides + IMP321 + Montanide) | Biological: 2 vaccine injections in 1 limb<br>* Participants receive the vaccine separated in 2 syringes with syringe 1 containing NA-17, MAGE-3.A2 and NY-ESO-1 peptides with IMP321/LAG3 ± Montanide, and syringe 2 containing Melan-A and MAGE-A3-DP4 peptides with IMP321/LAG-3 ± Montanide. The content of each syringe is injected s.c. in the same limb at about 5 cm distance from each other.<br>* Other names: vaccine 2: Melan-A, MAGE-A3-DP4 , IMP321, Montanide;|
| Experimental: 2 vaccine injections in distinct limbs<br>Groupe2: 2 vaccine injections in different limb should include 9 patients that were initially planned: patients received the same vaccine in 2 syringes injected each in a distinct limb (vaccine 1: NY-ESO-1, MAGE-3.A2, NA-17 peptides + IMP321 + Montanide) (vaccine 2: Melan-A, MAGE-A3-DP4 peptides + IMP321 + Montanide) | Biological: 2 vaccine injections in distinct limbs<br>* Participants receive the vaccine separated in 2 syringes with syringe 1 containing NA-17, MAGE-3.A2 and NY-ESO-1 peptides with IMP321/LAG3 ± Montanide, and syringe 2 containing Melan-A and MAGE-A3-DP4 peptides with IMP321/LAG-3 ± Montanide.The content of each syringe is injected s.c. in different limbs.<br>* Other names: vaccine 2: Melan-A, MAGE-A3-DP4, IMP321, Montanide);|
| Experimental: 2 vaccine injections in distinct limbs<br>Groupe3: 2 vaccine injections in distinct limb should include 9 patients that were initially planned; due to premature trial termination, no patients could be enrolled. Patients of this group should have received the same vaccine but without the MHC class II peptide (MAGE-A3) | Biological: 2 vaccine injections in distinct limbs<br> <br> * Other names: vaccine 2: Melan-A, NA-17, IMP321, Montanide;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Ex Vivo Frequency of Melan-A Specific CD8+T Cells | Cellular immunity was evaluated through the activation and the expansion of Melan-A-specific CD8+ cytotoxic T lymphocytes. Their frequency was measured in the peripheral blood mononuclear cells (PBMC) directly ex vivo (i.e. without prior in vitro expansion) by multicolor flow cytometry with Melan-A ELA tetramers. 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. | Melan-A specific CD8+ T cells were measured in PBMC collected at the end of Cycle 1 (Week 7), end of Cycle 2 (Week 25), end of Cycle 3 (Week 40), and Follow-up (6 to 18 months after the end of Cycle 3). | 
| In Vitro Frequency of MAGE A3.DP4-specific CD4+ T Cells Producing Interferon-gamma (IFN-γ) | The activation of peptide-specific CD4+ T cells was analyzed in vitro before and after vaccination by Intracellular Cytokine Staining (ICS). From each patient, total CD4+ T-cells were stimulated in the presence of peptide MAGE-A3.DP4 LP (MAGE-A3243-258 peptide presented by autologous cells). After 10 days, cell cultures were challenged for 4h with the peptide or left unchallenged. Specific CD4+ T cells responses were identified via detection of IFN-γ producing cells. | MAGE A3.DP4 specific CD4+ T cells producing IFN-γ were measured in PBMC collected at the end of Cycle 1 (Week 7), end of Cycle 2 (Week 25), end of Cycle 3 (Week 40), and Follow-up (6 to 18 months after the end of Cycle 3). | 
| In Vitro Frequency of MAGE A3.DP4-specific CD4+ T Cells Producing Tumor Necrosis Factor-alpha (TNF-α) | The activation of peptide-specific CD4+ T cells was analyzed in vitro before and after vaccination by ICS. From each patient, total CD4+ T-cells were stimulated in the presence of peptide MAGE-A3.DP4 LP (MAGE-A3243-258 peptide presented by autologous cells). After 10 days, cell cultures were challenged for 4h with the peptide or left unchallenged. Specific CD4+ T cells responses were identified via detection of TNF-α producing cells. | MAGE A3.DP4 specific CD4+ T-cells producing TNF-α were measured in PBMC collected at the end of Cycle 1 (Week 7), end of Cycle 2 (Week 25), end of Cycle 3 (Week 40), and Follow-up (6 to 18 months after the end of Cycle 3). | 
| In Vitro Frequency of MAGE A3.DP4-specific CD4+ T Cells | Frequencies of specific MAGE-A3.DP4-specific CD4+ T cells were quantified by flow cytometry using class II tetramers after 10 days of in vitro stimulation. The fold change for each time point compared to baseline was calculated as: MAGE-A3.DP4-specific CD4+ T cell frequency at the time point/ MAGE-A3.DP4-specific CD4+ T cell frequency at baseline. | MAGE A3.DP4 specific CD4+ T cells were measured in PBMC collected at the end of Cycle 1 (Week 7), end of Cycle 2 (Week 25), end of Cycle 3 (Week 40) and Follow-up (6 to 18 months after the end of Cycle 3). | 
| In Vitro Frequency of Melan-A-specific CD8+T Cells After Stimulation | After 12 days of in vitro stimulation with Melan-A peptides, CD8+ T cells were analyzed by flow cytometry using tetramer staining. 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. | In vitro stimulated Melan-A specific CD8+ T cells were measured in PBMC collected at the end of Cycle 1 (Week 7), end of Cycle 2 (Week 25), end of Cycle 3 (Week 40) and Follow-up (6 to 18 months after the end of Cycle 3). | 
| In Vitro Frequency of NY-ESO-1-specific CD8+ T Cells | After 12 days of in vitro stimulation with NY-ESO-1 peptide, CD8+ T cells were analyzed by flow cytometry using tetramer staining. 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. | In vitro stimulated NY-EYO-1 specific CD8+ T cells were measured in PBMC collected at the end of Cycle 1 (Week 7), end of Cycle 2 (Week 25), end of Cycle 3 (Week 40), and Follow-up (6 to 18 months after the end of Cycle 3). | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Tumor Response | The assessment of the baseline disease status was performed, using CT (Computed Tomography) scan or PET (Positron Emission Tomography)/ CT scan, at screening visit or within 8 weeks preceding the screening visit. Imagery examinations occurred after the end of each vaccination cycle. 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. Progressive 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. | Change from baseline in tumor response at the end of Cycle 1 (Week 7), end of Cycle 2 (Week 25) and end of Cycle 3 (Week 40) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Melanoma, Stage II-IV, Immunotherapy, Vaccination, HLA class I and II tumor-specific peptides, IMP321, Montanide ISA-51