Trial Outcomes & Findings for Immunotherapy of HLA-A2 Positive Stage II-IV Melanoma Patients (NCT NCT01308294)

16 patients were selected between June 15, 2010 and January 24, 2013. Among them 10 patients were included in the group 1 and 6 in the group 2. Since January 2013, 11 patients have been prescreened but could not be included in the study. On April 15, 2014, the decision had to taken to abort the study (16 patients included, while 28 were planned).

An unexplained high percentage, i.e. about 90% of the 11 prescreened patients were HLA-A2 negative. We assume that this was due to the increasing ethnic diversity of patients. The consequence of the time delay was that some of the peptides expired definitively.

Immunotherapy of HLA-A2 Positive Stage II-IV Melanoma Patients

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.

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.

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.

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.

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.

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.

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.