This clinical trial was approved by the University of Louisville Human Studies Committee. Only patients with distant metastases from cutaneous or mucosal melanoma or melanoma of unknown primary were eligible for inclusion. All patients fulfilled the following criteria: (i) primary tumor must have been documented by histopathologic analysis; (ii) metastatic disease must have been documented by radiologic examinations (CT scan or PET scan) with bidimensional measurements; and (iii) disease recurrences occurring greater than five years after the original diagnosis must have been biopsy proven.
All patients were subjected to fusion PET/CT or CT imaging within one month prior to receiving the first dose of DAB/IL2 and within one month after receiving the last dose of DAB/IL2. DAB/IL2 was administered as follows: 12 μg/kg, IV over 30 minutes every 24 hours for 4 doses (cycles repeated every 21 days). All patients had renal function tests, blood counts, and a thorough physical examination, including neurological examination, prior to each cycle of DAB/IL2. The endpoint definitions were as follows:
Clinical complete response (CR)
Disappearance of all evidence of tumor. The patient must be free of all symptoms of cancer.
Partial response (PR)
30% or greater decrease in the sum of the longest diameter of target lesions, taking as reference the baseline sum longest diameter.
Progressive disease (PD)
At least 20% increase in the sum of the longest diameter of target lesions, taking as reference the baseline sum longest diameter, or the appearance of new lesions and/or unequivocal progression of existing non-target lesion.
Stable disease (SD)
Neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum longest diameter since the treatment started.
Monocyte, granulocyte, lymphocyte and T cell subset quantification
Whole blood (50 ml) was collected in heparinized tubes and the absolute lymphocyte, granulocyte and monocyte peripheral blood concentrations were determined with a Sysmex XE-2100 Automated Hematology Analyzer. PBMCs were then isolated by centrifugation through Accuspin System Histopaque 1077 and washed twice with PBS.
In order to determine the percentage of CD4+, CD4+/CD25-, CD4+/CD25+, CD4+/CD25HI, CD4+/CD25HI/Foxp3-, and CD4+/CD25HI/Foxp3+ T cells within the lymphocyte gate (based on forward/side scatter profile), we incubated the total PBMCs with PE-anti-Foxp3, FITC-anti-CD4, and APC-anti-CD25 (eBioscience). 100 μl of PBMCs (1 × 106) were added to 20 μl of an anti-CD4/and-CD25 cocktail (1 μg anti-CD4 and 0.125 μg anti-CD25; eBioscience) and incubated for 30 minutes in the dark at 4°C and then washed in cold PBS. After decanting, the cell pellet was resuspended in residual buffer and 1 ml of freshly prepared eBioscience Fixation/Permeabilization Buffer was added to each sample and incubated at 4°C for 60 minutes in the dark. 2 ml of Permeabilization Buffer was used for washing followed by centrifugation and decanting of supernatant. 20 μl anti-human Foxp3 (PCH101) antibody or 20 μl rat IgG2b isotype control was added to resuspended cells and incubated at 4°C for 30 minutes in the dark. Cells were washed twice in 2 ml Permeabilization Buffer. Small lymphocytes were gated according to forward/side-scatter profiles and data was collected on a FACSCalibur flow cytometer within 1 hour after staining, and then analyzed with Cell Quest software (Becton Dickinson).
In order to detect the percentage of total CD8+ cells, and MART1-, gp100- and tyrosinase-specific CD8+ T cells within the lymphocyte gate (based on forward/side scatter profile), 106 PBMCs in 200 μl of flow cytometry staining buffer were incubated at 25°C for 30 minutes in the dark with 1.0 μg of APC-labeled tetramer (MART-1, gp100 or tyrosinase; Immunomics, Beckman Coulter) and 0.25 μg CD8-PE monoclonal antibody (R&D Systems). Small lymphocytes were gated according to forward/side-scatter profiles and then the percentage of tetramer+CD8+ cells was determined. Data was collected on a FACSCalibur flow cytometer within 1 hour after staining, and analyzed with Cell Quest software (Becton Dickinson).
The absolute concentrations of CD4+, CD4+/CD25-, CD4+/CD25+, CD4+/CD25HI, CD4+/CD25HI/Foxp3-, CD4+/CD25HI/Foxp3+, CD8+, CD8+/HLA-A2*0201-MART1-binding, CD8+/HLA-A2*0201-gp100-binding and CD8+/HLA-A2*0201-tyrosinase-binding cells were quantified by determining the percentage of fluorescence-positive cells within the forward/side scatter lymphocyte gate (as detailed above), and then multiplying this percentage by the absolute lymphocyte concentration determined using the Sysmex XE-2100 Automated Hematology Analyzer. The percent control of each sample was calculated by dividing the T cell subset absolute cell concentration on the indicated day of treatment with the cell concentration on day 0 prior to DAB/IL2 administration (× 100).
DAB/IL2 enzyme linked immunosorbent assay
Human plasma samples were tested for the presence of IgG specific for DAB/IL2 by enzyme linked immunosorbent assay (ELISA). The assay was carried out as follows: 96-well microtest polystyrene assay plates (BD) were coated (100 μL/well) with either Tris-NaCl pH 8.5 solution (30 mL 5 M NaCl + 50 mL 1 M Tris + 920 mL water) or DAB/IL2 (Ligand) diluted to 2 μg/mL in Tris-NaCl solution. After incubating overnight at 37°C, the plates were washed two times with Tris-NaCl solution. 300 μl PBS/BSA (30 mL PBS + 300 mg BSA; Sigma) was then added to each well and the plates were incubated for one hour at 37°C, followed by three washes with Tris-NaCl solution. 100 μl of test sera, diluted 1:500 in PBS/BSA solution, was then added to each well. After incubating at 37°C for two hours, the plates were washed three times with Tris-NaCl + 0.05% Tween (300 mL Tris-NaCl + 150 μl Tween). 100 μl of rabbit anti-human IgG HRP-conjugated antibody (Pierce), diluted 1:50,000 in PBS/BSA, was then added to each well and the plates incubated at 37°C for one hour, followed by three washes with Tris-NaCl + 0.05% Tween and two washes with DH2O. 100 μl of TMB substrate (Pierce) was added to each well. After five minutes, the reaction was stopped with 1N HCL (100 μl/well) and the plates were read at 450 nm.
Five μm sections of formalin-fixed and paraffin-embedded tumor tissue were mounted on charged glass slides and dried at 58°C for 60 minutes. Slides were first deparaffinized with xylene then incubated with a high temperature epitope retrieval solution (20 min) and hydrogen peroxide (H2O2) (for 10 min) to block endogenous peroxidases. The sections were incubated with primary antibody (anti-CD8, 1:50, Dako; anti-CD4, 1:50, Novocastra; anti-HLA Class I [HLA-A, B, C], 1:500, clone EMR8-5, MBL International) for 15 min, followed by a post-primary antibody and a polymer horse-radish-peroxidase linked detection system (each for 8 min, Define, Leica Microsystems). The sections were developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution (Invitrogen) for 10 min and nuclei counterstained with hematoxylin (Dako) for 7 min. PBS washes were performed between all steps. The slides were neutralized in ammonia water, dehydrated in graded alcohols (100%, 95%, and 80% ethanol [vol/vol] in H2O), cleared in xylene and coverslips attached with Permount (Fisher Scientific).
For MART-1 staining, slides were deparaffinized (with xylene), hydrated with distilled water and then placed in citrate buffer (Dako) in a 72°C oven overnight for antigen retrieval. Following treatment with H2O2, slides were incubated in MART-1 primary antibody (1:40, Signet) for 25 min then in LSAB2 biotinylated link antibody (Dako) for 20 min followed by a streptavidin-peroxidase reaction using DAB as a chromogen. Slides were finally counterstained in hematoxylin and then neutralized, dehydrated and coverslips attached as above. Double staining was accomplished by first staining for CD8 (as above) using DAB as the chromogen followed by washing and staining for MART-1 using the alkaline phosphatase system (Leica) omitting the deparaffinization and retrieval steps. Brown staining from the DAB indicated CD8+ T cells and red staining from the alkaline phosphatase indicated MART1+ cells. Both positive and negative controls were stained with the specimens.
CRL-11174 human melanoma cells (ATCC) were cultured in 1 ml of Dulbecco's Modified Eagle Medium (DMEM) (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) and 50 μg/mL gentamicin sulfate (Invitrogen, Carlsbad, CA) (2.5 × 105 cells/well, 6-well plate). DAB/IL2 (Ligand Pharmaceuticals) or PBS was added to the culture (0.05–5 μg/ml) and, after 48 hours, live and dead cells were enumerated by the addition of trypan blue and direct visualization using light microscopy.