Background: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study to evaluate safety and immune responses, with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic melanoma cell lines (Apo-Nec).

Methods: PBMC were obtained from leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs (iDCs) were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines with 5, 10, 15, or 20 x106 DC/Apo-Nec per vaccine, two weeks apart.

Results: The vaccine was well tolerated in all patients. Toxicity to vaccine was mild, and the toxicity-limiting dose has not been reached. We found that 42.3 ±13.7 % melanoma patients´ iDCs were able to phagocyte Apo-Nec cells wich induced DCs maturation, as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II compared to iDCs. Also, after phagocytosis, a 75.2 ±16 % reduction in Dextran-FITC endocytosis was observed compared to iDCs. CCR7 was upregulated upon Apo-Nec phagocytosis in DCs from all patients and accordingly in vitro DC/Apo-Nec cells were able to migrate towards MIP-3 beta. The DTH score increased significatively in the patients after the first vaccination and slightly decreased by the fourth vaccine (Mann-Whitney Test, p<0.05). For patient #1 a positive DTH reaction was detected to her own tumor irradiated cells. The presence of CD8+ T lymphocytes specific to gp100 and Melan A/MART-1 Ags were studied by tetramers binding in HLA-A*0201 patients (7 /15 patients) before and after vaccination. Two patients who remain NED increased Ags their specific T lymphocytes after vaccination. No humoral responses to Apo-Nec cells were detected. With a mean follow-up of 44.5 months post-surgery, the stage IIC pt is NED, 7/8 stage III pts are NED and 7/7 stage IV patients have progressed.

Conclussions: We conclude that DC/Apo-Nec vaccine is well tolerated, it induces specific immunity against melanoma Ags and in stage III patients it may prolong disease-free survival, affording protection from relapse in an adjuvant setting.

Inclusion Criteria:

• histologically confirmed cutaneous melanoma stages IIB, IIC, III or IV (AJCC)
• pts with minimal or non-detectable disease (NED) after surgery as asserted by CAT scans. Melanoma pts with unknown primary tumor site could be included in the study
• life expectancy > 6 months
• ages:from 15 to 60 years
• performance status (ECOG) 0 or 1
• pts with stage III disease had to be previously treated with IFN-alpha, and either finished the treatment or suspended it due to disease progression, toxicity or other clinical reasons. Alternatively, those pts who had not started IFN-alpha within six months after surgery could be included in this study
• a suitable venous access for the leukapheresis procedure
• laboratory eligibility criteria included: hemoglobin > 10 gr %; WBC count > 4800/mm3, platelets > 150.000/mm3, total and direct bilirubin, serum oxalacetic transaminase and glutamic pyruvic transaminase < 1.5 fold the upper normal value; LDH < 450 mU/ml
• absence of pregnancy, with serum βHCG determined one week before vaccination in premenopausal women
• serum creatinine < 1.4 mg %
• no chemotherapy, radiotherapy or any biological treatments during the previous month
• no concurrent medication with corticosteroids or NSAIDs
• l no active brain metastases m- normal ECG
• all pts gave written informed consent before inclusion in the Study.

Exclusion Criteria:

• Ocular melanoma or melanoma of mucosa
• Active brain metastases
• Other previous carcinoma (with the exeption of cervical or in situ basal cells carcinoma adequately treated)
• Pregnant or breast-feeding women
• Cardiac Arythmia, severe heart disease.
• Bacterial, mycotic or viral serious infections ( > grade 2 according to NCI common toxicity criteria)
• HIV, B or C Hepatitis previous infection
• Primary or secondary immunodeficiencies
• Other diseases that require treatment with regular corticoids or non steroids anti-inflammatory drugs or COX-2 inhibitors