Patients with stage III or stage IV melanoma will undergo surgical resection of a melanotic lesion.

Patients will undergo surgical removal of a metastatic lesion.

Day 3: change medium and start selection for stably transduced cells

Monocytes are isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch- Gladbach,Germany).

After the centrifugation (10’ at 5000 rpm) the supernatant is transferred into a pre-spun (5’ at 1500 rpm) 50 ml Phase Lock Gel tube (Eppendorf 0032 005.330) and 4 ml of chloroform (Fischer BP1145-1) are added into the falcon.

Cells are subsequently separated into monocytes and lymphocytes by elutriation in a closed system (Elutra). The two most monocyte-rich fractions collected contained > 80% monocytes, the fraction richest in lymphocytes contained > 90% lymphocytes and viability in both fractions was > 95%.

Patients will undergo leukapheresis.

Leukapheresis will be performed on the following day.

A HLA-A2 positive healthy donor undergoes leukapheresis.

HLA-A1 and/or HLA-A2 positive patients with stage III/IV melanoma undergo leukapheresis.

CD8 T cells are purified using MACS cell separation.

PBMC are isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM).

Day 4: harvesting (virus-containing)

Sources for tumor antigens as well as monocytes and T cells will be isolated from the leukapheresis product by elutriation.

Elutriation of the leukapheresis product.

The samples are centrifugated at 5000 rpm in an tabletop centrifuge.

After the spin (10’ at 3,000 rpm), the supernatants are precipitated in RNase-free centrifuge tube by adding 1/10 volume 3 M NaAcetate (pH 5.3) and 2.5 volumes of cold ETOH 100% overnight at -20°C. Centrifugation (30’ at 12000 rpm).