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).

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

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.

Day 4: harvesting (virus-containing)

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

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

A HLA-A2 positive healthy donor undergoes leukapheresis.

Leukapheresis will be performed on the following day.

Patients will undergo leukapheresis.

CD8 T cells are purified using MACS cell separation.

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).

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%.

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.

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

Patients will undergo surgical removal of a metastatic lesion.

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