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Our institution is conducting a phase II trial of dendritic cells (DC) cocultivated with autologous tumor cells (ATC) as active specific immunotherapy for metastatic melanoma patients. This study examines possible humoral responses against the patient’s ATC as well as the adjuvant used in our study (GM-CSF) before and after 3 weekly injections of a DC based vaccination. Monocyte derived DC were generated from gradient separated pheresis product using plastic flasks and AIM-V supplemented with 1,000 IU/ml each of GM-CSF and IL-4. Short term ATC lines were established from surgical resections prior to generation of the DC. ATC were cocultivated overnight with the newly generated DC and the cells recovered the following morning. Patients are injected S.C. with approximately 107 DC/ATC given weeks 1, 2, 3, 8, 12, 16, 20 and 24 with adjuvant GM-CSF. At week 0 (prior to receiving first vaccination) patients are subjected to a delayed type hypersensitivity (DTH) test against their ATC which was repeated at week 4 with serum samples also obtained at weeks 0 and 4. The serum samples were stored at −80° C until assayed. Tumor lysate derived from ATC and GM-CSF were applied to a 14% Tris-glycine gel and subjected to electropheresis. The proteins were then electroblotted onto 2 PVDF membranes and the membranes probed with patient serum from weeks 0 and 4 respectively. The results at week 4 were compared to the results obtained at week 0. To date, the serum from 20 patients has been evaluated. At week 0, all 20 patients displayed anti-tumor lysate bands (average of 9 ± 3 bands with no clear trend in molecular weight comparisons from patient to patient) with 11/20 showing no increase in reactive bands at week 4, 1/20 demonstrating a decrease and 8/20 demonstrating at least one additional reactive band (average 10 ± 2). At week 0, 11/20 patients demonstrate GM-CSF antibody reactivity which was maintained or increased at week 4. Of the 9 patients who were negative against GM-CSF at week 0, 7/9 maintained no response at week 4 while 2/9 patients gained antibody responsiveness against GM-CSF by week 4 for a total of 13/20 patients demonstrating antibody reactivity against GM-CSF at week 4. In addition, all 20 patients displayed no positive DTH at week 0 with 2/20 converting at week 4. Interestingly, the 2/20 DTH converters did not display additional antibody reactive bands at week 4 but did display GM-CSF reactivity. In conclusion, antibody reactivity against ATC is observed in all patients at week 0 with 8/20 patients demonstrating increased antibody responsiveness by week 4. GM-CSF antibody reactivity is observed in a majority of these patients prior to therapy with the responsiveness increasing at week 4. The clinical effects of these differences is unclear but does suggest that at least in some cases, immune modulation is occurring.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]