Mononuclear cells from peripheral blood will be purified by Ficoll-Hypaque 
gradient centrifugation. Cells will be cultured with autologous tumor cells, 
IL-2 (20-1000 units/ml) , and autologous or allogeneic stimulator cells for 7- 
14 days. The following assays to detect the presence of cellular immunity 
will be performed: 
Peripheral blood lymphocytes (PBL) will be cultured for 48 hours with 
autologous tumor cells, autologous normal cells, allogeneic tumor cells, or 
100 nM OKT3 (a positive control) . Cell proliferation will be measured by the 
"5 
uptake by H-thymidine added to the cultures for the last six hours of the 
incubation period. These assays will be performed with PBL obtained from 
patients immediately before each immunization and 2 weeks after the fourth 
injections . 
When autologous tumor cell lines are available, the cytotoxic response of PBL 
toward ^Cr-labeled autologous tumor will be measured. 
9.3 The immunocompetence of patients with metastatic disease may be 
diminished. To assess this possibility, delayed type hypersensitivity (DTH) 
reactions toward autologous tumor (5,000 rads) and against (5000 rads) UISO- 
MEL-4-IL-2 cells before and after the course of immunizations. It is 
recognized that generation of a DTH response to the cells does not indicate 
the presence of a cellular anti-melanoma response capable of controlling tumor 
growth in vivo. 
9.4 As additional determinant of the induction of an immune response to the 
cellular immunogen the titer of cytokines in the peripheral blood following 
the administration of allogeneic vaccine will be measured. Although the 
appearance of cytokines in the serum is a non-specific indicator of immune 
stimulation, such monitors provide a valuable means of determining the host 
response to the immunization. They may be aid in determining which of the two 
doses of immunogen induces an immune response of greatest magnitude. Levels 
of TNF-alpha, IL-2, IL-6, and soluble IL-2 receptor will be measured by ELISA 
in serum samples obtained before and 24 hours following injection. 
9.5 Interleukin-2 levels in patients' sera may also be increased as a result 
of IL-2 production by UISO-H-Mel-4-IL-2 cells. Sera obtained from patients 1, 
2, 8, and 15 days after immunization will be assayed for IL-2 titers. A 
commercially available (Centocor) RIA assay kit is used for this purpose. 
9.6 Tumor evaluation will be performed within 2 weeks of the first 
immunization and during weeks 10 or 11 of each course. Tumor evaluation after 
the completion of a course of immunization will include a physical exam and a 
CT scan of the chest and abdomen. Additional studies that revealed measurable 
disease before the first injections will be repeated following a course of 
immunization . 
10.0 Criteria For A Therapeutic Response 
10.1 Complete Response (CR) : Disappearance of all clinical evidence of tumor 
by physical examination. X-ray, and CT scan for a minimum of four weeks. 
Recombinant DNA Research, Volume 18 
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