8.0 Evaluation during study. 
8.1 Physical examination before each immunization. 
8.2 CBC, platelet count, and differential before each immunization. 
8.3 Liver function tests (including bilirubin alkaline phosphatase, SGOT and 
lactic dehydrogenase) and kidney profile (including serum electrolytes, 
creatinine and BUN) before each odd-numbered immunization. 
8.4 Peripheral blood samples will be obtained for humoral and cellular immune 
assays before each immunization and at two weeks after the fourth 
immunization . 
8.5 Tumor evaluation will be performed during week eleven of each course of 
immunization. The evaluation consists of a physical examination, CT scans of 
the chest and abdomen, and a repetition of any other studies performed before 
the first immunization that revealed evidence of disease. 
8.6 Patients removed from study without disease progression must be have 
repeat tumor evaluations every eight weeks as described in section 8.5. In 
addition, the patients will undergo physical examination every 4 weeks after 
removal from study until progression of disease is documented. 
9.0 Measurement of an Effect 
9.1 Humoral Immune Response: The most direct method to measure an immune 
response to melanoma is detection of anti melanoma antibodies. Approximately 
one-third of patients with metastatic melanoma have antibodies that react with 
autologous tumor cells (43) . Most of these auto antibodies are IgM but on 
occasional IgG antibodies are present. Several antigens recognized by the 
auto antibodies react with the acidic glycolipids GM2 and GD2, and the 
melanosomal glycoprotein gp 75 (43) . The detection of IgG antibodies induced 
by immunization is also an indirect measurement of helper T cell responses. 
All patients will have peripheral blood sera obtained for serologic analysis 
of antibodies directed against both the IL-2-secreting cell-line (to include 
antibodies against GD2 and gp 75) and autologous tumor cells. In addition, as 
a control for a positive immune response, tests for the detection of 
antibodies directed toward allogeneic MHC Class I antigens expressed by the 
cell-line will be performed. IgM and IgG antibodies toward cell surface 
antigens expressed by UISO-MEL-4-IL-2 cells will be detected by quantitative 
immunofluorescence. The same approach will be used to detect the induction of 
antibodies toward autologous tumor. 
9.2 Cellular Immune Response: MHC-restricted cellular cytotoxicity toward 
autologous tumor will be evaluated in the immunized patients. Although 
cellular immune responses are a critical measure of immune responses to 
melanoma, we realize the following limitations: a) autologous tumor cells are 
necessary to detect responses; b) autologous normal cells and MHC-matched 
allogeneic cells are necessary to evaluate the specificity of responses; c) 
evidence of cytotoxic or helper responses are not evident without in vitro 
stimulation by autologous tumor cells. 
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