8.3 Chemical profile including liver function tests, bilirubin, creatinine, and 
LDH will be done every four weeks for two months, then prior to each 
vaccine. 
8.4 Peripheral blood will be obtained for immune assays (see section 6.9). Six 
green top tubes and two red top tubes will be obtained prior to the first 
vaccination and one to two weeks after the fourth vaccination. 
8.6 Chest X-ray will be repeated every 4 weeks for two months, then prior to 
each vaccination. 
8.7 If moderate toxicity (grade 3) is observed, pertinent tests will be 
performed more frequently than indicated in the table. 
9.0 EVALUATION OF IMMUNE RESPONSE 
9.1 Serological studies 
The most direct methods to measure immune responses to melanoma is 
detection of antibodies. Approximately one- third of patients with metastatic 
melanoma have antibodies that react with autologous tumor cells (reference 
45). Most of these autoantibodies are IgM but occasional antibodies are IgG 
class. Several antigens recognized by these autoantibodies have been defined at 
the molecular level, including the acidic glycolipids GM2 and GD2, and the 
melanosomal glycoprotein gp75. The immunizing cell line SK-MEL-29 
expresses GD2 and gp75. The detection of IgG antibodies induced by 
immunization is also a possible indirect measure of helper T cell responses, 
because induction of high affinity, mature IgG responses requires T cell help 
by CD4-positive T cells. All patients will have peripheral blood sera obtained 
for serologic analysis of antibodies directed against the SK-MEL-29 cell line 
(to include antibodies against GD2 and gp75) and autologous tumor cells when 
available. In addition, a control for a positive immune response with antibodies 
against allogeneic MHC class I and II antigens expressed by SK-MEL-29 will 
be done. It is expected that most patients will develop strong IgG responses 
against class I and II alloantigens expressed by SK-MEL-29. The development 
of IgG antibodies will provide an indirect measure of induction of a T cell help 
response against SK-MEL-29. Antibodies against cell surface and intracellular 
antigen will be detected by: 
A. Immunoprecipitation of 1 % NP40 cell lysates of SK-MEL-29 
metabolically labeled with 35 -methionine. 
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Recombinant DNA Research, Volume 15 
