prohibited. All ancillary medications used for symptomatic therapy should be 
accurately recorded on the case report forms. 
VI. Study Endpoints and Monitoring - (Appendix III) 
1. The major laboratory endpoint of this study is to determine whether the 
B7-trans f ected melanoma cell lines induce cell-mediated T cell responses 
against the antigen (s) presented by HLA-A2 and HLA-A1. Lymphocytes will be 
obtained from 4 possible sites: the vaccine injection site, the draining lymph 
nodes, PBL, or an accessible tumor. If possible, lymphocytes will be tested 
directly against the targets, or will first be co-cultured with low-dose IL-2 
and with the immunizing cell line or matched allogeneic HLA-A2 or HLA-A1 
melanoma cell lines that do not share any of the alio determinants of the 
immunizing transfected cell line. Co-cultures with B7 trans f ectants of the 
melanoma cell lines will also be considered. 
The targets will be the immunizing cell lines (wild type and B7 transfected) , 
several HLA-A2 or HLA-A1 melanoma cell lines not sharing the allogeneic 
determinants of the immunizing cell line, HLA-A2 or HLA-A1 non-melanoma tumor 
cell lines, and perhaps autologous EBV transformed -lymphoblastoid cell lines 
(EBV-LCL) . Measures of reactivity will include cytotoxicity in 4 or 18 hour 
chromium release assays or cytokine production (interferon-gamma and TNF) . 
Frequency of CTL will be determined by limiting dilution assays (25) . In 
addition, proliferation assays will be performed against the panel of targets, 
in some cases after up-regulation of class II antigens with interferon-gamma. 
If antigen-specific CTL are isolated, a variety of experiments will be 
performed to learn how to optimally expand the cells in vitro. Although not 
a primary endpoint of the current study, these laboratory efforts will be 
designed to generate data for the next generation of clinical protocols. 
PBL samples will be obtained x 3 prior to treatment, then at 7, 14 (prior to 
the second injection), 21, 28 (prior to the third injection), 42, 56, 84, 112, 
140 and 168 days after the first injection, then every scheduled visit in 
patients who remain on study with stable or responding disease. LN and biopsy 
sample times are described above. Tumor biopsies when available will be 
obtained 2-4 weeks after the 4th or subsequent injections. 
Skin test reactivity to the non-transfected cell line DM-150 and will be 
tested at the time of administration of the 4th vaccination with the B7- 
transfected cell line. 10^ lethally irradiated cells will be used and will be 
injected in the opposite extremity. The area of induration will be measured 
Recombinant DNA Research, Volume 18 
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