cells cultured with the B7-trans fectants developed CTL activity, which was 
observed against both the B7- t rans fected and the parental tumor cell line. 
Furthermore, T cell adhesion to B7-expressing melanoma cell lines was 
significantly enhanced compared to the parental tumor cell lines and appeared 
to involve LFA-l/ICAM-1 interactions. 
In preparation for the clinical trial, a master cell bank consisting of 10 
vials (each vial containing 2-5 x 10 6 cells) for each B7-trans fected cell line 
(DM150/B7-8, DM13/B7-7, and DM93/B7-3) has been established. A working cell 
bank of 50 vials (each vial containing 2-5 x 10^ cells) will be established 
from 1 vial of each master cell bank. One vial of each master cell bank will 
be tested for mycoplasma, general sterility (fungal and bacterial cultures), 
and contamination by retroviruses (reverse transcriptase assay) . When a 
patient is identified and has signed an informed consent, one vial of the 
working cell bank for the designated cell line will be thawed and placed in 
culture containing fetal bovine serum. The culture medium will also contain 
penicillin and streptomycin. Cells will be expanded to the appropriate number 
in sterile flasks. Cell expansion is predicted to require no more than 3 
weeks, and will be performed in a laboratory dedicated to expansion of human 
cells (a LAK lab) . After expansion to the desired cell number, the tumor cell 
line will receive 20,000 rad (lethal irradiation) prior to administration to 
the patient. A sample of the final product will be examined microscopically 
prior to administration. 
HLA-A2 patients will receive inoculation every 2 weeks x 3, then monthly using 
one cell line at a time in the following order: DM150/B7-8, DM13/B7-7, and 
DM93/B7-3. By rotating administration of these lethally irradiated melanoma 
cell lines to HLA-A2 melanoma patients, we hope to induce a potent in vivo 
CD8+ CTL response to the common melanoma antigen (s) presented by HLA-A2 while 
minimizing the response to allogeneic determinants. Since only 1 of our cell 
lines expresses HLA-A1 and MAGE 1 and 3, patients who are HLA-A1 will receive 
only the DM150-B7 cell line although the same schedule will be maintained. 
Dose escalation will proceed separately for the HLA-A1 and HLA-A2 patients. 
Patients that express both HLA-A1 and HLA-A2 will be treated with the rotation 
schedule as noted for HLA-A2 patients, since there is preliminary evidence 
that antigens presented by HLA-A2 are immunodominant. Patients who are HLA-A2 
positive will not require a demonstration that their tumor expresses the 
tyrosinase gene, since Boon et al have shown expression of tyrosinase in 40/40 
melanoma tumors and have not detected mutations in the gene sequence. MAGE-1 
and 3 are detected in only 60-70% of patients that are HLA-A1 . All HLA-A1 
patients will be eligible for the study, however, since other as yet 
undetermined shared melanoma antigens may be presented by HLA-Al . 
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Recombinant DNA Research, Volume 18 
