Technical Abstract 
Scientific Abstract 
A phase I trial of B7-transfected allogeneic melanoma cell lines to induce cell-mediated immunity against 
tumor-associated antigens presented by HLA-A2 or HLA-A1 in patients with stage IV melanoma. 
BRMP 9401 
NCI Protocol T93-0161 
Biological Response Modifiers Program, DCT, NCI 
The scientific basis for this protocol is derived from two main points. Recent data indicate that two stimuli 
are required to activate T cells to proliferate and secrete cytokines. Signal one is provided by the T cell 
receptor, and the other requires interaction of other T cell surface receptors with their ligants on antigen 
presenting cells. The second point, is that in the case of human melanoma, tumors from many patients 
express the same tumor antigens, and that peptides derived from these tumor antigens are often presented 
on tumor cell surfaces by MHC class I molecules HLA-AI or A2. Thus, it may be possible to induce 
immunity against these shared melanoma antigens by vaccination with allogeneic tumor cell lines that 
express the tumor antigens and are HLA-AI or HLA-A2 positive. In order to study these questions in a 
clinical trial of advanced stage melanoma patients, we introduced the gene encoding the T cell co- 
stimulatory molecule B7 (CD28 ligand) into 3 human melanoma cell lines. These tumor lines are HLA-A2+ 
(one is HLA-AI +), express significant levels of LFA-3 and ICAM-I (and in one case HLA-DR) on their 
surfaces, and produce RNAs encoding the shared melanoma antigens MAGE-I and -3, and tyrosinase. We 
have examined the stimulatory capacity of parental tumor lines and B7-transfectants (DM150/B7-8, 
DM13/B7-7, and DM93/B7-4) in 7 day primary mixed lymphocyte cultures (MLC) with allogeneic human T 
cells obtained from PBL of normal donors or melanoma patients. In multiple experiments, parental tumor 
lines (which do not express detectable B7) fail to induce significant activation of allogeneic T cells as 
determined by lack of increased expression of HLA-DR or CD25 on T cell surfaces. In contrast, B7- 
transfected lines induce increased expression of HLA-DR and CD25 (in both CD4+ and CD8+ subsets), and 
a 5-10 fold increase in T cell number compared to cultures with parental cell lines. CTL induction in primary 
MLC was determined in this system by 7d co-culture of allogeneic T cells with the parental DM150 or 
DM150-B7 cell lines. While T cells cultured with DM150 exhibit only background levels of lytic activity, T 
cells cultured with DM150-B7 lyse this line, and also the unmodified parental line and the HLA-A2+ DM13 
cell line. TIL lines that recognize the shared melanoma antigen(s) presented by HLA-A2 lyse all three 
parental lines, providing functional data that these lines present shared melanoma antigens via the 
endogenous MHC class I pathway. These data indicate: 1. B7 is expressed by all three lines and is 
biologically functional as assessed by the ability to activate resting human T cells. 2. The cell lines 
express the genes encoding the known shared melanoma antigens and are lysed by HLA-A2 specific TIL 
derived from melanoma patients, demonstrating functional expression of the shared melanoma antigens 
by these lines. These studies form the preclinical basis for a vaccine trial in which patients will be 
vaccinated with lethally irradiated allogeneic melanoma cell lines genetically engineered to express human 
B7. The three lines will be injected subcutaneously at two week intervals for three vaccinations, followed 
by 3 injections at monthly intervals. The cell lines will be given on a rotating basis. Cohorts of patients will 
receive escalating doses of 1 0 7 , 1 0 8 , or 10 9 cells. This is a Phase I trial to determine the MTD of this 
therapy, but immunologic parameters such as generation of CTL precursors in peripheral blood and draining 
lymph nodes will also be monitored. 
Recombinant DNA Research, Volume 18 
[717] 
