demonstrated in a K1735 cell line that was further modified by insertion of a 
strong tumor antigen, the E7 gene product of human papillomavirus. 
Immunization with the E7+B7+ K1735 cell lines not only protected animals from 
a subsequent challenge of E7+K1735 tumor, but also delayed growth of a 
simultaneously placed E7+K1735 tumor in the opposite flank and of an 
established 4-day old E7+K1735 tumor. Depletion experiments indicated that 
antitumor effects were mediated by CD8+ T lymphocytes. Immunization with the 
B7+E7+ transfected cells also induced specific CTL activity against the 
corresponding B7-E7+ tumor in vivo. 
These experiments demonstrate that if a strong antigen is present (ie E7 ) on 
the tumor, immunization with the corresponding B7-transfected tumor cell line 
may result in regression of established tumor. Even if the tumor expresses 
only weak tumor-associated antigens (ie the parental K1735 cell line) , it is 
still possible to demonstrate that immunization wit.: a B7-trans fected tumor 
cell line will result in an antigen-specific CD8 + T lymphocyte response. In 
the latter case, additional manipulations such as systemic administration of 
cytokines, or isolation, in vitro expansion and adoptive transfer of the CTL 
will probably be necessary to produce regression of advanced tumors. 
Description and preclinical studies with three allogeneic melanoma cell lines 
transfected with B7 
Three allogeneic melanoma cell lines were obtained from Dr. Timothy Darrow 
of Duke University. The cell lines were clonal culture isolates from surgical 
specimens obtained from three unrelated melanoma patients. The cell lines and 
haplotypes are as described below: 
1. DM- 150 : HLA-A1, A2, B8, HLA-DR positive 
2. DM- 13 : HLA-A2, A31, B18, (B13, or B40), HLA-DR negative 
3. DM- 9 3 : HLA-A2 , A19 (or Aw33) , B8, B49, HLA-DR negative 
All three cell lines express moderate levels of LFA-3 (CD58) and ICAM-1 
(CD54). ICAM-1 and HLA class I and class II molecules are upregulated by 48- 
hour in vitro culture with interferon-gamma. 
The cDNA for human B7 was isolated by reverse transcriptase/PCR cloning using 
RNA from the human Burkitt's lymphoma cell line Raji. This PCR product is 970 
base pairs in length and encodes the complete protein. The gene was cloned 
into the Xho I site of vector BCMG-neo, downstream of the cytomegalovirus 
(CMV) promoter/enhancer, to generate the plasmid BCMGNeo-B7 (Appendix I). 
Sequences within the BCMG-neo encoding early region genes from the bovine 
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Recombinant DNA Research, Volume 18 
