Biomedical subjects
Publications and source records attributed to F L Graham.
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We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCD1, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region 1a and the N-terminal region of early region 1b are essential for DNA-mediated transformation.
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Transfection of the costimulatory molecule B7-1 (CD80) into murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this study was to test autologous lymphocyte responses against freshly resected human cancers infected in vitro with an adenovirus vector expressing the B7-1 molecule (AdB7). Resected tumors (sarcomas, adenocarcinomas, melanomas, and multiple myelomas) were disaggregated into single-cell suspensions and divided into three groups: (a) native, noninfected tumor cells (TM); (b) AdB7-infected, B7-1(+) tumor cells (TMB7); and (c) control Addl70.3-infected, B7-1(-) tumor cells (TMAd). B7-1 expression was verified by flow cytometry. Autologous peripheral blood lymphocytes from these patients were tested for proliferative and cytotoxic activity against the three tumor groups. There was an increased lymphocyte-proliferative response against B7-1(+) tumor cells, particularly in the presence of interleukin-12 (IL-12) or low-dose IL-2. B7-1(+) tumor cells were also killed more efficiently than B7-1(-) tumor cells in natural killer cell-mediated cytotoxicity assays, and this was most significant when lymphocytes had been pretreated with IL-12. Human natural killer cells were found to express CD28, a receptor for B7-1. The high efficiency of AdB7-mediated gene transfer and the augmented B7-1-mediated lymphocyte responses suggest that AdB7 vectors may be effective in human cancer immunotherapy.
The toxic effects of tumor necrosis factor alpha (TNFalpha) have greatly limited its use in tumor therapy. Recently, clear evidence has been obtained linking the p55 TNF receptor (TNFR) to the induction of systemic toxicity. We have generated a p75 murine TNFR (mTNFR)-specific mutant of mTNFalpha (D142N-A144R), cloned this gene into a recombinant adenovirus vector (Ad-75), and studied its efficacy for tumor immunotherapy of a murine transgenic breast cancer model. Cell culture supernatants from Ad-75-transduced cells showed no cytotoxic activity on L929 cells, but retained the ability to induce proliferation of a murine T-cell line (CT6); this activity was not blocked by soluble p55 mTNFR. Furthermore, it was shown that the mutant form of mTNFalpha was able to coimmunoprecipitate only with the p75 mTNFR and not with the p55 mTNFR. Tumors injected with Ad-75 became necrotic, and mice injected with < or =1 x 10(9) plaque-forming units showed no mortality, whereas both wild-type murine and human TNF vectors induced lethality at doses of 1 and 5 x 10(8) plaque-forming units. All Ad-TNF vectors induced partial or permanent tumor regressions, with cured mice showing immune memory against the tumor. These results demonstrate that a p75 mTNFR agonist expressed from a recombinant adenovirus vector does not induce mortality at doses that cause tumor regression.