A p75 tumor necrosis factor receptor-specific mutant of murine tumor necrosis factor α expressed from an adenovirus vector induces an antitumor response with reduced toxicity

Abstract

The toxic effects of tumor necrosis factor ␣ (TNF␣) 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 mTNF␣ (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 mTNF␣ 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 Յ1 ϫ 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 ϫ 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.

Transgene expression of α tumor necrosis factor with mutations D142N and A144R under control of human telomerase reverse transcriptase promoter eradicates well-established tumors and induces long-term antitumor immunity

“…The mouse mortality was then examined as described earlier. 12 To assess the therapeutic effect, the recombinant adenoviruses (Ad-V TERT mTNF-a or AdV TERT pLpA) at 2 Â 10 9 PFU in a volume of 10-20 ml of PBS were i.t. injected into tumors when tumors reached B6 mm in diameter.…”

“…These include septic-like shock syndrome, hypotension, malaise, diarrhea, leukothrombopenia, anorexia, and so on. [8][9][10] The p55 TNF-a receptor (TNFR) is primarily responsible for signaling in variety of responses including cytotoxicity 11,12 and cytokine secretion, 13 whereas the p75 TNFR is responsible for lymphoproliferative signals and activation of T cells. 12,14,15 To induce high local concentration of TNF-a while limiting systemic use of a high dose of TNF-a, isolated limb perfusion with TNF-a has been applied with less systemic toxicity.…”

“…[8][9][10] The p55 TNF-a receptor (TNFR) is primarily responsible for signaling in variety of responses including cytotoxicity 11,12 and cytokine secretion, 13 whereas the p75 TNFR is responsible for lymphoproliferative signals and activation of T cells. 12,14,15 To induce high local concentration of TNF-a while limiting systemic use of a high dose of TNF-a, isolated limb perfusion with TNF-a has been applied with less systemic toxicity. 16,17 These results prompted a number of studies aimed at decreasing TNF-a toxicity without modifying its antitumor properties, thereby allowing its use not only for regional treatments but also for systemic therapy.…”

“…22 To reduce the toxicity derived from the binding of murine TNF-a to its p55 receptor, point mutations at D142N and A144R of TNF-a protein generated by site-directed mutagenesis resulted in the loss of binding of mutant TNF-a to its p55 receptor and reduced in vivo toxicity. 12 An alternative is the direct in vivo gene delivery mediated by adenoviruses (AdVs) expressing TNF-a. It has been reported that intratumoral (i.t.)…”

“…It has been reported that intratumoral (i.t.) administration of recombinant adenoviral vectors (AdVTNF-a) resulted in the transgene expression of TNF-a in tumors, detection of serum TNF-a within 1-2 days, 23 and regression of small palpable tumors within 2-3 days in a mouse model, 12,[23][24][25] but not well-established experimental tumors, though the tumor growth in these treated mice was much slower. 25 Telomerase is highly active in immortalized cell lines and tumors, but inactive in most somatic cells.…”