Abstract A110: Tumor targeting therapy of solid tumors by engineeredB. longumsecreting human TNF-alpha.

Tumor necrosis factor alpha (TNF-α) is a multifunctional cytokine playing key roles in apoptosis and cell survival as well as in inflammation and immunity. Since the human TNF-α (hTNF-α) gene was cloned in 1984, a range of clinical trials using recombinant hTNF-α (rhTNF-α) has been set up against a variety of solid tumors, but most of the studies failed because of its instability in the body and severe adverse events. The current use of rhTNF-α in cancer is for the regional treatment of locally advanced solid tumors, and it has been demonstrated that rhTNF-α, by isolated limb perfusion setting, acts synergistically with cytostatic drugs on soft tissue sarcomas and metastatic melanomas, resulting in excellent clinical responses. Thus, TNF-α can be expected the great antitumor activity without adverse events when delivered only to the tumor sites. We previously reported that a strain of nonpathogenic and anaerobic bacteria, Bifidobacterium longum (B. longum), selectively localized to solid tumors and proliferated within these sites after the systemic administration. In this study, we developed engineered B. longum (3BTNF) transformed with the plasmid encoding secreting hTNF-α, and examined the characteristics of the strain from the point of views of molecular cell biology and antitumor efficacy. The engineered strain of B. longum, 3BTNF cells secreted high amount of the protein into the culture medium, and the molecular size was 17 kDa which was the same as that of natural hTNF-α. The crude protein showed a cytotoxic activity against tumor cell lines in vitro, and the activity was neutralized by the goat anti-hTNF-α. The protein was confirmed to induce apoptosis in tumor cell lines with co-existence of adriamycin (ADM) through the caspase-3-dependent pathway. In order to demonstrate the antitumor effect, it was examined that 3BTNF was administered intravenously twice weekly for 4 times (Days 1, 4, 8 and 11) with supplement of maltose into nude mice bearing human breast carcinoma KPL-1 (n=6). A significant antitumor effect was observed, i.e., tumor growth inhibition (T/C%) was 48.3% on Day 22 (p=0.021). When the 3BTNF treatment was combined with ADM (Days 0 and 7), a marked effect was obtained, i.e., T/C 12.2% (p=0.0001) in 3BTNF plus ADM group or 27.1% in ADM alone group, respectively. Against another human cancer cell line, colorectal carcinoma HCT 116 cell, obvious antitumor effects were also observed by the treatment with 3BTNF alone and in combination with chemotherapy. A meaningful amount of hTNF- (mean, 1.3 ± 1.5 ng/g tumor) was detected in the tumor tissue in HCT 116 bearing mice, concomitantly detected with a large number of live 3BTNF (mean, 1.2E+7 ± 2.1E+7 cfu/g tumor) on Day 25. In contrast, hTNF-α in the serum was not detected. No serious side effect was noted after 3BTNF administration in these mice. Taken together, these findings indicate that the engineered B. longum secreting hTNF-α has attractive therapeutic potential as a new candidate of antitumor modality targeting hypoxic solid tumors. A part of this study was supported by the grant from NEDO (New Energy and Industrial Technology Development Organization). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A110.