Apoptosis-inducing Fas ligand (FasL) is a type II membrane protein, predominantly expressed in the activated T cells. FasL is cleaved by a putative metalloproteinase to produce a soluble form. Here, we blocked the shedding of human FasL by deleting its cleavage site. Although human Jurkat cells and mouse primary hepatocytes that express a low level of Fas were resistant to the soluble form of FasL, they were efficiently killed by membrane-bound FasL. Furthermore, soluble FasL inhibited cytotoxicity of the membrane-bound FasL. These results indicate that the membrane-bound form of FasL is the functional form and suggest that shedding of FasL is to prevent the killing of the healthy bystander cells by cytotoxic T cells.

The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD154 interaction. However, the mechanism leading to the production of sCD40 has never been investigated. Here, we show that the engagement of mCD40 on the surface of B lymphocytes by anti-CD40 antibody led to enhanced sCD40 release associated with decreased amounts of mCD40. This sCD40 production was not affected by vesicular traffic inhibitors but was completely blocked by a broad-spectrum synthetic metalloproteinase (MP) inhibitor (GM6001) or a membrane-anchored MP-specific inhibitor (dec-RVKR-cmk). Recombinant MP disintegrin tumor necrosis factor-␣ converting enzyme (TACE) cleaved the purified CD40 ectodomain/Fc chimeric protein in vitro, giving rise to an sCD40 form similar to that shed from B cell cultures. Moreover, spontaneous production of sCD40 by mCD40-transfected human embryonic kidney cells (constitutively expressing TACE) was enhanced by the overexpression of TACE and abrogated by co-transfection with a dominant-negative TACE mutant. These results provide strong evidence that sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. Given the antagonistic activity of sCD40 on the CD40/CD154 interaction, this shedding mechanism might represent an important negative feedback control of CD40 functions.

Tumour necrosis factor (TNF)-a-converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the proform of TNF-a. In this study, we constructed expression vectors for the membrane-bound full-length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF-a expression vector was introduced into human 293 cells, processing of TNF-a to its mature form was enhanced by coexpressing mTACE, and this processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF-a, although the culture medium of sTACE-transfected cells could cleave a peptide containing the TNF-a cleavage site. Fas ligand (FasL)-transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF-a indicated that both mTACE and sTACE could interact with the proform of TNF-a. In the same assay, neither mTACE nor sTACE interacted with FasL. The catalytic domain-lacking TACE mutant, which could also interact TNF-a, showed a dominant negative effect on not only TNF-a secretion but also FasL secretion. These results suggest that binding of the membrane-anchored but not the soluble form of TACE to TNF-a results in efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.

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An alternative option to avoid such a risk is to use a non-replicating oncolytic virus [44]. We found that a non-replicating oncolytic virus (HVJ-E: hemagglutinating virus of Japanenvelope) is able to induce cancer cell-specific apoptosis and immunity [45]. The induction of apoptosis and activation of dendritic cells in vitro, and anti-tumor activity in vivo are similar to the wild-type hemagglutinating virus of Japan (also known as Sendai virus, HVJ) [45]. The hemagglutinating virus of Japan was discovered in Sendai, Japan, in the 1950s [46]. It is a paramyxovirus with a minus-strand RNA genome. The virus has fusogenic activity [47, 48], and is used to prepare hybridoma cells for the production of monoclonal antibodies, and heterokaryons for chromosome analysis [49-51]. The hemagglutinating virus of Japan-envelope is an inactivated HVJ particle [52]. It is manufactured by a process similar to that used for whole virus particle vaccines. Good manufacturing practice (GMP)-regulated processes have been established in their production for use in preclinical and clinical studies [53]. We conducted dose-setting efficacy studies for HVJ-E in a murine cancer model in which dosedependent anti-cancer activity was observed. We also conducted safety studies following good laboratory practices (GLP), including pharmacological safety studies and toxicokinetic (TK) studies in rats and monkeys, as part of an investigational new drug (IND) application. Osaka University Hospital is currently conducting two investigational clinical studies with HVJ-E for the treatment of advanced melanoma and castration-resistant prostate cancer (CRPC) [54-56]. These clinical trials are the first human studies for HVJ-E and will reveal the safety and efficacy of the non-replicating virus (HVJ-E). Virotherapy with a non-replicating oncolytic virus is a new approach that is anticipated to provide a new strategy for cancer therapy. 2. A new strategy for cancer therapy Most cancers are still incurable and new approaches are required to improve the efficacy of cancer treatments. However, conventional cancer therapies are problematic. Chemotherapy with anti-cancer agents is useful in achieving tumor regression. However, the immune system, which is important in the removal of residual cancer cells, is also suppressed by these agents (Figure 1). Therefore, surviving cancer cells and cancer stem cells (CSC) eventually acquire drug resistance, resulting in tumor relapse (Figure 1) [57]. Thus, chemotherapy with cytotoxic drugs does not generally result in the necessary eradication of cancer cells required for long-term survival. Immune therapies for cancer offer a new approach to cancer treatment, and several products, including sipuleucel-T, are currently approved in advanced countries [58, 59]. The aim of these therapies is the removal of cancers by the immune system. Numerous cancer immune therapies are currently under evaluation in clinical studies. However, these agents are not potent because of lack of cytotoxic effect on cancer cells (Figure 1).