Toxicology and Pharmacokinetic Studies in Mice and Nonhuman Primates of the Nontoxic, Efficient, Targeted Hexameric FasL: CTLA4-FasL.

Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.

Fn14·TRAIL fusion protein is oligomerized by TWEAK into a superefficient TRAIL analog.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) demonstrates specific anti-cancer activity, but insufficient efficacy in patients. A fusion protein Fn14·TRAIL, that combines soluble TRAIL molecule with a specific TWEAK receptor Fn14, is a better apoptosis-inducer for hepatocellular carcinomas than soluble TRAIL. However, Fn14·TRAIL does not effectively induce apoptosis in tumors of the lymphoid origin. As malignant cell apoptosis is strongly enhanced by secondary oligomerization of TRAIL, we tested the hypothesis that soluble Fn14·TRAIL can be oligomerized and become more active by adding TWEAK, a cytokine secreted in the tumor environment. We revealed that TWEAK and Fn14·TRAIL spontaneously formed a stable complex that induced apoptosis of malignant lymphoblasts earlier and more efficiently than TRAIL. The TWEAK-modified Fn14·TRAIL oligomer bound to target cells and delivered apoptotic signaling via TRAIL receptors. The oligomer induced faster and stronger cleavage of procaspase-8, -9, and -3; BID; poly-ADP ribose polymerase; and RIP compared to TRAIL. The oligomer also reduced expression of the anti-apoptotic proteins c-FLIP short and cIAP-1. Our data indicate that Fn14·TRAIL can be converted into a highly effective TRAIL oligomer upon binding to TWEAK.

Highly efficient, in-vivo Fas-mediated apoptosis of B-cell lymphoma by hexameric CTLA4-FasL.

Non-Hodgkin lymphomas (NHLs) account for 4% of all malignancies. 5-year survival rate increased to 50% with new treatment modalities, however there is need for new effective treatment for the more aggressive, relapsing forms. Recently, CTLA4-FasL, that can bind to B7 and Fas receptor (Fas), was shown to induce robust apoptosis of cell lines originating from B cell lymphomas expressing both B7 and Fas, by activating pro-apoptotic signals in parallel to abrogating anti-apoptotic ones. The present study focuses on the unique properties of CTLA4-FasL as a potent apoptosis inducer of malignant cells in-vitro and in a xenograft model. CTLA4-FasL was found to naturally form a stable homo-hexamer. CTLA4-FasL induces robust apoptosis of a large variety of malignant cells while relatively sparing non-malignant ones, being more efficient when both receptors (B7 and Fas) are expressed on target cells. Even in non-B7 expressing cells, CTLA4-FasL exhibited better apoptotic activity than its parts, alone or in combination, however, only in B7 expressing cells apoptosis occurs at low concentrations and CTLA4-FasL induces activation of apoptotic signals and reduces anti-apoptotic ones. Importantly, CTLA4-FasL efficiently inhibited the growth of human B cell lineage tumors in a xenograft model, by provoking tumor cells' apoptosis. Thus, CTLA4-FasL, a natural homo-hexamer protein, induces robust apoptosis of malignant cells, in-vitro and in-vivo. In B-cell lymphoma, its potency stems from the combination of its synergistic effect of activating the caspases while abrogating the anti-apoptotic signaling, with its unique hexameric structure, making CTLA4-FasL a promising candidate for aggressive B cell lymphomas treatment.

Fn14•TRAIL effectively inhibits hepatocellular carcinoma growth.

BACKGROUND: New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL's capacity to inhibit HCC growth was tested. RESULTS: Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL's molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice. CONCLUSIONS: In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein's potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies.