Functional Characterization of a Chimeric Soluble Fas Ligand Polymer with In Vivo Anti-Tumor Activity

Abstract

Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.

Prognostic Value of Soluble Death Receptor Ligands in Patients with Transitional Cell Carcinoma of Bladder

“…Indeed, the sFasL from patients was found to be bioactive and present as high molecular weight multimers and aggregates [21]. Daburon et al [22] also reported that a chimeric sFasL in its oligomerized form is cytotoxic and displays antitumor activity. We can hypothesize that, in our case, sFasL from sera of patients with bladder carcinomas could exist in aggregated forms, explaining its cytotoxic effect.…”

Targeting of the Tumor Necrosis Factor Receptor Superfamily for Cancer Immunotherapy

“…More recently, a polymeric dodecamer FasL chimera was described in which FasL was fused to the homotypic aggregation domain of Leukemia Inhibitory Factor receptor gp1909. This multimeric FasL chimera also proved highly active in vitro , possessed tumoricidal activity in vivo , and had no liver toxicity at tumoricidal concentrations [59]. Thus, it is possible to design sFasL variants that possess a clear therapeutic window, at least in murine model systems.…”

Enhancing Production and Cytotoxic Activity of Polymeric Soluble FasL-Based Chimeric Proteins by Concomitant Expression of Soluble FasL

“…We did not observe any effect of co-expressed sFasL on the net production of trimeric sfFasL, likely because it is already produced at saturating levels when expressed alone in the optimized experimental conditions used. For pfFasL, which is polymeric [21], the production of this chimera is enhanced by up to 10 fold in the presence of sFasL. For more complex FasL-based chimeras, such as HLA-pfFasL and TCR-pfFasL, production is also improved, but to a lower 2 to 5 fold range.…”

“…The soluble FasL (sFasL) and the soluble polymeric FasL (pFasL) constructs were previously described [21]. Regarding the TCR-pFasL, two constructs were generated by fusing the extracellular regions of the γ4 TCR chain (aa 20 to 295) or of the δ5 TCR chain (aa 27 to 272) to the pFasL coding sequence as follows.…”

“…It is a fusion protein between human sFasL and an 81 amino acids subdomain of the human Leukemia Inhibitory Factor (LIF) receptor chain gp190 having a spontaneous propensity to self-associate. It displays a higher cytotoxic activity than the commercially available antibody-crosslinked Flag-tagged sFasL (sfFasL), and is able to delay tumor growth in a murine model of transplanted human tumor cells [21]. To further improve this recombinant death inducer, we focused on two complementary objectives.…”