Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.

A Fas agonist induces high levels of apoptosis in haematological malignancies.

We developed and tested a potent hexameric Fas agonist, termed MegaFasL, for its cytotoxic effects on a panel of human haematopoietic malignant cells and healthy human haematopoietic progenitor cells (CD34+CD38low). Results demonstrated that MegaFasL induced apoptosis in cell lines and primary cells representing multiple myeloma (MM), acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and Burkitt's lymphoma. Cells from a chronic myeloid leukaemia (CML) line and from patients with chronic lymphocytic leukaemia (CLL) were resistant. Furthermore, CD34+CD38low progenitor cells were also resistant to MegaFasL. The data indicate that MegaFasL could be a highly efficient therapeutic agent ex vivo or potentially in vivo.

The combination of chemotherapy and intraperitoneal MegaFas Ligand improves treatment of ovarian carcinoma.

OBJECTIVE: MegaFas Ligand (MFL) is a recombinant molecule that efficiently triggers apoptosis after binding to the Fas receptor expressed on target cells. The purpose of this study was to determine the potency of MFL in vitro and efficacy in vivo for intraperitoneal treatment of mice implanted with human ovarian carcinoma cells. METHODS: The potency of MFL was compared to that of other Fas agonists in a cytotoxicity assay on SKOV-3 cells. The potency of MFL was further determined by measuring apoptosis in combination with cisplatin. The efficacy of MFL was determined in vivo using peritoneal xenograft models of human ovarian carcinoma. RESULTS: In vitro, MFL induced significantly higher levels of apoptosis than other Fas agonists, and was able to overcome the resistance of the ovarian cancer cell line SKOV-3 to Fas agonist antibody. MFL exerted an enhanced cytotoxic effect when combined with platinum-based drugs, leading to significantly more apoptosis than by incubation with MFL or these drugs alone. Treatment of mice xenografted with SKOV-3 and HOC79 ovarian tumors by intraperitoneal administration of MFL alone or in combination with cisplatin resulted in a significant decrease in peritoneal tumor nodules and ascitic cells, and prolongation of survival as compared to non-treated mice. The beneficial effects of MFL treatment occurred in the absence of severe toxicity. CONCLUSION: MFL is a novel pro-apoptotic molecule that is able to efficiently induce apoptosis in ovarian cancer cells as well as to potentiate the activity of chemotherapeutic agents in vitro and in vivo.