Molecular and Therapeutic Characterization of Anti-ectodysplasin A Receptor (EDAR) Agonist Monoclonal Antibodies

Abstract

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.

Antibodies That Block or Activate Mouse B Cell Activating Factor of the Tumor Necrosis Factor (TNF) Family (BAFF), Respectively, Induce B Cell Depletion or B Cell Hyperplasia

“…6C). This kinetics is in line with the 10-day half-life of a mouse IgG1 administered in mice (26). When treatment was repeated every 2 weeks, circulating B cells were depleted to the level of BAFF KO mice by week 4 and thereafter remained at low levels up to the end of the experiment at 6 months (Fig.…”

“…Anti-mBAFF antibodies 5A8 and 1C9 (rat IgG1) (21), anti-EDAR mAb3 (26), and anti-EDA EctoD1 and EctoD3 (23) have been described previously. Anti-FLAG M2 antibody was from Sigma.…”

Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia

“…Plasmids and Recombinant Proteins-Plasmids used in this study were reported before (14,27,29,30) or constructed using standard molecular biology techniques (Table 1). A fully human form of Fc-EDA1 was kindly provided by Edimer Pharmaceuticals (Cambridge, MA).…”

“…Generation and Purification of Anti-EDA Monoclonal Antibodies-Eda-deficient Tabby mice were immunized with Fc-EDA1 as described previously for EDAR-Fc (30). Cells harvested from mice positive for anti-EDA antibodies were used to generate hybridoma.…”

“…Administration of Proteins to Eda-deficient and Wild Type Animals-Eda-deficient Tabby pups were treated on the day of birth, as described, with one intraperitoneal injection of Fc-EDA1 at 2.5 mg/kg that was preincubated for 1 h with anti-EDA antibodies EctoD1 (n ϭ 3) or EctoD2 (n ϭ 4) at 10 mg/kg (30). Examination of tail hairs and sweat tests were performed at weaning.…”