Identification of Commercially Available Antibodies that Block Ligand Binding by BMPR2

Osteoporosis, a disease of low bone mineral density, affects 10 million Americans and triggers significant health problems and considerable socioeconomic burdens. Current treatments for osteoporosis have significant limitations, necessitating identifying new treatment strategies via building a better understanding of the endogenous mechanisms regulating bone mass. A recent study demonstrated that removal of the BMP type 2 receptor (BMPR2) in skeletal progenitor cells of Bmpr2-cKO mice during embryonic development leads to reduced age-related bone loss by sustained elevation in bone formation rate. This present study sought to advance the translational potential of the genetic model by identifying antibodies that neutralize the ligand-binding function of the BMPR2 extracellular domain (BMPR2-ECD). This study first established a modified, cell-free immunoprecipitation assay wherein the ligand BMP2 was pulled-down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2 (found in the supernatant) was subsequently quantified by ELISA. This yielded a standard assay wherein approximately 2 ug BMPR2-ECD leads to a 70% reduction in BMP2 signal. Next, the neutralizing ability of 3F6, a mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was examined and was found to cause a dose-dependent inhibition of BMPR2-ECD ligand-binding. Given the ascites preparation of 3F6, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative-control ascites. Using these results as a guide, 1F12, another mouse monoclonal antibody raised against the ligand-binding region of BMPR2, was evaluated and was also found to neutralize the ligand-binding function of BMPR2-ECD. In contrast, no effect on ligand-binding function of BMPR2-ECD was observed with 9A10 even though this mouse monoclonal antibody is also raised against the ligand-binding region of BMPR2. These results provide proof-of-concept data for future studies evaluating inhibition of BMPR2 function in vivo as a means to reduce age-related bone loss

Identification of a bone morphogenetic protein type 2 receptor neutralizing antibody

The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFβ) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD)

A method to switch from oral dopamine agonists to rotigotine in patients with restless legs syndrome and mild augmentation.

BACKGROUND: We examined the short- and long-term efficacy and tolerability of a cross-titration algorithm from oral dopamine agonists to the rotigotine transdermal patch in patients dissatisfied with their restless legs syndrome (RLS) treatment, predominantly with mild augmentation. METHODS: Patients with RLS (n = 20) were recruited at a single site. The cross-titration consisted of decreasing oral dopaminergic agents (ropinirole by 1 mg or pramipexole by 0.25 mg) and increasing rotigotine by 1 mg every two days. Efficacy and adverse events (AEs) were assessed at one, three, six and 12 months after the switch. RESULTS: Patients had moderate-severe RLS symptoms at the baseline (mean international restless legs syndrome (IRLS) score 19.4 ± 5.5); 85% had augmentation and 45% reported afternoon RLS symptoms. The baseline mean pramipexole equivalent dose was 0.6 ± 0.3 mg. At Week 5, 85% (17/20) had successfully switched from their oral dopamine agonist to rotigotine (mean dose 2.5 ± 0.6 mg; change in IRLS score: -6.7 ± 8.4, p = 0.002); 14 patients were CGI-I responders (much or very much improved). Three patients withdrew due to lack of efficacy. Twelve months after cross-titration, 10 patients continued on rotigotine, of whom four required either higher doses of rotigotine or supplemental RLS medication compared with their optimal Week 5 dose; five patients withdrew due to AEs and two due to lack of efficacy. CONCLUSION: A cross-titration to rotigotine was efficacious after five weeks in 70% of patients dissatisfied with RLS treatment, most of whom had mild augmentation. At one year following the medication switch, 50% had discontinued rotigotine due to lack of continued efficacy or side effects

Identification of Commercially-Available Antibodies that Block Ligand-Binding by BMPR2

Osteoporosis, a disease of low bone mineral density, affects 10 million Americans and is a significant health problem and a considerable socioeconomic burden. Current treatments for osteoporosis have significant limitations, necessitating identifying new treatment strategies via building a better understanding of the endogenous mechanisms regulating bone mass. We recently demonstrated that removal of the BMP type 2 receptor, BMPR2, in skeletal progenitor cells of Bmpr2-cKO mice leads to reduced age-related bone loss due to a sustained elevation in bone formation rate. The molecular mechanism underlying this phenotype is being pursued in other work. In the present study, we sought to advance the translational potential of our genetic model by identifying antibodies that neutralize the ligand-binding function of the BMPR2 extracellular domain (BMPR2- ECD). We first established a modified, cell-free immunoprecipitation assay wherein the ligand BMP2 is pulled-down by BMPR2-ECD conjugated to Protein G beads; the unbound BMP2, which is found in the supernatant, is subsequently quantified by ELISA. This yielded a standard assay wherein approximately 2 ug BMPR2-ECD leads to 70% reduction in BMP2 signal. Next, we examined the neutralizing ability of 3F6, which is a mouse monoclonal antibody raised against the ligand-binding region of BMPR2, and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Given that the commercial availability of this antibody is as an ascites preparation, specificity of this assay was confirmed by demonstrating that ligand-binding activity of BMPR2-ECD is unchanged in the presence of non-specific, negative control ascites. Using these results as a guide, we then evaluated 1F12, which is another mouse monoclonal antibody raised against the ligand-binding region of BMPR2, and found that this antibody is also capable of neutralizing the ligand-binding function of BMPR2-ECD. These results provide proof-of-concept data for future studies evaluating inhibition of BMPR2 function in vivo as a means to reduce age-related bone loss

Identification of Commercially Available Antibodies That Block Ligand-Binding by BMPR2

The Bone Morphogenetic Protein (BMP) signaling pathway comprises the largest subdivision of the Transforming Growth Factor (TGFβ) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. When dysregulated, BMP signaling underlies numerous human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms by which BMP signaling occurs and is regulated is a highly important goal and may yield translational opportunities. Unfortunately, there are limited tools available to perform functional evaluation of this pathway and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. While significant progress has been made in developing pharmacological inhibitors for type 1 BMP receptors, specific inhibitors of type 2 receptor are poorly represented. For these reasons, we sought to identify and validate commercially available antibodies that neutralize the ligand-binding function of the Bone Morphogenetic Protein Receptor Type 2 (BMPR2) extracellular domain (ECD). Using a modified, cell-free immunoprecipitation assay quantified by ELISA, we examined the neutralizing ability of the mouse monoclonal antibody 3F6 and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. We extended the results by examining the ability of 3F6 to block endogenous BMPR2 function in a BMP-responsive cell line and found that, consistent with the results of our cell-free system, pre-treatment of HEK293T cells with 3F6 leads to reduced sensitivity in response to BMP pathway activation by BMP2. These results provide important proof-of-concept data for future studies interrogating BMPR2 function in numerous physiological and pathophysiological contexts