Anti-RANKL (Denosumab biosimilar)

Human IgG2 monoclonal antibody (mAb) against human RANKL (TNFSF11)

Anti-hRANKL-hIgG2 is the biosimilar of the clinical antibody denosumab. Anti-hRANKL-hIgG2 features a human IgG2 constant region (Fc) and the variable region of denosumab which targets the transmembrane and soluble forms of the human RANKL (receptor activator of nuclear factor-κB ligand) antigen. RANKL is also known as member 11 of the tumor necrosis factor ligand superfamily (TNFSF11) [1]. RANKL binding to its receptor RANK induces signaling in osteoclasts and is responsible for bone resorption. RANKL-RANK also plays a pivotal role in dendritic cell maturation and T-cell differentiation [2]. 

Denosumab is a fully human IgG2 mAb that blocks the interaction of hRANKL with its receptor RANK and thus the downstream signaling. Human IgG2 is the second most common antibody present in serum. It is resistant to cleavage by proteolytic enzymes, due to a short hinge region. This isotype displays low complement-dependent cytotoxicity (CDC), very low antibody-dependent cellular phagocytosis (ADCP), and very low antibody-dependent cellular cytotoxicity (ADCC).
Denosumab has been approved by the FDA for the treatment of osteoporosis and bone metastasis.

InvivoGen's Anti-hRANKL-hIgG2 is generated by recombinant DNA technology, produced in Chinese hamster ovary (CHO) cells, and purified by affinity chromatography (protein G).

More isotypes of this antibody are available and can be used for comparison of biological activities such as ADCC (see below or in the 'upon request' section). 

 Read our review on Antibody Isotypes

References:

1. Ahern E. et al., 2018. Roles of the RANKL-RANK axis in antitumour immunity -implications for therapy. Nat Rev Clin Oncol. 15(11):676-693.
2. Cheng ML. & Fong L., 2014. Effects of RANKL-Targeted Therapy in Immunity and Cancer. Front Oncol. 3:329.

Figures

Binding of Anti-hRANKL-hIgG2 mAb to coated human RANKL protein. Human RANKL (1 µg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of the  Anti-hRANKL-hIgG2 mAb (red curve) or the Anti-β-Gal-hIgG2 control antibody (grey curve) was performed for the capture step. An HRP-labelled anti-hIgG antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

Dose-dependent inhibition of HEK-Blue™ RANKL cells response using denosumab biosimilar. A serial dilution of denosumab, a biosimilar Anti-hRANKL-hIgG2 monoclonal antibody (mAb) was incubated with 10 ng/ml of recombinant human RANKL for 2 hours prior to the addition of the HEK-Blue™ RANKL cells. After overnight incubation, the NF-kB response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as a percentage of activity.

Specifications

Target: Human receptor activator of nuclear factor-κB ligand (RANKL, TNFSF11, CD254)

Clonality: Monoclonal antibody

Clone: Denosumab (anti-hRANKL-hIgG2, kappa)

Isotype: Human IgG2, kappa

Control: Human IgG2

Source: CHO cells

Formulation: 0.2 µm filtered solution in 68 mM sodium phosphate buffer (pH 7.4) with 91 mM glycine, 5% w/v saccharose, and stabilizing agents

Purity: Purified by affinity chromatography with protein G

Tested applications: ELISA, neutralization assay

Quality control:

• Binding of Anti-hRANKL-hIgG2 to human RANKL has been confirmed using ELISA and neutralizing cellular assays.
• The complete sequence of this antibody has been verified.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

Anti-hRANKL-hIgG2 purified monoclonal antibody is provided azide-free and lyophilized. It is available in two quantities:

• hrankl-mab2: 100 µg
• hrankl-mab2-1: 10 x 100 µg (1 mg)

Anti-hRANKL-hIgG2 is shipped at room temperature.

Upon receipt, store at -20°C.

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