Anti-CD20 (Rituximab biosimilar - IgG4 (S228P) isotype)

Human IgG4 (S228P) engineered monoclonal antibody (mAb) against human CD20

Anti-hCD20-hIgG4 (S228P) features the variable region of rituximab and a mutated constant region of the human IgG4 isotype reported to reduce Fab-arm exchange [1].

Rituximab is a mouse/human chimeric monoclonal antibody that targets the CD20 antigen found on the surface of malignant and normal B lymphocytes.

The binding of rituximab to CD20 results in cell destruction through different mechanisms including direct signaling of apoptosis, complement activation, and cell-mediated cytotoxicity.

Rituximab has been approved by the FDA to treat various lymphoid malignancies, including B-cell non-Hodgkin's lymphoma and B-cell chronic lymphocytic leukemia.

Anti-hCD20-hIgG4 (S228P) is an engineered human IgG4 isotype. IgG4 antibodies display low antibody-dependent cell-mediated cytotoxicity (ADCC) and no complement-dependent cytotoxicity (CDC).

IgG4s are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half molecule) with a heavy-light chain pair from another molecule, resulting in bispecific antibodies [1-2].

IgG4 molecules thereby lose their ability to cross-link antigens and to form immune complexes under most conditions [2]. Thus, mutations that prevent Fab-arm exchange in vivo should be considered when designing therapeutic IgG4. The constant region of Anti-hCD20-hIgG4 (S228P) contains the S228P mutation which reduces Fab-arm exchange by stabilizing the disulfides in the core-hinge of the IgG4 molecules [1].

Anti-hCD20-hIgG4 (S228P) was generated by recombinant DNA technology. It has been produced in Chinese hamster ovary (CHO) cells and purified by affinity chromatography with 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). InvivoGen also provides type I and type II anti-hCD20 mAbs featuring the variable regions of Ofatumumab or Obinutuzumab, with either native or engineered Fc regions.

References:

1. Labrijn AF. et al., 2009. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo. Nat Biotechnol. 27(8):767-71.
2. van der Neut Kolfschoten M. et al., 2007. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange. Science. 317(5844):1554-7.

Figures

Comparison of ADCC potency for native and engineered anti-human CD20 antibody isotypes: Raji-Null cells were incubated with gradient concentrations of Anti-hCD20 or Anti-β-galactosidase (β-gal) mAbs for 1 hour. Jurkat-Lucia™ NFAT-CD16 effector cells were then co-incubated with targets cells for 6 hours. NFAT activation, reflecting the induced ADCC response, was assessed by determining Lucia luciferase activity in the supernatant using QUANTI-Luc™. Percentages of the maximal response normalized to the IgG1 isotype are shown

Specifications

Target: Human CD20

Clonality: Engineered monoclonal antibody

Clone: Rituximab (Anti-hCD20-hIgG1, kappa)

Isotype: Human IgG4 (S228P), kappa

Control: Human IgG4 (S228P)

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 application: Flow cytometry and ADCC

Quality control:

• Binding of Anti-hCD20-hIgG4 (S228P) to human CD20 has been tested using flow cytometry.
• 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-hCD20-hIgG4 (S228P) purified monoclonal antibody is provided azide-free and lyophilized. It is available in two quantities:

• hcd20-mab14: 100 µg
• hcd20-mab14-03: 3 x 100 µg

Product is shipped at room temperature.

 Upon receipt, store lyophilized antibody at -20°C.