Anti-CD20 Antibody with Multimerized Fc Domains: A Novel Strategy To Deplete B Cells and Augment Treatment of Autoimmune Disease

Abstract

We developed a fully recombinant anti-CD20 protein derived from cDNA encoding one Fab domain, two IgG1 Fc regions, the IgG2 hinge, and an isoleucine zipper. This protein, called GB4542, contained both the homodimer and higher-order multimers. Binding studies revealed that GB4542 preferentially bound CD20+ cells yet also recognized CD20−FcγR+ PBMC. In contrast, a control mAb containing the identical Fab region, GB4500, failed to bind CD20−FcγR+ PBMC. Consistent with these findings, interactions between GB4542 and the canonical FcγRs had substantially lower KD values than correlate interfaces between GB4500 and these receptors. At low concentrations, GB4542 showed enhanced Ab-dependent cellular cytotoxicity, Ab-dependent cellular phagocytosis, and complement-dependent cytotoxicity compared with GB4500. However, at higher concentrations, an Fc analog of GB4542 inhibited anti-CD20 mAb–mediated B cell clearance through direct blocking of both Fc–FcγR interactions and C1q deposition on target cells. Furthermore, the higher-order multimer fraction of GB4542 demonstrated greater binding avidity with the canonical FcγRs and was associated with inhibitory effects observed in Ab-dependent cellular phagocytosis and complement-dependent cytotoxicity assays. These data suggest that GB4542 might have utility in the treatment of autoimmune diseases by combining both mAb-mediated B cell depletion and multimerized Fc-mediated tolerogenic effects.

A recombinant human IgG1 Fc multimer designed to mimic the active fraction of IVIG in autoimmunity

“…The in vitro blocking of IC binding to CHO-FcγR cells and phagocytosis assays were performed with GL-2045 produced by Gliknik Inc. All the in vivo experiments were tested using GL-2045 produced by Pfizer. G001 was prepared by Gliknik Inc. (51). The IVIG used throughout was Gamunex-C (lot no.…”

“…SDS-PAGE analysis. Nonreduced and reduced SDS-PAGE analyses of GL-2045 were performed as previously described (51).…”

“…CHO-huFcγRs were pretreated with G001 or GL-2045 at 10 and 100 μg/ml for 15 minutes at room temperature (51). Serum samples from 6 patients with RA (52), used as source of IC (rheumatoid factor IgG > 60 U/ml), were added directly to the culture to achieve a final concentration of 10% and incubated for 15 minutes at room temperature.…”

A fully recombinant human IgG1 Fc multimer (GL-2045) inhibits complement-mediated cytotoxicity and induces iC3b

“…The fractions of GL-2045 were prepared by gel filtration and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 16 IVIG was purchased from Atlantic Biologicals (Miami, FL). HAGG was made by heating 100 mg of IVIG (10 or 50 mg/mL) at 63°C for 30 minutes.…”

“…16 A 96-well plate was coated with C1q at 5 mg/mL overnight in phosphate-buffered saline, followed by plate washing and blocking. The plate was then incubated with serial dilutions of test compounds in blocking buffer for 45 minutes and washed 5 times.…”