CHO expression of a novel human recombinant IgG1 anti‐RhD antibody isolated by phage display

Abstract

Summary. Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones, LD-6-3 and LD-6-33, specific for RhD. These Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growthadapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

A single recombinant anti-RhD IgG prevents RhD immunization: association of RhD-positive red blood cell clearance rate with polymorphisms in the FcγRIIA and FcγIIIA genes

“…To avoid the necessity for hyperimmune plasma, a recombinant anti-RhD antibody has been developed (MonoRho) and produced in a stable Chinese hamster ovary (CHO) cell line, MDJ8s. 2 Comparison of MonoRho with a polyclonal anti-RhD product (Rhophylac) for specificity and Fc function gave similar results in vitro. The next major question concerns the clinical efficacy and safety of a single recombinant antibody compared with a polyclonal antibody preparation.…”

“…Its development was based on RhD-specific phage isolated from phage display libraries and subsequent construction of the full-length human IgG1. 2,9 MonoRho recognizes a discontinuous epitope on loops 3, 4, and 6 of the RhD protein (Miescher et al 2 ; and S.M., unpublished data, July 2002). Clinical material was produced in a 200-L batchfermentation process, purified, and supplied in ready-to-use syringes containing 300 g antibody in 1 mL solution.…”

“…2 Polyclonal anti-RhD (Rhophylac; ZLB Bioplasma, Bern, Switzerland) 10 was supplied in ready-to-use syringes containing 1500 international units (IU) (300 g) anti-RhD determined by AutoAnalyzer.…”

Combination of yeast hydrolysates to improve CHO cell growth and IgG production

“…Cell culture CHO-AMW cell line producing a recombinant IgG1 anti-human RhD antibody was provided by Dr. F. Wurm (EPFL-Lausanne) (Miescher et al 2000). Cells were adapted to grow in suspension in PF-BDM, a protein free and chemically defined medium (Schneider 1989), consisting in a 5:5:1 (v:v:v) mixture of Iscove's MDM (Eurobio, Courtaboeuf, France), Ham's F12 (Eurobio), and NCTC 135 (Eurobio) media.…”

Low-temperature pausing of cultivated mammalian cells

“…Recombinant CHO-Clone 161 cells expressing the enhanced green fluorescent protein (GFP) were grown as an adherent culture in DMEM/F12 medium in the presence of 2% FCS (Hunt et al, 1999). Suspension cultures of recombinant CHO-YIgG3 cells that express the enhanced yellow fluorescent protein (YFP) and a human IgG were maintained in spinner flasks stirred at 90 rpm in serum-free ProCHO5-CDM medium (Cambrex BioScience, Walkersville, MD) supplemented with 4 mM glutamine (Miescher et al, 2000;Hunt et al, 2002).…”