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

Abstract

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 showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. 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 growth-adapted 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.

Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin®bioreactors

“…Cell growth was accessed with the Packed Cell Volume method. The dual expression vector pXLGCHO-A3, containing the cDNAs coding for human anti-Rhesus D IgG1 heavy and light chain cloned in separate expression cassettes in a head-to-head orientation, was kindly provided by Excellgene SA [4]. Transfections are performed at a cell density of 5.5 million cells/mL at a volume of 5 mL by the direct addition of 15 µg of pDNA and 76 µg of linear 25 kDa Polyethylenimine (PEI, Polysciences, Eppenheim, Germany)…”

Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization

“…These MAB can rapidly eliminate D + erythrocytes from circulation [2], but they do not prevent D-sensitization (Table 1). On the other hand, all polyclonal antirhesus Ig, as well as BRAD-5 [9] and MonoRho MAB [13] are active in ADC test with lymphocytes and inhibit anti-D response. These indirect data suggest that antibodies with immunosuppressive effect induce hemolysis in the "lymphocytic" variant of ADC test and are not inactivated by normal Ig.…”

Rh and RHAG Blood Group Systems

“…Despite anti -D immunoglobulin prophylaxis, in England and Wales at least 500 fetuses develop haemolytic disease per year, and about 25 -30 babies die from HDFN [596] . Genetically engineered IgG anti -D molecules, secreted by insect or mammalian cell lines, have a functional Fc domain and behave normally in immunological functional assays [574,575] . Preimplantation genetic diagnosis has been applied in the management of pregnancy in couples where the woman has high levels of anti -D and the man is heterozygous for D -positivity [303] .…”