CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display
| Autor: | Eddy Kragten, Maria De Jesus, Radmila Moudry, Sylvia Miescher, Michel Kobr, Beda M. Stadler, Monique Vogel, Monique Zahn-Zabal, Hanspeter Amstutz, Martin A. Imboden, Nicolas Mermod, Florian M. Wurm, Johann Bichler, Igor Fisch |
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| Předmět: |
Phage display
Erythrocytes Rho(D) Immune Globulin Molecular Sequence Data CHO Cells Biology Rh Isoimmunization law.invention Immunoglobulin Fab Fragments/*genetics/isolation & purification Immunoglobulin Fab Fragments Biotechnology/*methods law Cricetinae Immunoglobulin G/*genetics Animals Humans Bacteriophages Cloning Molecular Antibody-dependent cell-mediated cytotoxicity Recombinant Proteins/metabolism Expression vector Rh Isoimmunization/prevention & control Base Sequence Chinese hamster ovary cell Molecular Rho(D) Immune Globulin/*metabolism Hematology Fragment crystallizable region Molecular biology Bromelains Recombinant Proteins Bromelains/pharmacology Immunoglobulin G biology.protein Recombinant DNA Antibody Rh blood group system Biotechnology Cloning |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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