Construction and characterization of a functional chimeric murine-human antibody directed against human fibrin fragment-D dimer
| Autor: | F. Bulens, Roger Lijnen, Desire Collen, Hilde Bernar, Anne-Mieke Vandamme, Luc Nelles |
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| Rok vydání: | 1991 |
| Předmět: |
medicine.drug_class
Macromolecular Substances Genetic Vectors Molecular Sequence Data Restriction Mapping Immunoglobulin Variable Region Immunoglobulin light chain Monoclonal antibody Transfection Biochemistry Epitope law.invention Cell Line Sepharose Fibrin Fibrinogen Degradation Products Mice law medicine Animals Humans biology Base Sequence Chimera Chinese hamster ovary cell Antibodies Monoclonal Molecular biology Recombinant Proteins Recombinant DNA biology.protein Immunoglobulin Light Chains Antibody Immunoglobulin Constant Regions Oligonucleotide Probes Fibrinolytic agent Plasmids |
| Zdroj: | European journal of biochemistry. 195(1) |
| ISSN: | 0014-2956 |
| Popis: | Fibrin-directed monoclonal antibodies may be clinically useful for in vitro thrombus imaging and for the targeting of fibrinolytic agents to blood clots. One such murine monoclonal antibody, (mAb-15C5), raised against the fragment-D dimer epitope of cross-linked human fibrin, was previously characterized [Holvoet, P., Stassen, J. M., Hashimoto, Y., Spriggs, D., Devos, P. & Collen, D. (1989) Thromb. Haemostasis 61, 307-313] has recently been cloned and expressed [Vandamme, A.-M., Bulens, F., Bernar, H., Nelles, L., Lijnen, H. R. & Collen, D. (1990) Eur. J. Biochem. 192, 767-775]. In order to reduce the immunogenicity of the murine mAb-15C5 in man, we have now constructed a murine--human chimera of mAb-15C5, by substituting the cDNA sequences encoding the constant regions of the murine kappa light chain and gamma 1 heavy chain by the corresponding human genomic sequences. Both chimeric murine--human Ig chains were cloned into two separately selectable expression vectors, which were contransfected into Chinese hamster ovary (CHO) cells. Murine--human chimeric mAb-15C5 (mAb-15C5Hu) was purified from the conditioned medium of selected cell lines by chromatography on Zn-chelating Sepharose, protein-A-Sepharose and on insolubilized antigen (fragment-D dimer), with a final yield of 29 micrograms/l and a recovery of 33%. SDS/PAGE without reduction revealed a homogeneous band with a mobility similar to that of natural mAb-15C5, whereas after reduction, both the heavy and the light chains had slightly slower mobilities than their natural counterparts. Expression in the presence of tunicamycin suggested that the differences in gamma 1-chain mobility were due to different N-glycosylation patterns. Immunoblotting of proteins from SDS gels showed immunological reactivity of recombinant mAb-15C5Hu with goat anti-(human IgG) IgG and of recombinant and natural murine mAb-15C5 with goat anti-(mouse IgG) IgG. Competitive binding revealed a comparable affinity of recombinant murine mAb-15C5, recombinant mAb-15C5Hu and natural mAb-15C5, for fragment-D dimer, indicating that recombinant mAb-15C5Hu was obtained in a functionally intact form. Thus, mAb-15C5Hu may constitute a useful alternative to mAb-15C5 for in vivo use in man. |
| Databáze: | OpenAIRE |
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