title: Construction and characterization of a functional chimeric murine-human antibody directed against human fibrin fragment-D dimer.
abstract: 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.
authors.affiliation: Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
authors.forename: F
authors.initials: F
authors.lastname: Bulens
authors.forename: A M
authors.initials: AM
authors.lastname: Vandamme
authors.forename: H
authors.initials: H
authors.lastname: Bernar
authors.forename: L
authors.initials: L
authors.lastname: Nelles
authors.forename: R H
authors.initials: RH
authors.lastname: Lijnen
authors.forename: D
authors.initials: D
authors.lastname: Collen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D002460:Cell Line; D002678:Chimera; D005338:Fibrin Fibrinogen Degradation Products / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D005338:Fibrin Fibrinogen Degradation Products; D007127:Immunoglobulin Constant Regions; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins