Expression of Recombinant Mouse/Human Chimeric Antibody Specific to Human GMP-140/P-selectin

• Published in: Journal of biochemistry (Tokyo) Vol. 115; no. 3; pp. 608 - 614
• Main Authors: Kurome, Toru, Katayama, Masahiko, Murakami, Keiko, Hashino, Kimikazu, Kamihagi, Kyoko, Yasumoto, Masazumi, Kato, Ikunoshin
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.03.1994
• Subjects: activation; Amino Acid Sequence; Animals; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody Specificity; Antigen-Antibody Reactions; Antigens, CD - immunology; Base Sequence; Binding, Competitive; CD62; chimerization; Cloning, Molecular; DNA, Complementary - chemistry; DNA, Complementary - genetics; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; Genetic Vectors; Humans; Immunohistochemistry; Mice; Molecular Sequence Data; monoclonal; P-Selectin; Plasmids; platelet; Platelet Membrane Glycoproteins - immunology; Recombinant Fusion Proteins
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a124383

To construct a mouse/human chimeric antibody, we cloned the genomic DNAs for Ig from a murine hybridoma that produces PL7–6 monoclonal antibody specific to human P-selectin and expressed them in SP2/0 myelomas using a series of pSV2 vectors. Transfected cells that produce the mouse/human chimeric anti-human P-selectin antibody were geneticin-selected and screened by an immunoassay using immobilized antigen. The chimeric antibody, cPL-2R1, expressed by the resultant clone has the murine Ig variable region and the human Ig constant region. The native antibody PL7–6 and the chimeric antibody cPL-2R1 react equally with purified P-selectin and thrombin-stimulated platelets. Competitive inhibition tests demonstrated that the native antibody PL7–6 and the chimeric antibody cPL-2R1 had identical affinity for purified human P-selectin. Thus, although human IgG1 constant region was substituted for the murine counterpart in this chimeric antibody, its specificity and binding affinity for P-selectin was not altered. This chimeric antibody may prove useful when employed in combination with imaging reagents or therapeutic drugs for targeting activated platelets or endothelium in patients with thrombosis or intravascular inflammation.