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    • 5. 发明专利
    • AT476503T
    • 2010-08-15
    • AT01123596
    • 2001-10-01
    • DEUTSCHES KREBSFORSCH
    • BREITLING FRANKMOLDENHAUER GERHARDPOUSTKA ANNEMARIEKUEHLWEIN THORSTENLUETTGAU SANDRA
    • C12N15/09C12N15/10C07K16/30C07K16/40C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12P21/08C40B40/02
    • Humanizing a hybridoma cell (A) so that the expressed antibody (Ab) is bound to the surface of the cell expressing it, so as to allow detection and selection of those cells that present a humanized constant Ab domain, comprises transfecting the hybridoma cell line with heavy and light chain constant domains, and expanding cells that express Ig heavy chain and kappa or lambda light chain constant regions. Humanizing a hybridoma cell (A) comprises: (a) transfecting the hybridoma cell line with a DNA sequence (I) that encodes one or more of the human constant immunoglobulin (Ig) G, M, A, D or E domains; (b) (I) is flanked by DNA sequences (II) that are homologous with chromosomal gene regions that flank the constant domains of the active heavy chain-encoding gene locus in (a); (c) one or a few cells are expanded that, on the basis of homologous recombination (HR), express an Ig heavy chain with a humanized constant region; (d) a DNA sequence (III), encoding a human constant kappa or lambda domain, is transfected into the cells, where (III) is flanked by sequences (IV) homologous with chromosomal gene regions that flank the constant domain of the active kappa or lambda gene locus in (a); and (e) one or a few cells are expanded that, on the basis of HR, express a kappa or lambda chain having a human constant region. The expressed Ab is bound to the surface of the cell expressing it, through covalent bonding of the membrane-bound splice variant of the heavy chain, making it possible to detect and select those cells that present a human constant antibody domain. Independent claims are also included for: (1) vector that contains one or more of the DNA sequences specified above; and (2) host cell that contains a vector of (1).