Natural human antibody

#This application is a 35 U.S.C. §371 filing of PCT/J98/04469, filed on Oct. 2, 1998.

#The present invention relates to a method of preparing natural humanized antibody and the natural humanized antibody obtained by said method of preparation. The present invention also relates to DNA encoding natural humanized antibody, an expression vector comprising said DNA, a host comprising said DNA, and a method of preparing natural humanized antibody from cells into which said DNA has been introduced.

#Mouse monoclonal antibodies can be relatively easily isolated by the widely used hybridoma technology (Kohler, G. and Milstein, C. Nature (1975) 256, 495–497). On the other hand, a similar technique for human hybridoma has yet to be widespread though it is expected to become so. Furthermore, there is a need for antibodies to human antigens in clinical applications, and therefore the generation of mouse monoclonal antibodies is indispensable for the development of antibody pharmaceuticals.

#FIG. 1 is a graph showing that the fluorescent intensity of chimeric anti-HM1.24 antibody is shifted similarly to that of mouse anti-HM1.24 antibody as compared to control antibody in the FCM analysis using a human myeloma cell line KPMM2.

#FIG. 2 is a graph showing that chimeric anti-HM1.24 antibody inhibits the binding of biotinylated mouse anti-HM1.24 antibody to the WISH cells in a dose-dependent manner similarly to that of mouse anti-HM1.24 antibody.

#FIG. 3 is a graph showing that chimeric anti-HM1.24 antibody has an increased cytotoxic activity to the RPMI 8226 cells with increasing E/T ratios whereas control IgG1 or mouse anti-HM1.24 antibody has no cytotoxic activity to the RPMI 8226 cells.

#1. Natural FR Sequence

#In order to produce antibodies to a variety of antigens from the genes comprising limited antibody variable regions, organisms have a mechanism of introducing random gene mutations (called somatic mutations) in the antibody variable regions. In theory this should form extremely diverse FR amino acid sequences, but in practice positions of amino acid residues more prone to the introduction of mutations and the kinds of amino acid residues appear to be limited to a certain degree as determined by structural analysis of many human antibody FRs for which actual structures have been elucidated.

#As used herein, the term FR refers to the FR that has been defined in Kabat, E. A. et al., Sequence of Proteins of Immunological Interest (1991). Thus, in the H chain, FR1 is amino acids No. 1 to 30, FR2 is amino acids No. 36 to 49, FR3 is amino acids No. 66 to 94, and FR4 is amino acids No. 103 to 113. On the other hand, in the L chain FR1 is amino acids No. 1 to 23, FR2 is amino acids No. 35 to 49, FR3 is amino acids No. 57 to 88, and FR4 is amino acids No. 98 to 107.