Method of modifying isoelectric point of antibody via amino acid substitution in cdr

Abstract

The present inventors provide methods for modifying the isoelectric point of an antibody while retaining its antigen-binding activity, comprising modifying the charge of at least one exposable amino acid residue on the surface of the complementarity determining region (CDR). The present invention also provides methods for purifying multispecific antibodies, comprising modifying isoelectric point, and methods for improving the plasma pharmacokinetics of antibodies, comprising modifying isoelectric point. The present invention further provides antibodies with a modified isoelectric point, pharmaceutical compositions comprising the antibodies as an active ingredient, and methods for producing the antibodies and compositions.

1 - 24 . (canceled) 
     
     
         25 . A method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide each of which comprises an antibody variable region, which comprises:
 (a) modifying a nucleic acid encoding a polypeptide so as to modify the charge of at least one exposable amino acid residue on the surface of the CDR region of the first polypeptide and second polypeptide, specifically modifying both or either one of a nucleic acid encoding the amino acid residues of the first polypeptide and a nucleic acid encoding the amino acid residues of the second polypeptide, so as to increase the difference between the isoelectric points of the first polypeptide and second polypeptide when compared to before modification;   (b) culturing a host cell to express the nucleic acids; and   (c) collecting a multispecific antibody from the host cell culture.   
     
     
         26 . The method of  claim 25 , wherein the step of collecting the multispecific polypeptide comprising the first polypeptide and second polypeptide from the host cell culture is achieved by a standard chromatography. 
     
     
         27 . The method of  claim 25 , wherein the nucleic acid is modified so that the peaks for homomultimer of the first polypeptide, homomultimer of the second polypeptide, and heteromultimer of the first polypeptide and second polypeptide are more clearly separated in a standard chromatographic analysis when compared to those before modification. 
     
     
         28 . The method of  claim 25 , wherein the multispecific polypeptide is a multispecific antibody. 
     
     
         29 . A multispecific antibody that is produced by the method of  claim 27 . 
     
     
         30 . The multispecific antibody of  claim 29 , which is a bispecific antibody. 
     
     
         31 - 35 . (canceled) 
     
     
         36 . A multispecific antibody comprising a first polypeptide and a second polypeptide, each of which comprises an antibody variable region, whose isoelectric points are different from each other, and at least one amino acid residue of the first polypeptide selected from amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region and positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat's numbering system is charged. 
     
     
         37 . The antibody of  claim 36 , wherein at least one amino acid residue of the second polypeptide selected from amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region and positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat's numbering system has no charge or has a charge opposite to the charge of the selected amino acid residue of the first polypeptide. 
     
     
         38 . The antibody of  claim 36 , wherein the amino acid residue having a charge and the amino acid residue having an opposite charge as a combination are each selected from the different group of:
 (a) glutamic acid (E) and aspartic acid (D); and   (b) lysine (K), arginine (R), and histidine (H).   
     
     
         39 . The multispecific antibody comprising a first polypeptide and a second polypeptide of  claim 36 , which gives separated peaks for the homomultimer of the first polypeptide and the homomultimer of the second polypeptide in a standard chromatographic analysis. 
     
     
         40 . A composition comprising a pharmaceutically acceptable carrier and the antibody of  claim 36 . 
     
     
         41 . A nucleic acid encoding a polypeptide that constitutes the antibody of  claim 36 . 
     
     
         42 . A host cell comprising the nucleic acid of  claim 41 . 
     
     
         43 . A method for producing an antibody, which comprises the steps of culturing the host cell of  claim 42  and collecting the polypeptide from the cell culture. 
     
     
         44 . (canceled) 
     
     
         45 . The method of  claim 25 , wherein said at least one exposable amino acid residue is selected from amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region and positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat's numbering system. 
     
     
         46 . A method for producing a multispecific polypeptide comprising a first polypeptide and a second polypeptide, each of which comprises an antibody variable region, wherein the method comprises the steps of:
 (a) modifying both or either one of a nucleic acid encoding the amino acid residues of the first polypeptide and a nucleic acid encoding the amino acid residues of the second polypeptide, so that the difference between the isoelectric point of the first polypeptide and that of the second polypeptide will be 1.0 or more, preferably 1.2 or more, and more preferably 1.5 or more;   (b) culturing host cells to express the nucleic acids; and   (c) collecting the multispecific antibody from the host cell culture.   
     
     
         47 . The method of  claim 46 , wherein the step of collecting the multispecific polypeptide comprising the first polypeptide and second polypeptide from the host cell culture is achieved by a standard chromatography. 
     
     
         48 . The method of  claim 46 , wherein the nucleic acid is modified so that the peaks for homomultimer of the first polypeptide, homomultimer of the second polypeptide, and heteromultimer of the first polypeptide and second polypeptide are more clearly separated in a standard chromatographic analysis when compared to those before modification. 
     
     
         49 . The method of  claim 46 , wherein the multispecific polypeptide is a multispecific antibody. 
     
     
         50 . The method of  claim 49 , wherein the multispecific antibody is a bispecific antibody. 
     
     
         51 . The method of  claim 46 , wherein the antibody variable region comprises at least one amino acid residue selected from amino acid residues at positions 31, 61, 62, 64, and 65 in the heavy chain variable region and positions 24, 27, 53, 54, and 55 in the light chain variable region according to Kabat's numbering system. 
     
     
         52 . A multispecific antibody that is produced by the method of  claim 48 . 
     
     
         53 . The multispecific antibody of  claim 52 , which is a bispecific antibody.