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1. CA2700701 - METHOD OF MODIFYING ISOELECTRIC POINT OF ANTIBODY VIA AMINO ACID SUBSTITUTION IN CDR

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CLAIMS:
1. A method for increasing any one of parameters selected from area under the concentration curve (AUC), mean retention time in plasma, and half-life in plasma (t1/2) of an IgG antibody as compared to the antibody before modification while retaining the antigenbinding activity of the variable region of the antibody, which comprises modifying the isoelectric point of the antibody by modifying the charge of at least one exposable amino acid residue on the surface of a complementarity determining region (CDR) of the antibody, wherein the charge of the amino acid residue is modified by amino acid substitution and said 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.
2. The method of claim 1, wherein the antibody is a chimeric antibody, humanized antibody, or human antibody.
3. The method of claim 1 or 2, wherein the antibody is a multispecific antibody that binds to at least two types of antigens.
4. The method of any one of claims 1 to 3, wherein the modification in the charge of the amino acid residue results in a change of 1.0 or more in the theoretical isoelectric point.
5. The method of any one of claims 1 to 4, wherein the modified amino acid is selected from the amino acid residues in either of groups (a) and (b) below: (a) glutamic acid (E) and aspartic acid (D); and (b) lysine (K), arginine (R), and histidine (H).
6. A method for producing an 1gG antibody with an increased parameter selected from area under the concentration curve (AUC), mean retention time in plasma, and halflife in plasma (t1/2) as compared to the antibody before modification while retaining the antigenbinding activity of the variable region of the antibody, which comprises: (a) modifying the isoelectric point of an antibody by modifying a nucleic acid encoding the antibody so as to modify the charge of at least one exposable amino acid residue on the surface of a complementarity determining region (CDR) of the antibody, wherein the charge of the amino acid residue is modified by amino acid substitution, and said 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, and:
(b) culturing a host cell to express the nucleic acid; and (c) collecting the IgG antibody from the host cell culture.
7. The method of claim 6, wherein the antibody is a chimeric antibody, humanized antibody, or human antibody.
8. The method of claim 6 or 7, wherein the antibody is a multispecific antibody that binds to at least two types of antigens.
9. The method of any one of claims 6 to 8, wherein the modification in the charge of the amino acid residue results in a change of 1.0 or more in the theoretical isoelectric point.
10. The method of any one of claims 6 to 9, wherein the modified amino acid is selected from the amino acid residues in either of groups (a) and (b) below: (a) glutamic acid (E) and aspartic acid (D); and (b) lysine (K), arginine (R), and histidine (H).
11. A method for producing a multispecific IgG antibody comprising a first polypeptide and a second polypeptide each of which comprises an antibody variable region, wherein the antibody has an increased parameter selected from area under the concentration curve (AUC), mean retention time in plasma, and half-life in plasma (t1/2) as compared to the antibody before modification while retaining the antigen-binding activity, which comprises: (a) modifying a nucleic acid(s) encoding a polypeptide(s) so as to modify the charge of at least one exposable amino acid residue on the surface of a complementarity determining region (CDR) of the first polypeptide and/or the second polypeptide, wherein the charge of the amino acid residue is modified by amino acid substitution, and said 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, 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 IgG antibody from the host cell culture.
12. The method of claim 11, wherein the step (c) of collecting the multispecific IgG antibody from the host cell culture is achieved by a standard chromatography.
13. The method of claim 11 or 12, wherein the nucleic acid is modified so that 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.
14. The method of any one of claims 11 to 13, wherein the multispecific IgG antibody is a bispecific IgG antibody.
15. A method for producing an IgG antibody with an increased parameter selected from area under the concentration curve (AUC), mean retention time in plasma, and halflife in plasma (t1/2)as compared to the antibody before modification while retaining the antigen-binding activity, which comprises a human-derived framework region (FR), a human constant region, and a complementarity determining region (CDR) selected from the group consisting of human-derived CDRs, nonhuman animal-derived CDRs, and synthetic CDRs, wherein the method comprises modifying the isoelectric point of the antibody by modifying the charge of at least one exposable amino acid residue on the surface of the CDR of the antibody, wherein the charge of the amino acid residue is modified by amino acid substitution, and said 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 and is different in the charge from the amino acid residue at the corresponding position in the CDR of the antibody before modification.
16. The method of claim 15, wherein the human constant region comprises a human Fc domain.
17. A method for producing an IgG antibody with an increased parameter selected from area under the concentration curve (AUC), mean retention time in plasma, and halflife in plasma (t1/2) when compared to that before amino acid modification while retaining the antigen-binding activity, wherein the method comprises modifying the isoelectric point of the antibody by modifying the charge of at least one amino acid residue on the surface of a complementarity determining region (CDR) of the antibody, wherein the charge of the amino acid residue is modified by amino acid substitution, and said 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.
18. The method of claim 17, wherein the modified amino acid is selected from the amino acid
residues in either of groups (a) and (b) below: (a) glutamic acid (E) and aspartic acid (D); and (b) lysine (K), arginine (R), and histidine (H).
19. The method of any one of claims 1 to 18, further comprising a step of measuring the isoelectric point of said antibody.