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1. AU2008304778 - Method of modifying isoelectric point of antibody via amino acid substitution in CDR

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CLAIMS
1. A method for modifying the isoelectric point of a polypeptide comprising an antibody variable region while retaining the antigen-binding activity of the variable region, which comprises modifying the charge of at least one exposable amino acid residue on the surface of complementarity determining region (CDR) of the polypeptide.
2. The method of claim 1, wherein the polypeptide comprising an antibody variable region further comprises an FcRn-binding domain.
3. The method of claim 1, wherein the polypeptide comprising an antibody variable region is an IgG antibody.
4.  The method of claim 1, wherein the polypeptide comprising an antibody variable region is a chimeric antibody, humanized antibody, or human antibody.
5. The method of claim 1, wherein the polypeptide comprising an antibody variable region is a multispecific polypeptide that binds to at least two types of antigens.
6.  The method of claim 1, wherein the charge of amino acid residue is modified by amino acid substitution.
7.  The method of claim 1, wherein the modification in the charge of amino acid residue results in a change of 1.0 or more in the theoretical isoelectric point.
8.  The method of claim 1, wherein the exposable amino acid residue on the surface of the CDR region is 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.
9.  A polypeptide comprising an antibody variable region with a modified isoelectric point, which is obtained by the method of any one of claims 1 to 8.
10.  A method for controlling the plasma pharmacokinetics of a polypeptide comprising an antibody variable region, which comprises modifying the isoelectric point of the polypeptide by the method of any one of claims I to 8.
11. The method of claim 10, wherein the control of pharmacokinetics refers to increase or decrease of any one of the parameters of clearance (CL) in plasma, area under the concentration curve (AUC), mean retention time in plasma, and half-life in plasma (t1/2).
12. A polypeptide comprising an antibody variable region whose pharmacokinetics in plasma is controlled, which is obtained by the method of claim 10.
13. A method for producing a polypeptide comprising an antibody variable region with a modified isoelectric point, 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 polypeptide;
(b) culturing a host cell to express the nucleic acid; and
(c) collecting the polypeptide comprising an antibody variable region from the host cell culture.
14. The method of claim 13, wherein the polypeptide comprising an antibody variable region further comprises an FcRn-binding domain.
15. The method of claim 13, wherein the polypeptide comprising an antibody variable region is an IgG antibody.
16. The method of claim 13, wherein the polypeptide comprising an antibody variable region is a chimeric antibody, humanized antibody, or human antibody.
17.  The method of claim 13, wherein the polypeptide comprising an antibody variable region is a multispecific polypeptide that binds to at least two types of antigens.
18.  The method of claim 13, wherein the charge of amino acid residue is modified by amino acid substitution.
19.  The method of claim 13, wherein the modification in the charge of amino acid residue results in a change of 1.0 or more in the theoretical isoelectric point.
20.  The method of claim 13, wherein the exposable amino acid residue on the surface of the CDR region is 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.
21. A polypeptide comprising an antibody variable region with a modified isoelectric point, which is obtained by the method of any one of claims 13 to 20.
22. A method for producing a polypeptide comprising an antibody variable region whose pharmacokinetics in plasma is controlled, which comprises modifying the isoelectric point of the polypeptide comprising an antibody variable region by the method of any one of claims 13 to 20.
23. The method claim 22, wherein the control of pharmacokinetics refers to increase or decrease of any one of the parameters of clearance (CL) in plasma, area under the concentration curve (AUC), mean retention time in plasma, and half-life in plasma (t11/2).
24. A polypeptide comprising an antibody variable region whose pharmacokinetics in plasma is controlled, which is produced by the method of 22.
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.         A multispecific antibody comprising a first polypeptide and a second polypeptide, 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.
32.         The antibody of claim 31, 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.
33.         The antibody of claim 31, 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).
34.         The multispecific antibody comprising a first polypeptide and a second polypeptide of claim 31, which gives separated peaks for the homomultimer of the first polypeptide and the homomultimer of the second polypeptide in a standard chromatographic analysis.
35.         A composition comprising a pharmaceutically acceptable carrier and the antibody of any one of claims 31 to 34.
36.          A nucleic acid encoding a polypeptide that constitutes the antibody of any one of claims 37. A host cell comprising the nucleic acid of claim 36.
38.         A method for producing the antibody of any one of claims 31 to 34, which comprises the steps of culturing the host cell of claim 37 and collecting the polypeptide from the cell culture.
Chugai Seiyaku Kabushiki Kaisha
Patent Attorneys for the Applicant/Nominated Person
SPRUSON & FERGUSON