Methods for Producing Polypeptides by Regulating Polypeptide Association
First Claim
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1. A method for producing a polypeptide comprising a mutation in an amino acid residue forming a polypeptide interface such that polypeptide association will be regulated, wherein the method comprises:
- (a) modifying a nucleic acid encoding an amino acid residue forming the polypeptide interface from the original nucleic acid, such that polypeptide association will be inhibited;
(b) culturing host cells such that said nucleic acid is expressed; and
(c) recovering said polypeptide from the host cell culture.
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Abstract
In the course of the present invention, it was discovered that one could regulate association between polypeptides by modifying amino acid residues that form the interface during the association to amino acids carrying the same type of charge. In this context, the present invention enables efficient formation of heterologous molecules. For example, the present invention can be suitably applied to the preparation of bispecific antibodies.
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Citations
110 Claims
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1. A method for producing a polypeptide comprising a mutation in an amino acid residue forming a polypeptide interface such that polypeptide association will be regulated, wherein the method comprises:
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(a) modifying a nucleic acid encoding an amino acid residue forming the polypeptide interface from the original nucleic acid, such that polypeptide association will be inhibited; (b) culturing host cells such that said nucleic acid is expressed; and (c) recovering said polypeptide from the host cell culture. - View Dependent Claims (3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24)
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2. A method for producing a heteromultimer comprising a mutation in an amino acid residue forming an interface between polypeptides such that heteromultimer association will be regulated, wherein the method comprises:
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(a) modifying a nucleic acid encoding an amino acid residue forming the interface between polypeptides from the original nucleic acid, such that the association between polypeptides will be inhibited; (b) culturing host cells such that said nucleic acid is expressed; and (c) recovering said heteromultimer from the host cell culture. - View Dependent Claims (4, 22, 23, 98)
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- 25. A mutant polypeptide, comprising a modification made to an amino acid residue forming an interface in the original polypeptide such that the association within said polypeptide is inhibited.
- 26. A heteromultimer, comprising a modification made to an amino acid residue forming an interface between the original polypeptides such that the association between said polypeptides is inhibited.
- 52. A method for regulating polypeptide association, which comprises modifying an amino acid residue forming an interface in the original polypeptide such that the association within the polypeptide is inhibited.
- 53. A method for regulating heteromultimer association, which comprises modifying amino acid residues forming an interface between the original polypeptides such that the association between the polypeptides is inhibited.
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75. An antibody comprising a heavy chain variable region and a light chain variable region, wherein the following amino acid residues of (1) and (2) carry the same type of charge:
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(1) an amino acid residue which is included in the heavy chain variable region and corresponds to position 39 in the amino acid sequence of SEQ ID NO;
6; and(2) an amino acid residue which is included in the light chain variable region and corresponds to position 44 in the amino acid sequence of SEQ ID NO;
8. - View Dependent Claims (78, 80, 81, 82, 83, 84, 85, 86)
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76. An antibody comprising a heavy chain variable region and a light chain variable region, wherein the following amino acid residues of (1) and (2) carry the same type of charge:
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(1) an amino acid residue which is included in the heavy chain variable region and corresponds to position 45 in the amino acid sequence of SEQ ID NO;
6; and(2) an amino acid residue which is included in the light chain variable region and corresponds to position 50 in the amino acid sequence of SEQ ID NO;
8. - View Dependent Claims (103, 104, 105, 106)
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77. An antibody comprising a heavy chain variable region and a light chain variable region, wherein either one of the following amino acid residues of (1) or (2) is a charged amino acid residue:
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(1) an amino acid residue which is included in the heavy chain variable region and corresponds to position 45 in the amino acid sequence of SEQ ID NO;
6; and(2) an amino acid residue which is included in the light chain variable region and corresponds to position 50 in the amino acid sequence of SEQ ID NO;
8. - View Dependent Claims (79, 107, 108, 109, 110)
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87. An antibody comprising two or more types of heavy chain CH3 regions, wherein one to three pairs of amino acid residues in a first heavy chain CH3 region is/are selected from the pair(s) of amino acid residues indicated in (1) to (3) that carry the same type of charge:
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(1) amino acid residues included in the heavy chain CH3 region at positions 356 and 439 according to the EU numbering system; (2) amino acid residues included in the heavy chain CH3 region at positions 357 and 370 according to the EU numbering system; and (3) amino acid residues included in the heavy chain CH3 region at positions 399 and 409 according to the EU numbering system. - View Dependent Claims (88, 89, 90, 91, 92, 93, 94, 95, 96, 97)
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Specification