Methods of modifying antibodies for purification of bispecific antibodies
AssigneeChugai Seiyaku Kabushiki Kaisha
InventorsTomoyuki Igawa, Hiroyuki Tsunoda
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Abstract
The present inventors devised methods for efficiently purifying bispecific antibodies using a chromatography column based on the difference in isoelectric points between the H chains of two types of antibodies, wherein the difference is introduced by modifying the amino acids present on the surface of the antibody variable regions of two types of antibodies that constitute a bispecific antibody. Furthermore, the inventors devised methods for efficiently purifying bispecific antibodies using a chromatography column by linking respective antigen binding sites (heavy chain variable regions) to the antibody constant regions having different isoelectric points, and then coexpressing these antibodies.
Claims53
A multispecific antibody comprising a first polypeptide and a second polypeptide,
- wherein the first polypeptide comprises a first heavy chain variable region and the second polypeptide comprises a second heavy chain variable region;
- wherein at least one position selected from positions 10, 12, 23, 39, 43, and 105 (Kabat numbering) in the first heavy chain variable region is occupied by a charged amino acid residue;
- wherein either the charged amino acid residue at each selected position of the first heavy chain variable region is independently selected from glutamic acid and aspartic acid, and the amino acid residue at the corresponding position(s) of the second heavy chain variable region is independently selected from lysine, arginine, and histidine;
- or the charged amino acid residue at each selected position of the first heavy chain variable region is independently selected from lysine, arginine, and histidine, and the amino acid residue at the corresponding position(s) of the second heavy chain variable region is independently selected from glutamic acid and aspartic acid;
- and
- wherein the isoelectric point of the first polypeptide is different from the isoelectric point of the second polypeptide.
A multispecific antibody comprising:
- a first polypeptide that comprises a first heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs:
- 7-13;
- and
- a second polypeptide that comprises a second heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs:
- 14-16.
A multispecific antibody comprising:
- a first polypeptide that comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
- 11,
- a second polypeptide that comprises a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
- 16, and
- a third polypeptide and a fourth polypeptide, each of which comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:
- 17.
A multispecific antibody comprising:
- a first polypeptide that comprises a first heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
- 12,
- a second polypeptide that comprises a second heavy chain variable region comprising the amino acid sequence of SEQ ID NO:
- 16, and
- a third polypeptide and a fourth polypeptide, each of which comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:
- 18.
A multispecific antibody comprising a first polypeptide and a second polypeptide,
- wherein the first polypeptide comprises a first heavy chain variable region and first heavy chain constant region, and the second polypeptide comprises a second heavy chain variable region and second heavy chain constant region;
- wherein at least one position selected from positions 10, 12, 23, 39, 43, and 105 (Kabat numbering) in the first heavy chain variable region is occupied by a charged amino acid residue;
- wherein one of the heavy chain constant regions is an IgG1 constant region and the other is either IgG4 or IgG2;
- and
- wherein the isoelectric point of the first polypeptide is different from the isoelectric point of the second polypeptide.
A multispecific antibody comprising a first polypeptide and a second polypeptide,
- wherein the first polypeptide comprises a first heavy chain variable region and the second polypeptide comprises a second heavy chain variable region;
- wherein at least one position selected from Kabat numbering positions 10, 12, 23, 39, 43, and 105 in the first heavy chain variable region is occupied by a lysine, arginine, or histidine residue;
- wherein each of positions 10, 12, 23, 39, 43, and 105 in the first heavy chain variable region that is occupied by a charged residue is occupied by a lysine, arginine, or histidine residue;
- wherein at least one position selected from Kabat numbering positions 1, 3, 5, 8, 10, 12, 13, 15, 16, 19, 23, 25, 26, 39, 42, 43, 44, 46, 68, 71, 72, 73, 75, 76, 81, 82b, 83, 85, 86, 97, 105, 108, 110, and 112 that is occupied by a lysine, arginine, or histidine residue in the first heavy chain variable region is occupied by a glutamic acid or aspartic acid residue in the second heavy chain variable region;
- and
- wherein the isoelectric point of the first polypeptide is different from the isoelectric point of the second polypeptide.
A multispecific antibody comprising a first polypeptide and a second polypeptide,
- wherein the first polypeptide comprises a first heavy chain variable region and first heavy chain constant region, and the second polypeptide comprises a second heavy chain variable region and second heavy chain constant region;
- wherein at least one position selected from the Kabat numbering positions 10, 12, 23, 39, 43, and 105 in the first heavy chain variable region is occupied by a charged amino acid residue;
- wherein at least one of the heavy chain constant regions is an IgG4 constant region;
- and
- wherein the isoelectric point of the first polypeptide is different from the isoelectric point of the second polypeptide.
Dependencies are inferred from the claim text.