Method for making multispecific antibodies having heteromultimeric and common components
First Claim
1. A method of preparing a bispecific antibody that specifically binds to a first and a second antigen, wherein said first and second antigens are different from one another, said method comprising:
- (a) providing a first nucleic acid molecule encoding a first polypeptide comprising the variable region of a light chain of an antibody that binds said first antigen;
(b) providing a second nucleic acid molecule encoding a second polypeptide comprising the variable region of a heavy chain of said antibody that binds said first antigen;
(c) identifying and/or providing a third nucleic acid molecule encoding a third polypeptide comprising the variable region of a heavy chain, which, when paired with the light chain of said antibody that binds said first antigen, specifically binds to said second antigen;
(c) culturing a host cell comprising said first, second, and third nucleic acid molecules under conditions that permit formation of said bispecific antibody; and
(d) recovering said bispecific antibody.
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Abstract
The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method provides a multispecific antibody having a common light chain associated with each heteromeric polypeptide having an antibody binding domain. Additionally the method futher involves introducing into the multispecific antibody a specific and complementary interaction at the interface of a first polypeptide and the interface of a second polypeptide, so as to promote heteromultimer formation and hinder homomultimer formation; and/or a free thiol-containing residue at the interface of a first polypeptide and a corresponding free thiol-containing residue in the interface of a second polypeptide, such that a non-naturally occurring disulfide bond is formed between the first and second polypeptide. The method allows for the enhanced formation of the desired heteromultimer relative to undesired heteromultimers and homomultimers.
212 Citations
38 Claims
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1. A method of preparing a bispecific antibody that specifically binds to a first and a second antigen, wherein said first and second antigens are different from one another, said method comprising:
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(a) providing a first nucleic acid molecule encoding a first polypeptide comprising the variable region of a light chain of an antibody that binds said first antigen; (b) providing a second nucleic acid molecule encoding a second polypeptide comprising the variable region of a heavy chain of said antibody that binds said first antigen; (c) identifying and/or providing a third nucleic acid molecule encoding a third polypeptide comprising the variable region of a heavy chain, which, when paired with the light chain of said antibody that binds said first antigen, specifically binds to said second antigen; (c) culturing a host cell comprising said first, second, and third nucleic acid molecules under conditions that permit formation of said bispecific antibody; and (d) recovering said bispecific antibody. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of preparing a bispecific antibody that specifically binds to a first and a second antigen, wherein said first and second antigens are different from one another, said method comprising:
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(a) identifying a first nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of the variable region of a light chain of an antibody that binds said first antigen, wherein the amino acid sequence of said light chain of an antibody that binds said first antigen is identical to the amino acid sequence of a light chain of another antibody that binds said second antigen; (b) providing said first nucleic acid molecule; (c) providing a second nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of the variable region of a heavy chain of said antibody that binds said first antigen; (d) providing a third nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of the variable region of a heavy chain of said antibody that binds said second antigen; (e) culturing a host cell comprising said first, second, and third nucleic acid molecules under conditions that permit formation of said bispecific antibody; and (d) recovering said bispecific antibody. - View Dependent Claims (15)
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16. A method of preparing a bispecific antibody that specifically binds to a first and a second antigen, wherein said first and second antigens are different from one another, said method comprising:
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(a) identifying a first nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of the variable region of a light chain of an antibody that binds said first antigen, wherein the amino acid sequence of said light chain of an antibody that binds said first antigen is identical to the amino acid sequence of a light chain of another antibody that binds said second antigen; (b) providing a second nucleic acid molecule encoding a polypeptide comprising a light chain variable region comprising the three complementarity-determining regions (CDRs) of the light chain of said antibody that binds said first antigen, which, when paired with a heavy chain of said antibody that binds said first antigen specifically binds to said first antigen, and when paired with a heavy chain of said other antibody that binds said second antigen, specifically binds to said second antigen; (c) providing a third nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of the variable region of a heavy chain of said antibody that binds said first antigen; (d) providing a fourth nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of the variable region of a heavy chain of said other antibody that binds said second antigen; (e) culturing a host cell comprising said second, third, and fourth nucleic acid molecules under conditions that permit formation of said bispecific antibody; and (d) recovering said bispecific antibody.
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17. An isolated host cell comprising:
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(a) a first nucleic acid molecule encoding a first polypeptide comprising the variable region of a light chain of a first antibody that binds a first antigen; (b) a second nucleic acid molecule encoding a second polypeptide comprising the variable region of a heavy chain of said first antibody; (c) a third nucleic acid molecule encoding a third polypeptide comprising the variable region of a heavy chain of a second antibody that binds a second antigen; wherein the first and second antigens are different from one another, and wherein said first polypeptide, when paired with said second polypeptide specifically binds to the first antigen, and when paired with said third polypeptide specifically binds to the second antigen, and wherein said first, second and third polypeptides interact to form a bispecific antibody that specifically binds to the first and second antigens. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. An isolated host cell comprising:
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(a) a first nucleic acid molecule encoding a first polypeptide comprising a light chain variable region comprising the three complementarity-determining regions (CDRs) of a light chain of an antibody that binds a first antigen; (b) a second nucleic acid molecule encoding a second polypeptide comprising the variable region of a heavy chain of the antibody that binds the first antigen; (c) a third nucleic acid molecule encoding a third polypeptide comprising the variable region of a heavy chain of an antibody that binds a second antigen; wherein the first and second antigens are different from one another, and wherein said first polypeptide, when paired with said second polypeptide specifically binds to the first antigen, and when paired with said third polypeptide specifically binds to the second antigen, and wherein said first, second and third polypeptides interact to form a bispecific antibody that specifically binds to the first and second antigens. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
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Specification