Multivalent and multispecific binding proteins, their manufacture and use
First Claim
1. A dimer comprising:
- (i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked directly to each other by an amide bond or by a polypeptide linker of from 1 to 5 amino acids; and
(ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked directly to each other by an amide bond or by a polypeptide linker of from 1 to 5 amino acids;
the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and
the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site;
said first antigen binding site being different from said second antigen binding site.
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Accused Products
Abstract
Polypeptides comprising a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the domains being linked but incapable of associating with each other to form an antigen binding site, associate to form antigen binding multimers, such as dimers, which may be multivalent or have multispecificity. The domains may be linked by a short peptide linker or may be joined directly together. Bispecific dimers may have longer linkers. Methods of preparation of the polypeptides and multimers and diverse repertoires thereof, and their display on the surface of bacteriophage for easy selection of binders of interest, are disclosed, along with many utilities.
459 Citations
85 Claims
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1. A dimer comprising:
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(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked directly to each other by an amide bond or by a polypeptide linker of from 1 to 5 amino acids; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked directly to each other by an amide bond or by a polypeptide linker of from 1 to 5 amino acids; the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A vector comprising a nucleotide sequence encoding a first polypeptide and a nucleotide sequence encoding a second polypeptide, each of the first and second encoded polypeptides comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first domain of each polypeptide being linked to the second domain of that polypeptide directly to each other by an amide bond or by a polypeptide linker of from 1 to 5 amino acids;
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the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site; the nucleotide sequence encoding the first polypeptide being linked to a nucleotide sequence encoding a signal sequence for export of the first polypeptide from a host cell upon expression; the nucleotide sequence encoding the second polypeptide being linked to
1) a nucleotide sequence encoding a signal sequence for export of the second polypeptide from a host cell upon expression and
2) a nucleotide sequence encoding a surface component of a filamentous bacteriophage for display of the second polypeptide on the surface of a bacteriophage particle upon expression, the vector being capable of being packaged within a bacteriophage particle. - View Dependent Claims (41)
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42. A vector comprising:
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(a) a nucleotide sequence encoding a first polypeptide, said first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, wherein the domains of the first polypeptide are linked directly by an amide bond or by a polypeptide linker of from 1 to 5 amino acids; (b) a nucleotide sequence encoding a second polypeptide, said second polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the domains of the second polypeptide are linked directly to an amide bond or by a polypeptide linker of from 1 to 5 amino acids;
wherein the first domain of the first polypeptide and the second domain of the second polypeptide associate to form a first antigen binding site, and wherein the second domain of the first polypeptide and the first domain of the second polypeptide associate to form a second antigen binding site;
said first antigen binding site being different from said second antigen binding site; and(c) a nucleotide sequence encoding a surface component of a filamentous bacteriophage;
whereinexpression of nucleotide sequence (a) produces a polypeptide ("A") comprising the first and second domains of said first polypeptide; expression of nucleotide sequence (b) produces a polypeptide ("B") comprising the first and second domains of said second polypeptide; expression of nucleotide sequence (b) together with nucleotide sequence (c) produces a polypeptide ("C") comprising the first and second domains of said second polypeptide fused to a surface component of a filamentous bacteriophage for display of the polypeptide (C) on the surface of bacteriophage particles; nucleotide sequence (b) being linked to nucleotide sequence (c) by a nucleic acid linkage, said linkage being selected from a nucleotide sequence including an intervening suppressible stop codon and a nucleotide sequence lacking an intervening suppressible stop codon and wherein said transfected host cells are cultured under conditions suitable for co-expression of the polypeptides A, B, and C from the host cell. - View Dependent Claims (43, 44)
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45. A dimer comprising:
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(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked directly to each other by an amide bond; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked directly to each other by an amide bond; the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54, 80)
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55. A vector comprising a nucleotide sequence encoding a first polypeptide and a nucleotide sequence encoding a second polypeptide each of the first and second encoded polypeptides comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first domain of each polypeptide being linked to the second domain of that polypeptide directly to each other by an amide bond;
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the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site; the nucleotide sequence encoding the first polypeptide being linked to a nucleotide sequence encoding a signal sequence for export of the first polypeptide from a host cell upon expression; the nucleotide sequence encoding the second polypeptide being linked to
1) a nucleotide sequence encoding a signal sequence for export of the second polypeptide from a host cell upon expression and
2) a nucleotide sequence encoding a surface component of a filamentous bacteriophage for display of the second polypeptide on the surface of a bacteriophage particle upon expression, the vector being capable of being packaged within a bacteriophage particle.
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56. A dimer comprising:
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(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker of 5 amino acids; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker of 5 amino acids; the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site. - View Dependent Claims (57, 58, 59, 60, 61, 62, 63)
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64. A vector comprising a nucleotide sequence encoding a first polypeptide and a nucleotide sequence encoding a second polypeptide each of the first and second encoded polypeptides comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first domain of each polypeptide being linked to the second domain of that polypeptide directly to each other by a polypeptide linker of 5 amino acids;
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the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site; the nucleotide sequence encoding the first polypeptide being linked to a nucleotide sequence encoding a signal sequence for export of the first polypeptide from a host cell upon expression; the nucleotide sequence encoding the second polypeptide being linked to
1) a nucleotide sequence encoding a signal sequence for export of the second polypeptide from a host cell upon expression and
2) a nucleotide sequence encoding a surface component of a filamentous bacteriophage for display of the second polypeptide on the surface of a bacteriophage particle upon expression, the vector being capable of being packaged within a bacteriophage particle.
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65. A dimer comprising:
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(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker of 4 amino acids; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker of 4 amino acids; the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site. - View Dependent Claims (66, 67, 68, 69, 70, 71, 73)
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72. A vector comprising a nucleotide sequence encoding a first polypeptide and a nucleotide sequence encoding a second polypeptide each of the first and second encoded polypeptides comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first domain of each polypeptide being linked to the second domain of that polypeptide directly to each other by a polypeptide linker of 4 amino acids;
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the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site; the nucleotide sequence encoding the first polypeptide being linked to a nucleotide sequence encoding a signal sequence for export of the first polypeptide from a host cell upon expression; the nucleotide sequence encoding the second polypeptide being linked to
1) a nucleotide sequence encoding a signal sequence for export of the second polypeptide from a host cell upon expression and
2) a nucleotide sequence encoding a surface component of a filamentous bacteriophage for display of the second polypeptide on the surface of a bacteriophage particle upon expression, the vector being capable of being packaged within a bacteriophage particle.
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74. A dimer comprising:
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(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker of 3 amino acids; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker of 3 amino acids; the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site. - View Dependent Claims (77, 78, 79, 81, 82, 83, 84)
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75. A dimer comprising:
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(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker of 2 amino acids; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker of 2 amino acids; the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site.
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76. A dimer comprising:
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(i) a first polypeptide comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the first polypeptide being linked by a polypeptide linker of one amino acid; and (ii) a second polypeptide which has a first domain, which comprises a binding region of an immunoglobulin heavy chain variable region, a second domain, which comprises a binding region of an immunoglobulin light chain variable region, the first and second domains of the second polypeptide being linked by a polypeptide linker of one amino acid; the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; and the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site.
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85. A vector comprising a nucleotide sequence encoding a first polypeptide and a nucleotide sequence encoding a second polypeptide each of the first and second encoded polypeptides comprising a first domain which comprises a binding region of an immunoglobulin heavy chain variable region, and a second domain which comprises a binding region of an immunoglobulin light chain variable region, the first domain of each polypeptide being linked to the second domain of that polypeptide directly to each other by a polypeptide linker of from 1 to 3 amino acids;
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the first domain of the first polypeptide and the second domain of the second polypeptide associating to form a first antigen binding site; the second domain of the first polypeptide and the first domain of the second polypeptide associating to form a second antigen binding site; said first antigen binding site being different from said second antigen binding site; the nucleotide sequence encoding the first polypeptide being linked to a nucleotide sequence encoding a signal sequence for export of the first polypeptide from a host cell upon expression; the nucleotide sequence encoding the second polypeptide being linked to
1) a nucleotide sequence encoding a signal sequence for export of the second polypeptide from a host cell upon expression and
2) a nucleotide sequence encoding a surface component of a filamentous bacteriophage for display of the second polypeptide on the surface of a bacteriophage particle upon expression, the vector being capable of being packaged within a bacteriophage particle.
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Specification