Multivalent and multispecific binding proteins and their use

• US 6,492,123 B1
• Filed: 09/03/1998
• Issued: 12/10/2002
• Est. Priority Date: 12/04/1992
• Status: Expired due to Fees

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Holliger, Kaspar-Philip, Malmqvist, Magnus, Marks, James David, Griffiths, Andrew David, Winter, Gregory Paul, Pope, Anthony Richard, McGuinness, Brian Timothy, Hoogenboom, Hendricus Renerus Jacobus Matheus, Prospero, Terence Derek
• Primary Examiner(s): Huff, Sheela
• Assistant Examiner(s): HELMS, LARRY RONALD
• Application Number:
  US09/146,979
• Time in Patent Office: 1,559 Days
• Field of Search: 435/7.1, 435/69.1, 435/69.7, 435/69.8, 435/320.1, 435/328, 514/2, 530/387.1, 530/412, 530/413, 530/387.3, 536/23.4, 536/23.53, 536/24.1, 424/133.1, 424/136.1, 424/135.1
• US Class Current: 435/7.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/1063 env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: C07K 16/2809 against the T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 16/3007 Carcino-embryonic Antigens
• CPC Class Codes: C07K 16/3061 Blood cells
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 16/4283 against an allotypic or isotypic determinant on Ig
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/624 Disulfide-stabilized antibody (dsFv)
• CPC Class Codes: C07K 2317/626 Diabody or triabody
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218

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.

Claims

1. A dimer of

(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;

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;

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;

the polypeptide linker of the first polypeptide being of a length such that said first and second domains of said first polypeptide are incapable of associating with each other to form an antigen binding site.

7. A dimer according to claim 1 wherein the first polypeptide is fused to additional amino acid residues at its N- or C-terminus.

8. A dimer according to claim 1 having an antigen binding site which binds to an antigen selected from the group consisting of:

a virus, human immunodeficiency virus 1 (HIV

     1), a hormone, a cytokine, tumour necrosis factor alpha (TNFα

), an antibody, the idiotope of an antibody, a cell surface protein, a Fc receptor, a tumour specific marker, and carcinoembryonic antigen (CEA).

9. A dimer according to claim 1, wherein the first domains of the first and second polypeptides are located N-terminal to the second domains of said first and second polypeptides.

10. A dimer according to claim 1, wherein the first domains of the first and second polypeptides are located C-terminal to the second domains of said first and second polypeptides.

11. A dimer according to claim 1 which binds to antigens on two different cell surfaces.

13. An assay for an antigen using a dimer according to claim 1 comprising the steps:

(a) incubating the dimer and an antigen, wherein said antigen binds to either or both the first and second antigen binding site of the dimer, under conditions which permit binding of the dimer to the antigen, and

(b) detecting the antigen-dimer binding.

19. A pharmaceutical preparation comprising, as its active ingredient, a dimer according to claim 1.

2. A dimer of

(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 having a length selected from the group consisting of 9, 8, 7 and 6 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;

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.

3. A dimer according to claim 2 wherein said polypeptide linker of the second polypeptide is a length selected from the group consisting of 9, 8, 7, 6, 5, 4, 3, 2 and 1 amino acids.

20. A dimer according to claim 2 wherein the first polypeptide is fused to additional amino acid residues at its N- or C-terminus.

21. A dimer according to claim 3 wherein the first polypeptide is fused to additional amino acid residues at its N- or C-terminus.

22. A dimer according to claim 2 having an antigen binding site which binds to an antigen selected from the group consisting of:

a virus, human immunodeficiency virus 1 (HIV

     1), a hormone, a cytokine,

tumour necrosis factor alpha (TNFα

), an antibody, the idiotope of an antibody, a cell surface protein, a Fc receptor, a tumour specific marker, and carcinoembryonic antigen (CEA).

23. A dimer according to claim 3 having an antigen binding site which binds to an antigen selected from the group consisting of:

a virus, human immunodeficiency virus 1 (HIV

     1), a hormone, a cytokine,

tumour necrosis factor alpha (TNFα

), an antibody, the idiotope of an antibody, a cell surface protein, a Fc receptor, a tumour specific marker, and carcinoembryonic antigen (CEA).

24. A dimer according to claim 2, wherein the first domains of the first and second polypeptides are located N-terminal to the second domains of said first and second polypeptides.

25. A dimer according to claim 3, wherein the first domains of the first and second polypeptides are located N-terminal to the second domains of said first and second polypeptides.

26. A dimer according to claim 2, wherein the first domains of the first and second polypeptides are located C-terminal to the second domains of said first and second polypeptides.

27. A dimer according to claim 3, wherein the first domains of the first and second polypeptides are located C-terminal to the second domains of said first and second polypeptides.

28. A dimer according to claim 2 which binds to antigens on two different cell surfaces.

29. A dimer according to claim 3 which binds to antigens on two different cell surfaces.

30. An assay for an antigen using a dimer according to claim 2 comprising the steps:

(a) incubating the dimer and an antigen, wherein said antigen binds to either or both the first and second antigen binding site of the dimer, under conditions which permit binding of the dimer to the antigen, and

(b) detecting the antigen-dimer binding.

31. An assay for an antigen using a dimer according to claim 3 comprising the steps:

(a) incubating the dimer and an antigen, wherein said antigen binds to either or both the first and second antigen binding site of the dimer, under conditions which permit binding of the dimer to the antigen, and

(b) detecting the antigen-dimer binding.

32. A pharmaceutical preparation comprising, as its active ingredient, a dimer according to claim 2.

33. A pharmaceutical preparation comprising, as its active ingredient, a dimer according to claim 3.

4. A dimer of

(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;

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 having a length selected from the group consisting of 9, 8, 7 and 6 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;

the polypeptide linker of the first polypeptide being of a length such that said first and second domains of said first polypeptide are incapable of associating with each other to form an antigen binding site.

5. A dimer of

(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 6 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 6 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.

6. A dimer of

(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 7 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 7 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.

12. A population of dimers wherein each dimer is of

(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;

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;

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;

the polypeptide linker of the first polypeptide being of a length such that said first and second domains of said first polypeptide are incapable of associating with each other to form an antigen binding site, wherein said population has a diverse repertoire of antigen binding activities.

14. An assay according to claim 12 which is a diagnostic assay.

15. An assay according to claim 12 comprising the crosslinking of two antigens.

16. An assay according to claim 12 comprising agglutination of two cells.

17. An assay according to claim 12 wherein one antigen is on a surface and the other antigen is in solution.

18. An assay for an antigen using a dimer comprising the steps:

(a) incubating the dimer and an antigen, wherein said antigen binds to either or both of the first and second antigen binding sites of the dimer, under conditions which permit binding of the dimer to the antigen, and

(b) detecting the antigen-dimer binding using surface plasmon resonance;

said dimer being of

(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; 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;

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;

the polypeptide linker of the first polypeptide being of a length such that said first and second domains of said first polypeptide are incapable of associating with each other to form an antigen binding site.