Multivalent and multispecific binding proteins, their manufacture and use

• US 20040058400A1
• Filed: 09/20/2002
• Published: 03/25/2004
• Est. Priority Date: 12/04/1992
• Status: Active Grant

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Malmqvist, Magnus, Marks, James David, Griffiths, Andrew David, Winter, Gregory Paul, Pope, Anthony Richard, McGuinness, Brian Timothy, Hoogenboom, Hendricus Renerus Jacobus Matheus, Holliger, Kaspar-Philipp, Prospero, Terence Derek
• Granted Patent: US 7,122,646 B2
• Time in Patent Office: Days
• US Class Current: 435/7.23
• 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 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 being linked but incapable of associating with each other to form an antigen binding site.

2. A polypeptide according to claim 1 wherein the first and second domains are linked without any intervening amino acid residues.

3. A polypeptide according to claim 1 wherein the first and second domains are linked by a peptide linker.

4. A polypeptide according to any one of claims 1 to 3 wherein the binding region of an immunoglobulin heavy chain variable region and the binding region of an immunoglobulin light chain variable region are, when in association with complementary light or heavy chain binding region respectively, able to bind the same epitope as each other.

5. A polypeptide according to any one of claims 1 to 4 wherein the binding region of an immunoglobulin heavy chain variable region and the binding region of an immunoglobulin light chain variable region are, when in association with complementary light or heavy chain binding region respectively, able to bind different epitopes from each other.

6. A polypeptide according to any one of claims 1 to 5 fused to additional amino acid residues.

7. A polypeptide according to claim 6 wherein the additional amino acid residues are a peptide tag.

8. A polypeptide according to claim 6 wherein the additional amino acid residues are a signal sequence.

9. A polypeptide according to claim 6 wherein the additional amino acid residues are a polypeptide domain.

10. A polypeptide according to claim 9 wherein the polypeptide domain is a surface component of a filamentous bacteriophage.

11. A polypeptide according to claim 10 wherein the surface component is GIII.

12. A polypeptide according to any one of claims 1 to 11 whose binding region of an immunoglobulin heavy chain variable region is in association with a binding region of an immunoglobulin light chain variable region and whose binding region of an immunoglobulin light chain variable region is in association with a binding region of an immunoglobulin heavy chain variable region, each association forming an antigen binding site.

13. A diverse repertoire of polypeptides, each of which is according to any one of claims 1 to 12.

14. A dimer of 2 polypeptides according to claim 12.

15. A dimer according to claim 14 wherein each polypeptide is different.

16. A dimer according to claim 15 wherein the N-terminal to C-terminal order of the first and second domains is the same in each polypeptide.

17. A dimer according to claim 14 wherein each polypeptide is the same.

18. A diverse repertoire of dimers according to any one of claims 14 to 17.

19. Nucleic acid comprising a sequence encoding a polypeptide according to any one of claims 1 to 11.

20. Nucleic acid according to claim 19 comprising nucleic acid encoding an RNA splice site between nucleic acid encoding the first domain and nucleic acid encoding the second domain.

21. Nucleic acid according to claim 19 or claim 20 comprising, between nucleic acid encoding the first domain and nucleic acid encoding the second domain, nucleic acid encoding a site for recombination in vivo or in vitro.

22. A vector comprising nucleic acid according to any one of claims 19 to 21.

23. A vector according to claim 22 comprising nucleic acid necessary for expression of the polypeptide.

24. A vector according to claim 23 comprising nucleic acid for secretion of the polypeptide upon expression.

25. A vector according to any one of claims 22 to 24 comprising nucleic acid encoding two said polypeptides, a first polypeptide and a second polypeptide.

26. A vector according to claim 25 comprising, between nucleic acid encoding the first polypeptide and nucleic acid encoding the second polypeptide, nucleic acid comprising a site for recombination in vivo or in vitro.

28. A host cell transfected with a vector according to any one of claims 23 to 27.

29. A method of making of making a polypeptide according to any one of claims 1 to 11 which comprises culturing a host cell according to claim 28 under conditions for expression of the polypeptide from the host cell.

30. A method according to claim 29 wherein the polypeptide is recovered from the host cell medium.

31. A method according to claim 29 wherein the polypeptide is recovered from the host cell.

32. A method according to claim 31 wherein the polypeptide or a dimer of two of said polypeptides is refolded from denaturing conditions.

33. A method according to claim 31 wherein the polypeptide is recovered from the host cell periplasm.

34. A method according to any one of claims 30 to 33 wherein a dimer of two of said polypeptides is selected by binding with antigen.

37. A method of making a dimer of two polypeptides, each 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 being linked but incapable of associating with each other to form an antigen binding site, which method comprises culturing a host cell according to claim 31 under the conditions for co-expression of the polypeptides A and B from the host cell.

38. Use of a polypeptide according to claim 12 in a homogeneous or heterogeneous assay for antigen.

51. A polypeptide according to claim 12 or a dimer according to any one of claims 14 to 17, 39, 40 to 45 having an antigen binding site which binds to any of the following:

a cell surface protein;

a Fc receptor;

a tumour specific marker;

CEA;

a virus;

HIV 1;

a small chemical molecule;

a hormone;

a cytokine;

TNFα

;

an antibody;

the idiotope of an antibody.

52. A dimer according to any one of claims 14 to 17, 39, 40 to 45 wherein the binding of an antigen to one antigen binding site of the dimer affects the binding of an antigen to the other binding site of the dimer.

53. A dimer according to any one of claims 14 to 17 wherein alterations of the linker lead to an improvement in antigen binding affinity of at least one of the binding sites of the dimer.

54. An assay for an antigen using a polypeptide according to claim 12 or a dimer according to any one of claims 14 to 17, 39, 40 to 45.

55. An assay according to claim 54 which is diagnostic.

56. An assay according to claim 54 involving the crosslinking of two antigens.

57. An assay according to claim 56 involving agglutination of two cells.

58. An assay according to claim 56 wherein one antigen is on a surface and the other antigen is in solution.

59. An assay according to claim 57 involving detection using surface plasmon resonance.

60. A polypeptide according to claim 12 or a dimer according to any one of claims 14 to 17, 39, 40 to 45 which binds to antigens on two different cell surfaces.

61. A pharmaceutical preparation comprising a polypeptide according to any one of claims 1 to 12 or a dimer according to any one of claims 14 to 17, 39, 40 to 45.

27. A vector comprising nucleic acid encoding a first polypeptide and nucleic acid encoding a second polypeptide, each of the 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 but incapable of associating with it to form an antigen binding site, the nucleic acid encoding the first polypeptide being linked to nucleic acid encoding a signal sequence for export of the first polypeptide from a host cell upon expression, the nucleic acid encoding the second polypeptide being linked to nucleic acid encoding a signal sequence for export of the second polypeptide from a host cell upon expression and nucleic acid 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.

35. A vector comprising:

(a) nucleic acid encoding a first polypeptide domain, which comprises a binding region of an immunoglobulin heavy chain variable region, and a second polypeptide domain, which comprises a binding, region of an immunoglobulin light chain variable region; and

(b) nucleic acid encoding a surface component of a filamentous bacteriophage;

expression of nucleic acid (a) producing a polypeptide (“

A”

) comprising the first and second domains linked but incapable of associating with each other to form an antigen binding site;

expression of nucleic acid (a) together with nucleic acid (b) producing a polypeptide (“

B”

) comprising the first and second domains linked but incapable of associating with each other to form an antigen binding site, fused to a surface component of a filamentous bacteriophage for display of the polypeptide (B) on the surface of bacteriophage particles;

nucleic acid (b) being linked to nucleic acid (a) by nucleic acid including an intervening suppressible stop codon.

36. A host cell transfected with a vector according to claim 35 and capable of providing conditions for co-expression of polypeptides A and B.

39. 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 domains of the first polypeptide being linked but incapable of associating with each other to form an antigen binding site;

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, and a polypeptide linker linking the first and second domains and allowing association of the domains of the second polypeptide with each other to form an antigen binding site;

the first domain of the first polypeptide and the second domain of the second polypeptide associating to form an antigen binding site; and

the second domain of the first polypeptide and the first domain of the second polypeptide associating to form an antigen binding site.

41. A dimer according to claim 39 or claim 40 wherein one of the polypeptides is fused to additional amino acid residues.

42. A dimer according to claim 41 wherein the additional amino acid residues are a peptide tag.

43. A dimer according to claim 41 wherein the additional amino acid residues are a polypeptide domain.

44. A dimer according to claim 43 wherein the polypeptide domain is a surface component of a filamentous bacteriophage.

45. A dimer according to claim 44 wherein the surface component is GIII.

46. A vector comprising nucleic acid which encodes first and second polypeptides which are able to form a dimer according to any one of claims 39 to 45.

47. A vector according to claim 46 comprising nucleic acid necessary for expression of the polypeptides.

48. A host cell transfected with a vector according to claim 47.

49. A method of which comprises:

culturing a host cell according to claim 48 under conditions for expression of said first and second polypeptides, and

recovering a dimer which has antigen bispecificity.

50. A method according to claim 49 wherein the dimer is selected by binding with antigen.

40. A dimer of

(i) a first 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, and a polypeptide linker linking the first and second domains and allowing association of the domains with each other to form an antigen binding site;

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, and a polypeptide linker linking the first and second domains and allowing association of the domains with each other to form an antigen binding site;

the first domain of the first polypeptide and the second domain of the second polypeptide associating to form an antigen binding site which has a first binding specificity; and

the second domain of the first polypeptide and the first domain of the second polypeptide associating to form an antigen binding site which has a second binding specificity.