Single-chain antagonist polypeptides

• US 20040014948A1
• Filed: 05/23/2003
• Published: 01/22/2004
• Est. Priority Date: 10/07/1999
• Status: Abandoned Application

• Current Assignee: Maxygen APS
• Original Assignee: Maxygen APS
• Inventors: Soni, Bobby, Schambye, Hans Thalsgard, Jeppesen, Claus Bekker, Nissen, Torben Lauesgaard, Andersen, Kim Vilbour, Okkels, Jens Sigurd, Halkier, Torben, van den Hazel, Bart
• Application Number:
  US10/444,691
• Publication Number:
  US 20040014948A1
• Time in Patent Office: Days
• US Class Current: 530/388.22
• 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: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The invention relates to a single-chain oilgomeric polypeptide antagonist which binds to an extracellular ligand-binding domain of a cellular receptor of a type requiring binding of an oligomeric ligand to two or more receptor subunits to be activated, the polypeptide comprising at least two, typically structurally homologous, receptor-binding sites of which at least one is capable of binding to a ligand-binding domain of the cellular receptor and at least one is incapable of effectively binding to a ligand-binding domain of the cellular receptor, whereby the single-chain oligomeric polypeptide is capable of binding to the receptor, but incapable of activating the receptor; as well as to nucleotide sequences encoding such single-chain oligomeric polypeptides, expression vectors comprising such a nucleotide sequence, recombinant host cells comprising such a nucleotide sequence or expression vector, methods for producing the nucleotide sequences and polypeptides, pharmaceutical compositions comprising the single-chain oligomeric polypeptides, and use of the single-chain oligomeric polypeptides for the production of medicaments and in therapy. A preferred single-chain antagonist according to the invention is a TNF-α antagonist.

Claims

1. A single-chain oligomeric polypeptide which binds to an extracellular ligand-binding domain of a cellular receptor of a type requiring binding of an oligomeric ligand to two or more receptor subunits to be activated, the polypeptide comprising at least three receptor-binding sites of which at least one is capable of binding to a ligand-binding domain of the cellular receptor and at least one is incapable of effectively binding to a ligand-binding domain of the cellular receptor, whereby the single-chain oligomeric polypeptide is capable of binding to the receptor, but incapable of activating the receptor.

3. The polypeptide of claim 1, wherein the receptor-binding sites are located in different, symmetrical regions of the polypeptide.

4. The polypeptide of claim 1, wherein each receptor-binding site includes one or more amino acid residues originating from a first monomer and one or more amino acid residues originating from a second monomer of the oligomeric polypeptide.

5. The polypeptide of claim 1, wherein the receptor-binding sites are located at interfaces between monomeric constituents of the oligomeric polypeptide.

6. The polypeptide of claim 1, wherein at least two of the receptor-binding sites are structurally homologous.

8. The polypeptide of claim 1, which is a trimer.

9. The polypeptide of claim 8, wherein at least two of the receptor-binding sites are structurally homologous.

10. The polypeptide of claim 8, which comprises two receptor-binding sites capable of binding to a ligand-binding domain of the cellular receptor and one modified receptor-binding site which is incapable of effectively binding to the ligand-binding domain of the cellular receptor.

11. The polypeptide of claim 8, which comprises one receptor-binding site capable of binding to a ligand-binding domain of the cellular receptor and two modified receptor-binding sites which are incapable of effectively binding to a ligand-binding domain of the cellular receptor.

12. The polypeptide of claim 10, wherein the modified receptor-binding site is incapable of effectively binding to the ligand-binding domain of the cellular receptor due to steric hindrance.

13. The polypeptide of claim 12, wherein the modified receptor-binding site is blocked by a non-polypeptide moiety.

14. The polypeptide of claim 10, wherein the modified receptor-binding site differs from its parent receptor-binding site by deletion, substitution and/or insertion of one or more amino acid residues at one or more positions of said parent receptor-binding site.

15. The polypeptide of claim 1, which is modified by introduction of an amino acid residue comprising an attachment group for a non-polypeptide moiety in such a manner that a non-polypeptide moiety conjugated to said attachment group blocks a receptor-binding site.

16. The polypeptide of claim 15, wherein the amino acid residue comprising an attachment group for a non-polypeptide moiety is introduced in a receptor-binding site.

17. The polypeptide of claim 16, wherein the amino acid residue comprising an attachment group for a non-polypeptide moiety is a cysteine residue or an asparagine residue.

18. The polypeptide of claim 15, wherein the non-polypeptide moiety is an oligosaccharide moiety or a polymer.

19. The polypeptide of claim 1, comprising first and second monomers that are each modified in at least one position forming part of the same receptor-binding site.

20. The polypeptide of claim 1, which binds to a cellular receptor selected from the group consisting of cytokine receptors, growth factor receptors, protein-tyrosine kinase receptors, death domain receptors, including the TNF receptor family, and serine-threonine kinase receptors.

21. The polypeptide of claim 20, wherein the cytokine is selected from the group consisting of IFN-γ

, lymphotoxin-α

, lymphotoxin-β

, IL-10, IL-16, FAS ligand, TRAIL, CD40 ligand, CD30 ligand, CD27 ligand, OX40 ligand, APRIL, 4-1BB ligand, TRANCE and OPGL.

22. The polypeptide of claim 21, which is an OPGL antagonist.

23. The polypeptide of claim 20, wherein the growth factor is selected from the group consisting of VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF, PlGF, TGF-β

1, TGF-β

2, TGF-β

3, TGF-β

4, bone morphogenetic protein 2 (BMP-2), BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, growth differentiating factor 1 (GDF-1), GDF-5, GDF-8 (myostatin), GDF-10, Muellerian inhibiting factor, inhibin A, inhibin B, activin A and activin AB.

24. The polypeptide of claim 1, comprising at least one receptor-binding site with at least one modification that results in increased receptor-binding activity of said modified receptor-binding site compared to a corresponding polypeptide without said modification.

25. The polypeptide of claim 24 which is a trimer with one or two receptor-binding sites having increased receptor-binding activity.

26. The polypeptide of claim 1, wherein the monomers are linked by a linker peptide.

27. The polypeptide of claim 26, wherein the linker peptide is a sequence of at least one and not more than about 30 amino acid residues.

28. The polypeptide of claim 26, wherein the linker peptide comprises one or more amino acid residues selected from the group consisting of Gly, Ser, Ala and Thr.

29. The polypeptide of claim 1 which is a single-chain trimeric TNF receptor antagonist.

30. The polypeptide of claim 29, wherein the TNF receptor is the TNF-R1, the TNF-R2 or both of the TNF-R1 and TNF-R2.

31. The polypeptide of claim 29, wherein each of the three monomers comprised in the single-chain trimeric polypeptide is derived from a cytokine ligand of the TNF family.

32. The polypeptide of claim 29, which comprises at least one TNF-α

monomer.

33. The polypeptide of claim 32, which comprises one TNF-α

monomer and two monomers derived from the same or different cytokine ligand(s) of the TNF family other than TNF-α

, or two TNF-α

monomers and one monomer derived from a cytokine ligand of the TNF family other than TNF-α

.

34. The polypeptide of claim 29, comprising three TNF-α

monomers.

35. The polypeptide of claim 34, wherein receptor-binding sites 1 and 3 are inactive and receptor-binding site 2 is active.

36. The polypeptide of claim 29, which comprises at least one monomer derived from a cytokine ligand of the TNF family other than TNF-α

.

37. The polypeptide of claim 36, which comprises three monomers derived from the same or different cytokine ligands of the TNF family other than TNF-α

.

38. The polypeptide of claim 31, wherein the cytokine ligand of the TNF family other than TNF-α

is selected from the group consisting of LT-α and

LT-β

.

39. The polypeptide of claim 38, which comprises a) one LT-α

monomer, one LT-β

monomer and one TNF-α

monomer;

b) two LT-α

monomers and either one LT-β

monomer or one TNF-α

monomer;

or c) two LT-β

monomers and either one LT-α

monomer or one TNF-α

monomer.

40. The polypeptide of claim 32, wherein a receptor-binding site of TNF-α

is substituted by a receptor-binding site of a cytokine ligand of the TNF family other than TNF-α

.

41. The polypeptide of claim 34, which comprises a first, second and third TNF-α

monomer which are linked via a peptide bond or a peptide linker so as to form a trimer, which polypeptide comprises a first, a second and a third receptor-binding site located at interfaces between the monomers, wherein

(a) said first and/or second TNF-α

monomer is modified in an amino acid residue constituting part of the first receptor-binding site, and/or wherein

(b) said second and/or third TNF-α

monomer is modified in an amino acid residue constituting part of the second receptor-binding site, and/or wherein

(c) said third and/or first TNF-α

monomer is modified in an amino acid residue constituting part of the third receptor-binding site,

such that the polypeptide comprises one or two active receptor-binding sites capable of binding to a ligand-binding domain of a TNF receptor and one or two inactive receptor-binding sites.

42. The polypeptide of claim 41, wherein:

(a) said first TNF-α

monomer is modified by substitution of one or more amino acid residues in positions 53, 71, 72, 73, 74, 75, 77, 82, 84, 85, 86, 87, 88, 89, 91, 97, 125, 127, 137 or 138, and/or wherein said second TNF-α

monomer is modified by substitution of one or more amino acid residues in positions 6, 17, 20, 21, 23, 29, 30, 31, 32, 33, 34, 35, 63, 65, 66, 67, 110, 111, 112, 113, 114, 115, 140, 142, 143, 144, 145, 146, 147 or 149; and

/or

(b) said second TNF-α

monomer is modified by substitution of one or more amino acid residues in positions 53, 71, 72, 73, 74, 75, 77, 82, 84, 85, 86, 87, 88, 89, 91, 97, 125, 127, 137 or 138, and/or wherein said third TNF-α

monomer is modified by substitution of one or more amino acid residues in positions 6, 17, 20, 21, 23, 29, 30, 31, 32, 33, 34, 35, 63, 65, 66, 67, 110, 111, 112, 113, 114, 115, 140, 142, 143, 144, 145, 146, 147 or 149; and

/or

(c) said third TNF-α

monomer is modified by substitution of one or more amino acid residues in positions 53, 71, 72, 73, 74, 75, 77, 82, 84, 85, 86, 87, 88, 89, 91, 97, 125, 127, 137 or 138, and/or wherein said first TNF-α

monomer is modified by substitution of one or more amino acid residues in positions 6, 17, 20, 21, 23, 29, 30, 31, 32, 33, 34, 35, 63, 65, 66, 67, 110, 111, 112, 113, 114, 115, 140, 142, 143, 144, 145, 146, 147 or 149;

with the proviso that the modified polypeptide comprises one or two active receptor-binding sites capable of binding to a ligand-binding domain of a TNF receptor.

43. The polypeptide of claim 42, wherein:

(a) said first monomer is modified by substitution of one or more amino acid residues in positions 75, 87, 91 or 97, and/or said second monomer is modified by substitution of one or more amino acid residues in positions 20, 32, 65, 143 or 146; and

/or

(b) said second monomer is modified by substitution of one or more amino acid residues in positions 75, 87, 91 or 97 and/or said third monomer is modified by substitution of one or more amino acid residues in positions 20, 32, 65, 143 or 146; and

/or

(c) said third monomer is modified by substitution of one or more amino acid residues in positions 75, 87, 91 or 97 and/or said first monomer is modified by substitution of one or more amino acid residues in position 20, 32, 65, 143 or 146;

with the proviso that the modified polypeptide comprises one or two active receptor-binding sites capable of binding to a ligand-binding domain of a TNF receptor.

44. The polypeptide of claim 29, wherein at least one N-glycosylation site has been introduced into the amino acid sequence of the polypeptide in such a manner that the resulting polypeptide, when expressed in a glycosylating host cell, comprises an oligosaccharide moiety which is positioned at said N-glycosylation site so as to render at least one receptor-binding site inactive by means of steric hindrance.

45. The polypeptide of claim 29, comprising at least one receptor-binding site with a higher binding affinity to a ligand binding domain of a TNF receptor relative to wild-type human TNF-α

.

46. The polypeptide of claim 45, wherein said at least one receptor-binding site has a higher binding affinity towards the p55 receptor, the p75 receptor or both of these relative to a corresponding receptor-binding site of a corresponding unmodified single-chain trimeric polypeptide.

47. The polypeptide of claim 29, wherein 1-10 amino acid residues at the N-terminal part of at least one of the monomers are deleted relative to wild-type human TNF-α

.

48. The polypeptide of claim 29, comprising a linker peptide as shown SEQ ID NO:

2.

49. The polypeptide of claim 29, which has substantially no agonist effect on at least one TNF receptor.

50. The polypeptide of claim 49, which has substantially no agonist effect on either of the TNF receptors TNF-R1 and TNF-R2.

51. The polypeptide of claim 29, which has a partial agonist effect on at least one TNF receptor.

52. The polypeptide of claim 29, which has a serum or in vivo half-life comparable to that of the ligand which it is intended to block.

53. The polypeptide of claim 29, which has a serum or in vivo half-life that is not more than about 10 times that of human TNF-α

.

54. A nucleotide sequence encoding a single-chain oligomeric polypeptide according to claim 1.

55. An expression vector comprising a nucleotide sequence according to claim 54.

56. A recombinant host cell comprising a nucleotide sequence according to claim 54 or an expression vector comprising said nucleotide sequence.

57. A method for producing a nucleotide sequence according to claim 54, wherein a single nucleotide sequence encoding the single-chain polypeptide is subjected to mutagenesis so as to render at least one receptor-binding site of the polypeptide encoded by said nucleotide sequence incapable of effectively binding to a ligand-binding domain of the cellular receptor.

58. The method of claim 57, wherein the mutagenesis comprises at least one DNA shuffling cycle.

59. A method for preparing a single-chain oligomeric polypeptide according to claim 1, comprising culturing a recombinant host cell comprising a single nucleotide sequence encoding said polypeptide in a suitable culture medium under conditions permitting expression of the nucleotide sequence and recovering the resulting polypeptide from the cell culture.

60. The method of claim 59, which further comprises conjugating the polypeptide to a non-polypeptide moiety.

61. A pharmaceutical composition comprising a single-chain oligomeric polypeptide according to claim 1 together with at least one pharmaceutically acceptable excipient or vehicle.

62. A method for inhibiting activation of a cellular receptor that requires binding of an oligomeric ligand to two or more receptor subunits to be activated, comprising administering to a patient in need thereof a therapeutically effective dose of a single-chain oligomeric polypeptide according to claim 1.

63. A method for preventing or treating a disease or condition involving undesirable signal transduction from or undesirable activation of a TNF-α

receptor, or for counteracting undesirable effects of endogenous or exogenous TNF-α

, comprising administering to a patient in need thereof a therapeutically effective dose of a TNF receptor antagonist polypeptide according to claim 29.

64. The method of claim 63, wherein the disease or condition is an inflammatory disease or condition.

65. The method of claim 64, wherein the inflammatory disease or condition is selected from the group consisting of rheumatoid arthritis, Crohn'"'"'s disease, systemic lupus erythematosus, Sjö

gren'"'"'s disease, cachexia, diabetes mellitus, septic shock, myastenia gravis, juvenile arthritis, athero sclerosis, myocardial infarction, psoriasis, psoriasis arthritis, morbus Still, Wegener'"'"'s granulomatosis, uveitis, anchylosing spondylitis, acute inflammatory conditions, post-surgical stress, and brain damage.

2. A single-chain oligomeric polypeptide which binds to an extracellular ligand-binding domain of a cellular receptor of a type requiring binding of an oligomeric ligand to two or more receptor subunits to be activated, the polypeptide comprising at least two structurally homologous receptor-binding sites of which at least one is capable of binding to a ligand-binding domain of the cellular receptor and at least one is incapable of effectively binding to a ligand-binding domain of the cellular receptor, whereby the single-chain oligomeric polypeptide is capable of binding to the receptor, but incapable of activating the receptor.

7. The polypeptide of claim 2, which is a dimer.