STABILIZED SOLUBLE PREFUSION RSV F POLYPEPTIDES

• US 20160102123A1
• Filed: 04/24/2014
• Published: 04/14/2016
• Est. Priority Date: 04/25/2013
• Status: Active Grant

• Current Assignee: Janssen Vaccines & Prevention B.V. (Johnson & Johnson)
• Original Assignee: Crucell Holland B.V (Johnson & Johnson)
• Inventors: Langedijk, Johannes Petrus Maria, Krarup, Anders
• Granted Patent: US 10,899,800 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/55561 CpG containing adjuvants; Oligonucleotide containing adjuvants
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/155 Paramyxoviridae, e.g. parainfluenza virus
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/135 Respiratory syncytial virus
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C07K 2319/73 containing coiled-coiled motif (leucine zippers)
• CPC Class Codes: C12N 2760/18522 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2760/18534 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

Described are stable pre-fusion respiratory syncitial virus (RSV) F polypeptides, immunogenic compositions comprising the polypeptides and uses thereof for the prevention and/or treatment of RSV infection.

Claims

1. A recombinant pre-fusion respiratory syncytial virus (RSV) Fusion (F) polypeptide, comprising:

at least one epitope that is specific to the pre-fusion conformation F protein, wherein the at least one epitope is recognized by

a pre-fusion specific monoclonal antibody comprising;

a heavy chain CDR1 region of SEQ ID NO;

54,

a heavy chain CDR2 region of SEQ ID NO;

55,

a heavy chain CDR3 region of SEQ ID NO;

56, and

a light chain CDR1 region of SEQ ID NO;

62,

a light chain CDR2 region of SEQ ID NO;

63, and

a light chain CDR3 region of SEQ ID NO;

64, and/or

a pre-fusion specific monoclonal antibody comprising;

a heavy chain CDR1 region of SEQ ID NO;

58,

a heavy chain CDR2 region of SEQ ID NO;

59,

a heavy chain CDR3 region of SEQ ID NO;

60 and

a light chain CDR1 region of SEQ ID NO;

66,

a light chain CDR2 region of SEQ ID NO;

67, and

a light chain CDR3 region of SEQ ID NO;

68.

2. Pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide is trimeric.

3. The pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide comprises a mutation of the amino acid residue on position 67 and/or a mutation of the amino acid residue on position 215.

4. The pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide comprises a F1 domain and a F2 domain, and a linking sequence comprising from 1 to 10 amino acid residues, linking said F1 domain to said F2 domain.

5. The pre-fusion RSV F polypeptide of claim 1, comprising a truncated F1 domain and a F2 domain, and a linking sequence comprising 1 to 10 amino acid residues, linking said F1 to said F2 domain, wherein polypeptide comprises at least one stabilizing mutation in the F1 and/or F2 domain as compared to the RSV F1 and/or F2 domain in the wild-type RSV F protein, wherein the at least one stabilizing mutation is selected from the group consisting of:

(a) a mutation of amino acid residue N/T on position 67.

(b) a mutation of amino acid residue S on position 215.

6. The pre-fusion RSV F polypeptide of claim 1, wherein the at least one stabilizing mutation is selected from the group consisting of:

(a) a mutation of amino acid residue N/T on position 67 into I; and

(b) a mutation of amino acid residue S on position 215 into P.

7. The pre-fusion RSV F polypeptide according to claim 5, wherein the polypeptide comprises a heterologous trimerization domain linked to said truncated F1 domain.

8. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide comprises at least one further mutation, wherein said mutation is selected from the group consisting of:

(a) a mutation of the amino acid residue on position 46;

(b) a mutation of the amino acid residue on position 77;

(c) a mutation of the amino acid residue on position 80;

(d) a mutation of the amino acid residue on position 92;

(e) a mutation of the amino acid residue on position 175;

(f) a mutation of the amino acid residue on position 184;

(g) a mutation of the amino acid residue on position 185;

(h) a mutation of the amino acid residue on position 201;

(i) a mutation of the amino acid residue on position 209;

(j) a mutation of the amino acid residue on position 421;

(k) a mutation of the amino acid residue on position 426;

(l) a mutation of the amino acid residue on position 465;

(m) a mutation of the amino acid residue on position 486;

(n) a mutation of the amino acid residue on position 487; and

(o) a mutation of the amino acid residue on position 508.

9. Pre-fusion RSV F polypeptide according to claim 8, wherein the at least one further mutation is selected from the group consisting of:

(a) a mutation of the amino acid residue S on position 46 into G;

(b) a mutation of the amino acid residue K on position 77 into E;

(c) a mutation of the amino acid residue K on position 80 into E;

(d) a mutation of the amino acid residue E on position 92 into D;

(e) a mutation of the amino acid residue N on position 175 into P;

(f) a mutation of the amino acid residue G on position 184 into N;

(g) a mutation of the amino acid residue V on position 185 into N;

(h) a mutation of the amino acid residue K on position 201 into Q;

(i) a mutation of the amino acid residue K on position 209 into Q;

(j) a mutation of the amino acid residue K on position 421 into N;

(k) a mutation of the amino acid residue N on position 426 into S;

(l) a mutation of the amino acid residue K on position 465 into E or Q;

(m) a mutation of the amino acid residue D on position 486 into N;

(n) a mutation of the amino acid residue E on position 487 into Q, N or I; and

(o) a mutation of the amino acid residue K on position 508 into E.

10. The pre-fusion RSV F polypeptide according to claim 1, wherein the polypeptide comprises at least two mutations.

11. Pre-fusion RSV F polypeptide according to claim 10, wherein the at least two mutations comprise a mutation of amino acid residue N/T on position 67 into I;

and a mutation of amino acid residue S on position 215 into P.

12. Pre-fusion RSV F polypeptide according to claim 11, wherein the polypeptide comprises at least one further mutation selected from the group consisting of:

(a) a mutation of the amino acid residue S on position 46 into G;

(b) a mutation of the amino acid residue K on position 77 into E;

(c) a mutation of the amino acid residue K on position 80 into E;

(d) a mutation of the amino acid residue E on position 92 into D;

(e) a mutation of the amino acid residue N on position 175 into P;

(f) a mutation of the amino acid residue G on position 184 into N;

(g) a mutation of the amino acid residue V on position 185 into N;

(h) a mutation of the amino acid residue K on position 201 into Q;

(i) a mutation of the amino acid residue K on position 209 into Q;

(j) a mutation of the amino acid residue K on position 421 into N;

(k) a mutation of the amino acid residue N on position 426 into S;

(l) a mutation of the amino acid residue K on position 465 into E or Q;

(m) a mutation of the amino acid residue D on position 486 into N;

(n) a mutation of the amino acid residue E on position 487 into Q, N or I; and

(o) a mutation of the amino acid residue K on position 508 into E.

13. The pre-fusion RSV F polypeptide of claim 1, wherein the heterologous trimerization domain comprises SEQ ID NO:

3.

14. Pre-fusion RSV F polypeptide according to claim 13, wherein the trimerization domain is linked to amino acid residue 495 of the RSV F protein.

15. The pre-fusion RSV F polypeptide of claim 1, wherein the heterologous trimerization domain comprises SEQ ID NO:

4.

16. Pre-fusion RSV F polypeptide according to claim 15, wherein the trimerization domain is linked to amino acid residue 513 of the RSV F protein.

17. The pre-fusion RSV F polypeptide of claim 1, wherein the linker between the F1 and the F2 domain comprises 5 amino acid residues.

18. The pre-fusion RSV F polypeptide of claim 17, wherein the linker comprises SEQ ID NO:

5.

19. The pre-fusion RSV F polypeptide of claim 1, wherein the F1 domain and/or the F2 domain are from an RSV A strain.

20. The pre-fusion RSV F polypeptide of claim 1, wherein the F1 domain and/or the F2 domain are from an RSV B strain.

21. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable for at least 30 minutes at 55°

C.

22. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable after storage at 4°

C. for at least 30 days.

23. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide comprises a peptide selected from the group consisting of SEQ ID NO:

21-SEQ ID NO;

52 and 71-89.

24. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide does not comprise a HIS-Tag.

25. A nucleic acid molecule encoding the pre-fusion RSV F polypeptide of claim 1.

26. The nucleic acid molecule of claim 25, wherein the nucleic acid molecule has been codon-optimized for expression in mammalian cells.

27. A vector comprising the nucleic acid molecule of claim 25.

28. A composition comprising:

the pre-fusion RSV F polypeptide of claim 1, and

a pharmaceutically acceptable carrier or excipient.

29. A method of inducing an immune response against RSV F protein in a subject, the method comprising:

administering to the subject the pre-fusion RSV F polypeptide of claim 1 in an amount to induce an immune response against RSV F protein in the subject.

30. A vaccine comprising:

the pre-fusion RSV F polypeptide of claim 1.

31. A method of prophylaxing and/or treating RSV infection in a subject, the method comprising:

administering to the subject the pre-fusion RSV F polypeptide of claim 1, to prophylax against and/or treat the subject for RSV infection.

47. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable for at least thirty minutes at 58°

C.

48. The pre-fusion RSV F polypeptide of claim 47, wherein the polypeptide is stable for at least thirty minutes at 60°

C.

49. The pre-fusion RSV F polypeptide of claim 1, wherein the polypeptide is stable after storage at 4°

C. for at least sixty days.

50. The pre-fusion RSV F polypeptide of claim 49, wherein the polypeptide is stable after storage at 4°

C. for at least six months.

51. The pre-fusion RSV F polypeptide of claim 50, wherein the polypeptide is stable after storage at 4°

C. for at least one year.

52. A vector comprising the nucleic acid molecule of claim 26.

32. A method for stabilizing the pre-fusion conformation of an RSV F polypeptide, the method comprising:

introducing one or more mutations in a RSV F1 and/or F2 domain, as compared to the wild-type RSV F1 and/or F2 domain, wherein the one or more mutations are selected from the group consisting of;

(a) a stabilizing mutation in a region that locks the HRA domain from hinging adjacent to the conserved 69-212 disulfide bridge, said region comprising the amino acid residues 66-68 and 214-216,

(b) a mutation in the helix comprising the amino acid residues 76-98 at the C-terminus of the F2 domain;

(c) a mutation that reduces the negative charge repulsion between the top of the HRB stem region comprising amino acids 486, 487; and

(d) a stabilizing mutation in the HRA region.

33. Method according to claim 32, wherein the mutation in the HRA hinge region is at position 67.

34. The method according to claim 32, wherein the mutation in the HRA hinge region is at position 215.

35. The method according to claim 32, wherein the mutation in the HRA hinge region is at position 66 or 68, and/or at position 214 or 216.

36. Method according to claim 32, wherein the mutation in the helix is at position 77.

37. Method according to claim 32, wherein the mutation in the helix is at position 80.

38. The method according to claim 36, wherein the amino acid residue at position 77 and/or 80 is changed into a negatively charged amino acid.

39. The method according to claim 32, wherein the mutation is at position 92.

40. Method according to claim 32, wherein the mutation that reduces the negative charge repulsion between the top of the HRB stem region comprising amino acids 486, 487, 489.

41. Method according to claim 40, wherein the mutation is at position 489.

42. Method according to claim 40, wherein the mutation is at position 486.

43. Method according to claim 32, wherein the mutation stabilizes the beta-turns between the amino acid residues 175-193.

44. Method according to claim 43, wherein the mutation is stabilizing the turn at position 175.

45. Method according to claim 43, wherein the mutation is stabilizing the turn at position 184-185.

46. A stabilized pre-fusion RSV F polypeptide, produced by the method according to claim 32.

53. The method according to claim 37, wherein the amino acid residue at position 80 is a negatively charged amino acid.