Claims
- 1. A modified adenovirus capsid protein,
the unmodified capsid protein binds to heparin sulfate proteoglycan (HSP); and the capsid protein comprises a mutation, whereby binding to heparin sulfate proteoglycan (HSP) is altered.
- 2. The modified protein of claim 1 that is a fiber protein
- 3. The capsid protein of claim 2, wherein the binding of the modified fiber protein is eliminated or reduced compared to the unmodified protein.
- 4. The modified protein of claim 2, wherein the binding of the modified fiber protein is eliminated or reduced compared to the unmodified protein.
- 5. The modified protein of claim 3 that comprises an insertion, deletion or replacement of amino acids.
- 6. The modified protein of claim 2, wherein the mutation alters the motif that binds to HSP, whereby HSP interaction is altered.
- 7. The modified protein of claim 6, motif is BBXB or BBBXXB, wherein the B is a basic amino acid and X is any amino acid.
- 8. The modified protein of claim 7, wherein the motif comprises the consensus sequence KKTK.
- 9. The modified protein of claim 2, wherein the fiber is a modified Ad5 or Ad2 fiber.
- 10. A modified protein of claim 2 that is a chimeric fiber protein, comprising portions of fiber proteins from at least two different adenoviruses, wherein:
a shaft or portion thereof is from a first adenovirus, whereby the resulting fiber does not bind to HSP or binds to HSP with reduced affinity compared to an unmodified fiber protein; a shaft or portion thereof from the first adenovirus does not bind to HSP or binds to HSP with reduced affinity compared to the second adenovirus; the second adenovirus binds to HSP; and the portion comprises a sufficient portion to alter HSP binding of the resulting protein.
- 11. The modified protein of claim 10, wherein the binding to HSP of the modified fiber protein is eliminated or reduced compared to the unmodified protein.
- 12. The modified protein of claim 10, wherein the remainder of the fiber protein is from the second adenovirus.
- 13. The modified protein of claim 2, further comprising one or more further modifications that reduce or eliminate interaction of the resulting fiber with one or more cell surface proteins in addition to HSP.
- 14. The modified protein of claim 13, further comprising a ligand, whereby the resulting fiber binds to a receptor for the ligand.
- 15. The modified protein of claim 14, wherein the ligand is included in the knob region.
- 16. The modified protein of claim 14, wherein the ligand is inserted or it replaces a portion of the fiber, whereby the resulting fiber binds to a receptor for the ligand.
- 17. A modified protein of claim 11, wherein affinity for HSP is reduced at least by an amount selected from among reduced 5-fold, 10-fold and 100-fold.
- 18. The modified protein of claim 11, wherein the first adenovirus is selected from the group consisting of subgroup B, D or F, and the second is of subgroup C.
- 19. The modified protein of claim 10, wherein the first adenovirus is selected from the group consisting of Ad3, Ad35, Ad7, Ad11, Ad16, Ad21, Ad34, Ad40, Ad41 and Ad46.
- 20. The modified protein of claim 18, wherein the second adenovirus is Ad5 or Ad2.
- 21. The modified protein of claim 19, wherein the second adenovirus is Ad5 or Ad2.
- 22. A modified protein of claim 1 selected from the group consisting of a fiber protein comprising:
the sequence of amino acids set forth in any of SEQ ID Nos. 52, 54, 56, 58, 62, 66, 70 and 72; or a sequence of amino acids having 90% sequence identity with a sequence of amino acids set forth in any of SEQ ID Nos. 52, 54, 56, 58, 62, 66, 70 and 72; or a sequence of amino acids encoded by a sequence of nucleotides that hybridizes under conditions of high stringency along at least 70% of its length to a sequence of nucleotides that encodes a sequence of amino acids set forth in any of SEQ ID Nos. 52, 54, 56, 58, 62, 66, 70 and 72.
- 23. A nucleic acid molecule encoding a modified protein of any of claims 1-3, 10, 11, 13 and 14.
- 24. The nucleic acid molecule of claim 23 that comprises a vector.
- 25. The nucleic acid molecule of claim 24 that is an adenovirus vector.
- 26. The vector of claim 25 that is an adenoviral vector from a subgroup B, C or D adenovirus.
- 27. A cell, comprising a nucleic acid molecule of claim 23.
- 28. The cell of claim 27 that is a eukaryotic cell.
- 29. A cell, comprising a nucleic acid molecule of claim 25, wherein:
the cell is a eukaryotic cell; and the cell in a packaging cell.
- 30. An adenoviral particle, comprising a modified protein of any of claims 1-3, 10, 11, 13 and 14, whereby binding of the viral particle to HSP is altered compared to a particle that expresses an unmodified fiber.
- 31. An adenoviral particle of claim 30, wherein a native receptor for the fiber is coxsackie-adenovirus receptor (CAR).
- 32. The adenoviral particle of claim 31, further comprising a mutation in the CAR-binding region of the capsid.
- 33. The adenoviral particle of claim 31, further comprising a mutation in the αv integrin-binding region of the capsid, whereby binding to the integrin is eliminated or reduced.
- 34. The adenoviral particle of claim 32, further comprising a mutation in the αv integrin-binding region of the capsid, whereby binding to the integrin is eliminated or reduced
- 35. The adenoviral particle of claim 31, wherein the CAR-binding region of the capsid modified is on a fiber knob.
- 36. The adenoviral particle of claim 35, wherein the fiber knob modification is in the AB loop or CD loop.
- 37. The adenoviral particle of claim 36, wherein the fiber knob modification is selected from the group consisting of KO1 and KO12.
- 38. The adenoviral particle of claim 32, wherein the adenovirus is a subgroup C, D or F adenovirus.
- 39. The adenoviral particle of claim 38, wherein the subgroup C virus is Ad2 or Ad5, the subgroup D virus is Ad46 and the subgroup F virus is Ad41.
- 40. The adenoviral vector of claim 25 that is an early generation adenoviral vector, a gutless adenoviral vector or a replication-conditional adenoviral vector.
- 41. The adenoviral vector of claim 40, wherein the replication-conditional adenoviral vector is an oncolytic adenoviral vector.
- 42. The adenoviral vector of claim 41, wherein the replication-conditional adenoviral vector is an oncolytic adenoviral vector.
- 43. The adenoviral vector of claim 25 that comprises heterologous nucleic acid.
- 44. The adenoviral vector of claim 43, wherein the heterologous nucleic acid encodes a polypeptide.
- 45. The adenoviral vector of claim 43, wherein the heterologous nucleic acid comprises or encodes a regulatory nucleic acid.
- 46. The adenoviral vector of claim 43, wherein the heterologous nucleic acid comprises or encodes a regulatory nucleic acid.
- 47. The adenoviral vector of claim 56, wherein the heterologous nucleic acid comprises or encodes a promoter or RNA.
- 48. The adenoviral vector of claim 47, wherein the promoter is a cell or tissue specific promoter.
- 49. The adenoviral vector of claim 47, wherein the promoter is operably linked to a gene of an adenovirus essential for replication.
- 50. The adenoviral vector of claim 48, wherein the tissue specific promoter is a tumor specific promoter.
- 51. The adenoviral vector of claim 44, wherein the polypeptide is a therapeutic polypeptide.
- 52. A method of expressing heterologous nucleic acid in a cell, comprising transducing the cell with an adenoviral vector of claim 44.
- 53. The method of claim 52, wherein:
the cell is a tumor cell; the adenoviral vector is an oncolytic vector; and the cell is killed.
- 54. The method of claim 52, wherein the cell is a mammalian cell.
- 55. The method of claim 54, wherein the cell is a primate cell.
- 56. The method of claim 55, wherein the cell is a human cell.
- 57. A method of reducing transduction of liver cells by an adenoviral particle, comprising reducing or eliminating binding of the particle to heparin sulfate proteoglycans (HSPs) on the liver cells.
- 58. A scale up method for the propagation of a detargeted adenoviral particle, comprising:
infecting a cell capable of replicating, maturing and packaging an adenoviral vector with a detargeted adenoviral vector in the presence of a reagent that results in entry of the adenoviral particle into the cell; culturing the infected cell under conditions suitable for growth, spread and propagation of the adenoviral vector; and recovering the resulting adenoviral particles.
- 59. The method of claim 58, wherein the reagent is a polycation.
- 60. The method of claim 59, wherein the polycation is selected from the group consisting of hexadimethrine bromide, polyethylenimine, protamine sulfate and poly-L-lysine.
- 61. The method of claim 58, wherein the reagent is a bifunctional protein that binds to the adenoviral particle and to a receptor on the cell.
- 62. The method of claim 61, wherein:
the bifunctional protein is selected from the group consisting of an anti-fiber antibody ligand fusion, an anti-fiber-Fab-FGF conjugate, an anti-penton-antibody ligand fusion, an anti-hexon antibody ligand fusion and a polylysine-peptide fusion, wherein the ligand is a ligand that binds to the receptor.
- 63. The method of claim 58, wherein the detargeted adenoviral particle expresses a modified capsid, whereby binding to at least one host cell receptor is reduced or eliminated compared with a wild-type adenovirus.
- 64. The method of claim 63, wherein the adenoviral particle is modified to eliminate or reduce binding with one host cell receptor.
- 65. The method of claim 63, wherein the adenoviral particle is modified to eliminate or reduce binding with two host cell receptors.
- 66. The method of claim 63, wherein the adenoviral particle is modified to eliminate or reduce binding with three host cell receptors.
- 67. The, method of claim 63, wherein the particle is modified with one or more mutations selected from the group consisting of mutations that reduce or eliminate interactions with one or more of αv integrins, coxsackie-adenovirus receptors (CAR) and heparin sulfate proteoglycans (HSP).
- 68. The method of claim 67, wherein the mutation is selected from the group consisting of PD1, KO1, KO12 and S*.
- 69. The modified protein of claim 2, wherein the mutation is in the shaft of a fiber.
- 70. A modified protein of claim 3, wherein affinity for HSP is reduced at least by an amount selected from among reduced 5-fold, 10-fold and 100-fold.
RELATED APPLICATIONS
[0001] Benefit of priority is claimed under 35 U.S.C. §119(e) to U.S. provisional application Serial No. 60/350,388, filed Jan. 24, 2002, entitled “FIBER SHAFT MODIFICATIONS FOR EFFICIENT TARGETING,” to Stevenson, Susan C., Kaleko, Michael, Smith, Theodore and Nemerow, Glen R., and to U.S. provisional application Serial No. 60/391,967, filed Jun. 26, 2002, entitled “FIBER SHAFT MODIFICATIONS FOR EFFICIENT TARGETING,” to Stevenson, Susan C., Kaleko, Michael, Smith, Theodore and Nemerow, Glen R. This application is also related to International PCT application No. (attorney docket number 22908-1236PC), filed the same day herewith, entitled “FIBER SHAFT MODIFICATIONS FOR EFFICIENT TARGETING,” to Stevenson, Susan C., Kaleko, Michael, Smith, Theodore and Nemerow, Glen R. The subject matter of each of these applications is incorporated by reference herein.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60350388 |
Jan 2002 |
US |
|
60391967 |
Jun 2002 |
US |