Chimeric molecules for cleavage in a treated host

• US 20030219402A1
• Filed: 02/14/2003
• Published: 11/27/2003
• Est. Priority Date: 02/14/2002
• Status: Abandoned Application

• Current Assignee: William J. Rutter
• Original Assignee: William J. Rutter
• Inventors: Rutter, William J.
• Application Number:
  US10/367,555
• Publication Number:
  US 20030219402A1
• Time in Patent Office: Days
• US Class Current: 424/85.1
• 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: A61K 47/51 the non-active ingredient being a modifying agent
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/485 Epidermal growth factor [EGF], i.e. urogastrone
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 2799/026 where the vector is derived from a baculovirus

Abstract

The present invention relates to chimeric molecules containing component molecules that are linked together in a non-naturally occurring manner where the linker contains at least one enzyme cleavage site, and the enzyme cleavage site is engineered to be cleaved by an enzyme in a treated subject. The present invention also relates to compositions and kits containing the chimeric molecules, methods of making the chimeric molecules in a production host, methods of using the present chimeric molecules for diagnostic, prophylactic, therapeutic, and nutritional purposes in subjects requiring such.

Claims

1. A method of delivery of a plurality of component molecules to a multi-cellular host, comprising the steps of:

(a) providing a composition comprising a chimeric molecule; and

(b) administering the chimeric molecule to the host to produce a treated host,

wherein the chimeric molecule comprises at least one first component molecule, at least one linker, and at least one second component molecule;

wherein the linker comprises an enzyme cleavage site and wherein at least a first linker is operably linked to a first component molecule and a second component molecule to produce a non-naturally occurring linkage and cleavage site between the first component molecule and second component molecule;

wherein the cleavage site is engineered for cleavage in vivo by a host enzyme and is not susceptible to cleavage in a production host;

wherein, upon cleavage of the chimeric molecule at the cleavage site, at least one of the component molecules is functionally active; and

wherein at least one of the first and second component molecules comprises one selected from the group consisting of a peptide, a protein, or an active fragment thereof.

2. The method of claim 1, wherein the cleavage site is engineered for cleavage in vivo by an enzyme in the gastrointestinal tract of the host.

3. The method of claim 1, wherein the enzyme is enterokinase, a matrix metalloproteinase, or a tissue type plasminogen activator.

4. The method of claim 1, wherein upon cleavage of the chimeric molecule at the enzyme cleavage site, at least two of the component molecules are functionally active.

5. The method of claim 1, wherein at least one of the component molecules is functionally active prior to cleavage of the chimeric molecule.

6. The method of claim 1, wherein the component molecules are non-inhibitory molecules.

7. The method of claim 1, wherein the component molecules are non-cytotoxic molecules.

8. The method of claim 1, wherein the first component molecule is the same as the second component molecule.

9. The method of claim 1, wherein the chimeric molecule has a formula:

A(x_iB_i)ⁿ, wherein A represents the first component molecule, x represents the linker, B represents the second component molecule, i and n are each a positive integer.

10. The method of claim 9, wherein the formula is selected from the group consisting of:

(a) A(x₁B₁);

(b) A(x₁B₁)(x₂B₂), wherein x₁ and x₂ may be the same or different, and B₁ and B₂ may be the same or different;

(c) A(x₁B₁)(x₂B₂)(x₃B₃), wherein x₁, x₂ and x₃ may each be the same or different, and B₁, B₂ and B₃ may each be the same or different;

(d) A(x₁B₁)(x₂B₂)(x₃B₃)(x₄B₄), wherein x₁, x₂, x₃ and x₄ may each be the same or different, and B₁, B₂, B₃ and B₄ may each be the same or different; and

(e) A(x₁B₁)(x₂B₂)(x₃B₃)(x₄B₄)(x₅B₅), wherein x₁, x₂, x₃, x₄ and x₅ may each be the same or different, and B₁, B₂, B₃, B₄ and B₅ may each be the same or different.

11. The method of claim 1, wherein the first component molecule is a peptide or protein or an active fragment thereof and at least one second component molecule is selected from the group consisting of:

peptides, proteins, nucleic acids, carbohydrates, synthetic polymers, plant products, fungal products, small molecule drugs, detectable molecules, haptens, ligands, anti-infectives, and analogs and fragments thereof.

12. The method of claim 1, wherein the chimeric molecule is a polyprotein.

13. The method of claim 9, wherein the chimeric molecule is a polyprotein.

14. The method of claim 10, wherein the chimeric molecule is a polyprotein.

15. The method of claim 10, wherein x₁, x₂, x₃, x₄ and x₅ are the same.

16. The method of claim 10, wherein B₁, B₂, B₃, B₄ and B₅ are the same.

17. The method of claim 1, wherein at least one of the component molecules is selected from the group consisting of:

antigens, soluble receptors, growth factors, cytokines, lymphokines, chemokines, enzymes, anti-infectives, prodrugs, toxins, and active fragments thereof.

18. The method of claim 1, wherein at least one of the component molecules is selected from the group consisting of:

soluble p75TNFα

receptor Fc fusion, human growth hormone, granulocyte colony stimulating factor (GCSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-α

2b, pegylated (PEG) interferon-α

, PEG-asparagase, PEG-adamase, anti-CO 17-1 A, hirudin, tissue type plasminogen activator, erythropoietin, human DNAase, IL-2, coagulation factor IX, IL-11, TNKase, activated protein C, PDGF, coagulation factor VIa, insulin, interferon α

-N3, interferon γ

1b, interferon α

consensus sequence, platelet activating factor acetyl hydrolase and active fragments thereof.

19. The method of claim 1, wherein the first component molecule is a peptide, protein or an active fragment thereof and the second component molecule is a chemical compound.

20. The method of claim 1, wherein at least one of the component molecules is an antibody.

21. The method of claim 1, wherein first component molecule is an antibody or an active fragment thereof and the second component molecule is other than an antibody.

22. The method of claim 1, wherein second component molecule is an antibody or an active fragment thereof and the first component molecule is other than an antibody.

23. The method of claim 1, wherein the first and second component molecules are each an antibody or an active fragment thereof.

24. The method of claim 1, wherein at least one of the component molecules is selected from the group consisting of anti-microbial peptides, proteins, analogs or active fragments thereof.

25. The method of claim 1, wherein at least one of the component molecules is a defensin, a lysozyme, or a lactoferrin.

26. The method of claim 1, wherein at least one of the component molecules is selected from the group consisting of human and non-human animal peptides, proteins, analogs and active fragments thereof.

27. The method of claim 1, wherein at least one of the component molecules is selected from the group consisting of plant peptides, proteins, analogs or active fragments thereof.

28. The method of claim 9, wherein at least one of the component molecules is selected from the group consisting of microbial peptides, proteins, analogs or active fragments thereof.

29. The method of claim 1, wherein at least one of the component molecules is selected from the group consisting of fish peptides, proteins, analogs or active fragments thereof.

30. The method of claim 9, wherein at least two of the components are selected from the group consisting of:

peptides, proteins, analogs or active fragments thereof.

31. The method of claim 1, wherein the peptide or protein is selected from the group consisting of:

IGF-I, EGF, PDGF, ITF, KGF, lactoferrin, lysozyme, fibrinogen, α

₁-antitrypsin, erythropoietin, hGH, tPA, interferon alpha, interferon beta, interferon gamma, consensus interferon, insulin, human chorionic gonadotropin, diphtheria protein, and anti-hemophilic factor.

32. The method of claim 1, wherein at least one of the component molecules is a hormone.

33. The method of claim 32, wherein the hormone is selected from the group consisting of:

testosterone, estrogen, and progesterone.

34. The method of claim 1, wherein at least one of the component molecules is selected from the group consisting of:

taxol or its analogs or derivatives, matrix metalloproteinase inhibitors, and anti-infectives.

35. The method of claim 9, wherein at least two of the component molecules are selected from the group consisting of the combinations:

lactoferrin/lactoferrin;

lactoferrin/lysozyme;

lysozyme/lysozyme;

lactoferrin/EGF;

EGF/EFG;

lactoferrin/ITF;

ITF/ITF;

ITF/EFG;

EGF/KGF;

KGF/KGF;

ITF/KGF;

KGF/PDGF;

PDGF/PDGF;

α

₁-antitrypsin/MMP inhibitor;

estrogen/progesterone;

antibody/antibody;

ITF/ITF; and

analogs, variants and derivatives thereof.

36. The method of claim 1, wherein administration of the chimeric molecule achieves a biological effect in the treated host, and the biological effect is diagnostic, prophylactic, therapeutic, anti-infective or nutritional.

37. The method of claim 1, wherein the chimeric molecule further comprises at least a fragment of an additional polypeptide, wherein the polypeptide is highly expressed in the production host.

38. The method of claim 1, wherein the cleavage site is engineered for cleavage in vivo, extracellularly in the treated host, other than at a cell surface.

39. The method of claim 38, wherein neither the first nor the second component molecule is interferon-beta.

40. The method of claim 1, wherein the cleavage site is engineered for cleavage in vivo in the treated host at a cell surface.

41. The method of claim 1, wherein the first component molecule is not an antibody or an antibody fragment.

42. The method of claim 1, wherein the cleavage site is engineered for cleavage by an endogenous treated host enzyme.

43. The method of claim 1, wherein the cleavage site is engineered for cleavage by an endogenous host enzyme selected from the group consisting of:

coagulation factors;

ADAMTS 4, 5;

Aggreganases 1, 2;

thrombin;

plasmin;

complement factors;

gastricin;

granule proteases;

matrix metalloproteinases;

membrane type matrix metalloproteinases;

type II transmembrane serine proteases;

ADAMs;

neprilysin;

tissue-type plasminogen activator, and caspases.

44. The method of claim 1, wherein the cleavage site is engineered for cleavage in vivo intracellularly by an enzyme in the treated host, and the combination of first and second component molecules is other than the combination of a protein transduction domain and a cytotoxic domain.

45. The method of claim 1, wherein the cleavage site is engineered for cleavage in vivo intracellularly by an enzyme in the treated host, and the cleavage site is not a viral pathogen activated cleavage site.

46. The method of claim 1, wherein the cleavage site is engineered for cleavage in vivo intracellularly by an enzyme in the treated host, and the second component is not a cytotoxic molecule.

47. The method of claim 1, wherein the chimeric molecule further comprises a leader sequence for directing secretion of the chimeric molecule in a production host or for directing storage of the chimeric molecule in the production host.

48. The method of claim 1, wherein the chimeric molecule comprises a targeting molecule for directing the chimeric molecule to a location for action in the treated host.

49. The method of claim 1, wherein the chimeric molecule further comprises a purification moiety that facilitates in vitro purification of the chimeric molecule after production from a production host.

50. The method of claim 1, wherein the linker comprises two cleavage sites and a spacer adjacent between the two cleavage sites.

51. The method of claim 1, wherein the chimeric molecule is a component of an edible product.

52. The method of claim 51, wherein the edible product is selected from the group consisting of milk, a plant, a seed, a microbial cell, and derivatives and extracts thereof.

53. The method of claim 51, wherein the edible product is a cereal grain.

54. The method of claim 1, wherein the chimeric molecule is administered orally, parenterally, or by inhalation.

55. The method of claim 1, wherein the chimeric molecule is administered parenterally by intravenous route, subcutaneous route, intraperitoneal route, intracardiac route, or transdermal route.

56. The method of claim 1, wherein the chimeric molecule is not a nucleic acid molecule.

57. The method of claim 1, wherein the chimeric molecule further comprises an additional molecule that is linked to the first component molecule, but not to the linker, wherein the additional molecule is highly expressed in the production host.

58. The method of claim 1, wherein at least one of the first or second component molecules is an antibody or an active fragment thereof and the antibody is selected from the group consisting of:

anti-IL8, anti-CD11a, anti-ICAM-3, anti-CD80, anti-CD2, anti-CD3, anti-complement C5, anti-TNFα

, anti-CD4, anti-α

4β

7, anti-CD40L (ligand), anti-VLA4, anti-CD64, anti-IL5, anti-IL4, anti-IgE, anti-CD23, anti-CD147, anti-CD25, anti-β

2 integrin, anti-CD 18, anti-TGF#2, anti-Factor VII, anti-II_bII_a receptor, anti-PDGFβ

R, anti-F protein (from RSV), anti-gp120 (from HIV), anti-Hep B, anti-CMV, anti-CD14, anti-VEFG, anti-CA125 (ovarian cancer), anti-17-1A (colorectal cell surface antigen), anti-anti-idiotypic GD3 epitope, anti-EGFR, anti-HER2/neu;

anti-α

Vβ

3 integrin, anti-CD52, anti-CD33, anti-CD20, anti-CD22, anti-HLA, and anti-HLA DR or an active fragment thereof.

59. The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.

60. A kit comprising a composition comprising a chimeric molecule and a package insert comprising instructions for administration of composition to a human or non-human animal treated host, wherein the chimeric molecule comprises at least one first component molecule, at least one linker, and at least one second component molecule;

wherein the linker comprises an enzyme cleavage site and wherein at least a first linker is operably linked to a first component molecule and a second component molecule to produce a non-naturally occurring linkage and cleavage site between the first component molecule and second component molecule;

wherein the cleavage site is engineered for cleavage in vivo by a treated host enzyme and is resistant to cleavage in any production host;

wherein, upon cleavage of the chimeric molecule at the cleavage site, at least one of the component molecules is functionally active; and

wherein at least one of the first and second component molecules comprises one selected from the group consisting of a peptide, a protein, or an analog or active fragment or derivative thereof.

61. The kit of claim 60, wherein the cleavage site is engineered for cleavage in vivo in the gastrointestinal tract of the treated host.

62. The kit of claim 60, wherein the cleavage site is engineered for cleavage in vivo by enterokinase.

63. The kit of claim 60, wherein the cleavage site is engineered for cleavage in vivo extracellularly in the treated host, other than at a cell surface.

64. The kit of claim 60, wherein the cleavage site is engineered for cleavage in vivo in the treated host, at a cell surface.

65. The kit of claim 60, wherein the cleavage site is engineered for cleavage in vivo intracellularly in the treated host by an endogenous host enzyme.

66. The kit of claim 65, wherein the combination of the first component molecule and second component molecule is not a combination of a protein transduction domain and a cytotoxic domain.

67. The kit of claim 60, wherein the cleavage site is engineered for cleavage in vivo intracellularly in the treated host, and wherein the cleavage site is not a viral pathogen activated cleavage site.

68. The kit of claim 60, wherein the cleavage site is engineered for cleavage in vivo intracellularly in the treated host, and the second component molecule is other than a cytotoxic molecule.

69. A chimeric molecule that comprises a formula:

A(x_iB_i)ⁿ, wherein A represents the first component molecule, x represents the linker, B represents the second component molecule, i and n are each a positive integer, and wherein the chimeric molecule comprises at least one first component molecule, at least one linker, and at least one second component molecule;

wherein the linker comprises an enzyme cleavage site and wherein at least a first linker is operably linked to a first component molecule and a second component molecule to produce a non-naturally occurring linkage and cleavage site between the first component molecule and second component molecule;

wherein the cleavage site is engineered for cleavage in vivo by a host enzyme and is not susceptible to cleavage in a production host;

wherein, upon cleavage of the chimeric molecule at the cleavage site, at least one of the component molecules is functionally active; and

wherein at least one of the first and second component molecules comprises one selected from the group consisting of a peptide, a protein, or an analog or active fragment or derivative thereof.

70. The chimeric molecule of claim 69, wherein the formula is selected from the group consisting of:

(a) A(x₁B₁);

(b) A(x₁B₁)(x₂B₂), wherein x, and x₂ may be the same or different, and B₁ and B₂ may be the same or different;

(c) A(x₁B₁)(x₂B₂)(x₃B₃), wherein x₁, x₂ and x₃ may each be the same or different, and B₁, B₂ and B₃ may each be the same or different;

(d) A(x₁B₁)(x₂B₂)(x₃B₃)(x₄B₄), wherein x₁, x₂, x₃ and x₄ may each be the same or different, and B₁, B₂, B₃ and B₄ may each be the same or different; and

(e) A(x₁B₁)(x₂B₂)(x₃B₃)(x₄B₄)(x₅Bs), wherein x₁, x₂, x₃, x₄ and x₅ may each be the same or different, and B₁, B₂, B₃, B₄ and B₅ may each be the same or different.

71. The chimeric molecule of claim 69, wherein the chimeric molecule is a polyprotein.

72. A nucleic acid molecule encoding the chimeric molecule of claim 71.

73. A vector comprising the nucleic acid molecule of claim 72.

74. A host cell comprising the nucleic acid molecule of claim 72.

75. A method for the preparation of a chimeric molecule in a production host for administration to a treated host comprising:

(a) providing a nucleic acid molecule that encodes a chimeric molecule;

(b) transforming a production host with the nucleic acid molecule;

(c) allowing the production host to produce the chimeric molecule;

(d) recovering the chimeric molecule from the production host; and

(e) performing quality control on the harvested chimeric molecule to meet regulatory approval;

wherein the chimeric molecule comprises component molecules including a first component molecule, a linker that comprises a cleavage site, and a second component molecule,

wherein at least one of the first and second component molecules comprises a peptide, a protein or an active fragment thereof;

wherein the linker is operably linked to the first and second component molecules to produce a non-naturally occurring linkage and cleavage site;

wherein the cleavage site is engineered for in vivo cleavage by a treated host enzyme.

76. The method of claim 75, wherein the enzyme is present in the gastrointestinal tract of the treated host.

77. The method of claim 75, wherein the enzyme is an enzyme that acts extracellularly in the treated host, but not at a cell surface.

78. The method of claim 75, wherein the enzyme is an enzyme that acts at a cell surface in the treated host.

79. The method of claim 75, wherein the enzyme is an enzyme that acts intracellularly in the treated host.

80. The method of claim 79, wherein the chimeric molecule is other than a combination of a protein transduction domain and a cytotoxic domain.

81. The method of claim 75, wherein the enzyme is an enzyme that acts intracellularly in the treated host and the cleavage site is not a viral pathogen activated cleavage site.

82. The method of claim 75, wherein the enzyme is an enzyme that acts intracellularly in the treated host and the second component molecule is other than a cytotoxic molecule.

83. The method of 75, wherein the production host is selected from the group consisting of:

a bacterial cell, a fungal cell, a mammalian cell, a plant cell, a plant seed, an insect cell, a plant, a fungus, and an animal.

84. A composition comprising a chimeric molecule and a pharmaceutically acceptable carrier for administration to a treated host, wherein the chimeric molecule

comprises component molecules including at least one first component molecule, at least one linker, and at least one second component molecule;

wherein the linker comprises an enzyme cleavage site and wherein at least a first linker is operably linked to a first component molecule and a second component molecule to produce a non-naturally occurring linkage and cleavage site between the first component molecule and second component molecule;

wherein the cleavage site is engineered for cleavage in vivo by a treated host enzyme and is resistant to cleavage in a production host;

wherein, upon cleavage of the chimeric molecule at the cleavage site, at least one of the component molecules is functionally active; and

wherein at least one of the first and second component molecules comprises one selected from the group consisting of a peptide, a protein, or an active fragment thereof.

85. The composition of claim 84, wherein the cleavage site is engineered for in vivo cleavage by an enzyme in the gastrointestinal tract of the treated host.

86. The composition of claim 84, wherein the enzyme is enterokinase.

87. The composition of claim 84, wherein the cleavage site is engineered for in vivo cleavage by an enzyme in an inflammatory tissue of the treated host.

88. The composition of claim 87, wherein the inflammatory tissue is inflammatory bowel or synovium.

89. The composition of claim 84, wherein the cleavage site is engineered for in vivo cleavage extracellularly in the treated host other than at a cell surface.

90. The composition of claim 84, wherein the cleavage site is engineered for in vivo cleavage in the treated host at a cell surface.

91. The composition of claim 84, wherein the cleavage site is engineered for in vivo cleavage intracellularly in the treated host by an endogenous treated host enzyme.

92. The composition of claim 84, wherein the cleavage site is engineered for in vivo cleavage intracellularly in the treated host and wherein the combination of the first and second component molecules is not a combination of a protein transduction domain and a cytotoxic domain.

93. The composition of claim 84, wherein the cleavage site is engineered for in vivo cleavage intracellularly in the treated host and wherein the second component molecule is not a cytotoxic molecule.

94. The composition of claim 84, wherein the composition is encapsulated.

95. The composition of claim 84, wherein one of the component molecules binds to an extracellular matrix in the treated host.

96. The composition of claim 84, wherein the chimeric molecules comprises two cleavage sites, one of which is engineered for cleavage in vitro after expression is a production host and the other is engineered for cleavage in vivo in the treated host.

97. The composition of claim 84, wherein the composition is formulated for oral delivery.

98. The composition of claim 84, wherein the composition is formulated for parenteral delivery.

99. The composition of claim 98, wherein parenteral delivery is selected from the group consisting of:

subcutaneous, intravenous, intra-arterial, intraventricular, intracranial, percutaneous and transdermal delivery

100. The composition of claim 84, wherein the composition is formulated for intranasal delivery or for inhalation.

101. The composition of claim 84, wherein the chimeric molecule is a vaccine.

102. The composition of claim 84, wherein the chimeric molecule comprises an adjuvant as one of the component molecules.

103. The composition of claim 101, wherein the vaccine comprises a component of a pathogenic organism.

104. The composition of claim 101, wherein the vaccine is a cancer vaccine, and the component molecules are molecules that are over-expressed in a cancer cell.

105. The use of a chimeric molecule in the preparation of a medicament for diagnosis, prophylaxis, treatment of a disease or condition, or for enhancement of nutrition in a subject in need of such, wherein the chimeric molecule comprises at least one first component molecule, at least one linker, and at least one second component molecule;

wherein the linker comprises an enzyme cleavage site and wherein at least a first linker is operably linked to a first component molecule and a second component molecule to produce a non-naturally occurring linkage and cleavage site between the first component molecule and second component molecule;

wherein the cleavage site is engineered for cleavage in vivo by a treated host enzyme and is not susceptible to cleavage in a production host;

wherein, upon cleavage of the chimeric molecule at the cleavage site, at least one of the component molecules is functionally active; and

wherein at least one of the first and second component molecule comprises one selected from the group consisting of a peptide, a protein, or an active fragment thereof.