Motif-grafted hybrid polypeptides and uses thereof

• US 20030215880A1
• Filed: 04/08/2003
• Published: 11/20/2003
• Est. Priority Date: 04/09/2002
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Burton, Dennis R., Moroncini, Gianluca, Williamson, R. Anthony
• Granted Patent: US 7,939,641 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 16/1063 env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2318/10 Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: G01N 33/6896 Neurological disorders, e.g. Alzheimer's disease

Abstract

Provided herein are hybrid polypeptides that specifically bind to a disease-associated isoform of a polypeptide involved in diseases of protein aggregation. The hybrid polypeptides can be used for diagnosis and treatment of such diseases. In a particular embodiment, a hybrid protein that specifically binds to the infectious form of a prion (PrP^Sc) is provided.

Claims

1. A hybrid polypeptide, comprising:

a polypeptide motif that contains a sufficient number of contiguous amino acid residues from a polypeptide associated with a disease of protein aggregation or conformation to bind to an aggregating form of the polypeptide or to a disease-associate conformer of the polyeptide; and

additional amino acids from a polypeptide other than the polypeptide from which the motif is derived, whereby

the resulting hybrid polypeptide binds with greater affinity to a disease causing or infectious conformer of the polypeptide that is the source of the polypeptide motif compared to a benign form of the polypeptide.

2. The polypeptide of claim 1 that is multimeric.

3. The polypeptide of claim 1, wherein additional amino acids comprise at least about 5 amino acids at the N-terminus and at least about 5 amino acids at the C-terminus of the motif portion.

4. The polypeptide of claim 1, wherein additional amino acids comprise at least about 15 amino acids at the N-terminus and at least about 15 amino acids at the C-terminus of the motif portion.

5. The polypeptide of claim 1 that is a dimer.

6. The polypeptide of claim 1 that is a trimer.

14. A hybrid molecule of claim 1 that binds with at least 10-fold greater affinity to a disease-related isoform of a protein than to a benign isoform thereof.

17. A hybrid molecules or polypeptide of any of claims 1, 7 and 9, wherein the polypeptide motif comprises all or a portion of a polypeptide selected from the group consisting of APP, Aβ

, α

1-antichymotrypsin, tau, non-Aβ

component, presenilin 1, presenilin 2, apoE, superoxide dismutase (SOD) and neurofilament, Pick body, α

-synuclein, tau in fibrils, amylin, IgGL-chain, transthyretin, procalcitonin, β

₂-microglobulin, atrial natriuretic factor, serum amyloid A, ApoAI, gelsolin, Huntington protein.

18. The polypeptide of any of claims 1, 7 and 9, wherein the disease-related protein is a prion protein.

19. The polypeptide of claim 18, wherein the protein is a prion from a animal selected from the group consisting of humans, hamsters, mice, rats, deer, sheep, goats, elk, kudu, horses, dogs, cats, camels and pigs.

20. The polypeptide of claim 19, wherein the disease is a genetic disease and the protein is a prion that is encoded by a mutant form of a prion-encoding allele.

24. The polypeptide of claim 18 that comprises residues that include at least one α

-helix from the PrP^c form of a prion.

26. The polypeptide of claim 1, claim 7 or claim 9 that binds with at least 10-fold greater affinity to a disease-related isoform of a polypeptide than to a benign isoform thereof.

28. The polypeptide of claim 1, claim 7 or claim 9, wherein the polypeptide motif comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 residues or up to a full length prion polypeptide presented in its native non-infectious conformation.

61. A method of detecting an isoform of polypeptide associated with a disease of protein aggregation, comprising:

contacting a sample suspected of containing the isoform with a hybrid polypeptide of any claim 1;

and

detecting binding of the polypeptide, whereby the isoform of the polypeptide associated with the disease is detected.

62. The method of claim 61, wherein the hybrid polypeptide is detectably labeled.

7. A hybrid molecule, comprising:

a scaffold; and

a polypeptide motif from a protein that is involved in a disease of protein aggregation or conformation, wherein;

the polypeptide motif includes residues from a target polypeptide that are involved in the aggregation reaction or that induce or are involved in the change in conformation of the polypeptide;

upon linkage of the polypeptide motif to or insertion into the scaffold the resulting hybrid polyeptide specifically binds as a monomeric or multimeric unit to a disease-associated form of the protein;

the disease is a disease of protein aggregation or conformation.

8. The hybrid molecule of claim 7, wherein the scaffold comprises amino acids.

9. A hybrid molecule of claim 7 that is a hybrid polypeptide, comprising:

a scaffold; and

a polypeptide motif not derived from the scaffold, wherein;

the polypeptide motif contains a sufficient number of contiguous amino acid residues from a polypeptide associated with a disease of protein aggregation to bind to the aggregating form of the polypeptide;

the polypeptide motif is inserted within the scaffold; and

the resulting hybrid polypeptide preferentially binds to a disease causing or infectious isoform of the polypeptide that is the source of the polypeptide motif compared to a benign form of the polypeptide.

10. A hybrid polypeptide of claim 9 that binds with at least 10-fold greater affinity to a disease-related isoform of a protein than to a benign isoform thereof.

11. A hybrid polypeptide of claim 10 that binds with at least 100-fold greater affinity to a disease-related isoform of a protein than to a benign isoform thereof.

12. A hybrid polypeptide of claim 9, wherein the disease is selected from the group consisting of amyloid diseases.

13. A hybrid polypeptide of claim 9, wherein the disease is selected from the group consisting of Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy, Alzheimer'"'"'s Disease, Type II Diabetes, Huntington'"'"'s Disease, immunoglobulin amyloidosis, reactive amyloidosis associated with chronic inflammatory disease, hereditary systemic amyloidosis associated with autosomal dominant inheritance of variant transthyretin gene, ALS, Pick'"'"'s Disease, Parkinson'"'"'s disease, Frontotemporal dementia, Diabetes Type II, Multiple myeloma, Plasma cell dyscrasias, Familial amyloidotic polynueuropathy, Medullary carcinoma of thyroid;

chronic renal failure, congestive heart failure, senile cardiac and systemic amyloidosis, chronic inflammation, atherosclerosis and familial amyloidosis.

15. A hybrid polyeptide of claim 7 or claim 9, wherein the scaffold includes a constant region from an IgG, IgM, IgA, IgD or IgE immunoglobulin.

16. A hybrid polyeptide of claim 7 or claim 9, wherein the scaffold is an immunoglobulin, an Fab, and F(ab)₂ or a single chain Fv.

21. The polypeptide of claim 7 or claim 9, wherein the scaffold comprises all or a sufficient portion of a protein selected from the group consisting of antibodies, enzymes, chromogenic proteins, fluorescent proteins and fragments thereof sufficient to present the polypeptide motif whereby the preferential or specific binding of the motif is retained.

22. The polypeptide of claim 21, wherein the scaffold comprises all or a portion of an enzyme, an antibody or a fluorescent or chromogenic polypeptide.

23. The polypeptide of claim 21, wherein the scaffold comprises all or a portion of an antibody.

49. A nucleic acid molecule encoding any of the polypeptides of any of claim 7 or claim 9.

50. A vector, comprising the nucleic acid molecule of claim 49.

51. The vector of claim 50 that is an expression vector.

52. The vector of claim 50 that is a eukaryotic vector.

53. The vector of claim 50 that includes a sequence of nucleotides that directs secretion of any polypeptide encoded by a sequence of nucleotides operatively linked thereto.

54. The vector of claim 50 that is a mammalian vector, a yeast vector or a bacterial vector.

56. A cell, comprising a vector of claim 50.

57. The cell of claim 56 that is a prokaryotic cell.

58. The cell of claim 56 that is a eukaryotic cell.

59. The cell of claim 56 that is selected from among a bacterial cell, a yeast cell, a plant cell, an insect cell and an animal cell.

60. The cell of claim 58 that is a mammalian cell.

94. A solid support comprising a plurality of polypeptides of any of claim 7 or claim 9.

95. A method of detecting cells that contain a protein conformer associated with a disease of protein aggregation, comprising:

contacting cells from an animal or tissue with a hybrid polypeptide of claim 7, wherein the hybrid polypeptide is detectably labeled or comprises a detectable scaffold; and

detecting labeled cells.

96. The method of claim 95, wherein the label is a fluorescent label

97. The method of claim 96, wherein detection is effected by flow cytometry or scanning cytometry.

98. The method of claim 95, wherein the cells are contacted with a plurality of different hybrid polyeptides.

99. The method of claim 98, wherein the hybrid polypeptides bind to distinct epitopes on a target polypeptide.

100. The method of claim 95, wherein the hybrid polypeptide comprises a detectable scaffold.

101. The method of claim 100, wherein the detectable scaffold comprises a luminescent protein or luminescent portion thereof.

102. The method of claim 101, wherein the luminescent protein is a fluorescent protein (FP).

103. The method of claim 102, wherein the FP is selected from the group consisting of a green FP, red FP, blue FP and variants thereof that have distinct emission spectra.

104. The method of claim 95, wherein the cells are prion-infected cells.

105. The method of claim 95, wherein the disease is selected from the group consisting of Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy, Alzheimer'"'"'s Disease, Type II Diabetes, Huntington'"'"'s Disease, immunoglobulin amyloidosis, reactive amyloidosis associated with chronic inflammatory disease, hereditary systemic amyloidosis associated with autosomal dominant inheritance of variant transthyretin gene, ALS, Pick'"'"'s Disease, Parkinson'"'"'s disease, Frontotemporal dementia, Diabetes Type II, Multiple myeloma, Plasma cell dyscrasias, Familial amyloidotic polynueuropathy, Medullary carcinoma of thyroid;

chronic renal failure, congestive heart failure, senile cardiac and systemic amyloidosis, chronic inflammation, atherosclerosis and familial amyloidosis.

25. An isolated substantially pure polypeptide that specifically binds to the infectious form of a prion protein.

27. The polypeptide of claim 25, wherein the polypeptide binds with an affinity of at least 10⁸ l/mol.

29. A polypeptide of claim 25 that comprises residues from a portion of a PrP that corresponds to residues 87-169 of a Syrian hamster prion polypeptide.

30. The polypeptide of claim 25 that comprises at least residues 121-131, 121-141, 121-136, 121-144, 121-158, 87-112, 87-118, 87-130, 126-158, 131-158, 136-158 or 141-158 of a prion polyeptide.

31. The polypeptide of claim 30, wherein the prion portion of the polypeptide consists essentially of residues 121-131, 121-141, 121-136, 121-144, 121-158, 87-112, 87-118, 87-130, 126-158, 131-158, 136-158, 141-158.

32. The polypeptide of claim 25 that comprises at least residues 136-158, 89-105, 89-112 or 95-112 of a prion polyeptide.

33. The polypeptide of claim 30, wherein the prion portion of the polypeptide consist essentially of residues 136-158, 89-105, 89-112 or 95-112 of a prion polyeptide.

34. The polypeptide of claim 25 that comprises residues that include at least one α

-helix from the PrP^c form of the prion.

35. The polypeptide of claim 25 that comprises antibody b12 or a fragment therein, wherein residues 121-158 or a binding portion thereof of a prion are inserted in place of residues 119-131 of SEQ ID No. 4.

36. The polypeptide of claim 25 that comprises antibody b12 or a fragment therein, wherein residues 87-112 or a binding portion thereof of a prion are inserted in place of residues 119-131 of SEQ ID No. 4.

37. The polypeptide of claim 35 that comprises the heavy and light chains of antibody b12, wherein the heavy chain comprises the sequence of amino acids of SEQ ID No. 4, and the light chain comprises the sequence of amino acids of SEQ ID No. 2.

38. A polypeptide, comprising at least 5, 10, 15, 20, 25, 30, 35 contiguous residues from the region of residues 119-158 of a prion polypeptide, wherein:

residues from the region are the only prion-derived residues in the polypeptide; and

the residues correspond upon alignment of the prion sequence with the Syrian hamster prion sequence to residues 119-158 of Syrian hamster set forth in SEQ ID No. 5.

39. The polypeptide claim 38, wherein the prion is an animal prion selected from the group consisting of humans, hamsters, mice, rats, deer, sheep, goats, elk, kudu, horses, dogs, cats, camels and pigs.

40. A hybrid immunoglobulin polypeptide, comprising a polypeptide motif not derived from an immunoglobulin molecule, wherein:

the polypeptide motif contains a sufficient number of contiguous amino acid residues from a polypeptide associated with a disease of protein aggregation to bind to the aggregating form of the polypeptide;

the polypeptide motif is inserted within the third complementarity-determining region (CDR) of the immunoglobulin molecule; and

the resulting hybrid immunoglobulin molecule preferentially binds to a disease causing or infectious isoform of the polypeptide that is the source of the polypeptide motif compared to a benign form of the polypeptide.

41. The polypeptide of claim 40 that contains at least 10, 15, 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more contiguous amino acid residues from the polypeptide associated with a disease of protein aggregation.

42. The polypeptide of claim 40, wherein the disease is selected from the group consisting of amyloid diseases.

43. The polypeptide of claim 40, wherein the polypeptide associated with a disease of protein aggregation is a prion.

44. The polypeptide of claim 41, wherein the disease is selected from the group consisting of Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy, Alzheimer'"'"'s Disease, Type II Diabetes, Huntington'"'"'s Disease, immunoglobulin amyloidosis, reactive amyloidosis associated with chronic inflammatory disease, hereditary systemic amyloidosis associated with autosomal dominant inheritance of variant transthyretin gene, ALS, Pick'"'"'s Disease, Parkinson'"'"'s disease, Frontotemporal dementia, Diabetes Type II, Multiple myeloma, Plasma cell dyscrasias, Familial amyloidotic polynueuropathy, Medullary carcinoma of thyroid;

chronic renal failure, congestive heart failure, senile cardiac and systemic amyloidosis, chronic inflammation, atherosclerosis and familial amyloidosis.

45. The polypeptide of claim 40, wherein the polypeptide motif comprises all or a portion of a polypeptide selected from the group consisting of APP, Aβ

, α

1-antichymotrypsin, tau, non-Aβ

component, presenilin 1, presenilin 2, apoE, superoxide dismutase (SOD) and neurofilament, Pick body, α

-synuclein, tau in fibrils, amylin, IgGL-chain, transthyretin, procalcitonin, β

₂-microglobulin, atrial natriuretic factor, serum amyloid A, ApoAI, gelsolin, Huntington protein.

46. A polypeptide of claim 40 that binds with at least 10-fold greater affinity to a disease-related isoform of a protein than to a benign isoform thereof.

47. A polypeptide of claim 40 that binds with at least 100-fold greater affinity to a disease-related isoform of a protein than to a benign isoform thereof.

48. A polypeptide claim 40, wherein them motif is from a prion polypeptide, and the prion is an animal prion selected from the group consisting of humans, hamsters, mice, rats, deer, sheep, goats, elk, kudu, horses, dogs, cats, camels and pigs.

55. The vector of claim 41 that is a viral vector, a Pichia vector or an E. coli vector.

67. A method of detecting a PrP^Sc form of a prion polypeptide, comprising:

contacting a sample containing a prion polypeptide with a polypeptide of claim 40, and

detecting binding to any PrP^Sc in the sample, thereby detecting the presence of PrP^Sc.

71. The method of claim 67, wherein the polypeptide contacted with the sample is a hybrid polypeptide that comprises:

a scaffold; and

a polypeptide motif not derived from the scaffold, wherein;

the polypeptide motif contains a sufficient number of contiguous amino acid residues from a polypeptide associated with a disease of protein aggregation to bind to the aggregating form of the polypeptide;

the polypeptide motif is inserted within the scaffold; and

the resulting hybrid polypeptide preferentially binds to a disease causing or infectious isoform of the polypeptide that is the source of the polypeptide motif compared to a benign form of the polypeptide.

72. The method of claim 67, wherein the polypeptide contacted with the sample is a hybrid polypeptide that comprises:

a scaffold; and

a polypeptide motif not derived from the scaffold, wherein;

the polypeptide motif contains a sufficient number of contiguous amino acid residues from a polypeptide associated with a disease of protein aggregation to bind to the aggregating form of the polypeptide;

the polypeptide motif is inserted within the scaffold;

the resulting hybrid polypeptide preferentially binds to a disease causing or infectious isoform of the polypeptide that is the source of the polypeptide motif compared to a benign form of the polypeptide; and

the polypeptide associated with a disease of protein aggregation is a prion.

73. The method of claim 71, wherein the scaffold comprises all or a portion of an enzyme, an antibody or a fluorescent or chromogenic molecule.

63. A method of detecting a PrP^Sc form of a prion polypeptide, comprising:

contacting a sample suspected of containing an infectious isoform of a prion polypeptide with polypeptide comprising a PrP^c form of a prion polypeptide or a portion thereof that binds to the infectious form; and

detecting binding to any PrP^Sc in the sample.

64. The method of claim 63, wherein the sample is a body fluid, a tissue or organ.

65. The method of claim 63, wherein the sample suspected of containing an infectious isoform of a prion polypeptide is contacted with polypeptide that consists essentially of all or at least about 20, 25, 30, 35, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more contiguous amino acid residues of a PrP^c form of a prion polypeptide.

66. The method of claim 63, wherein the prion is an animal prion selected from the group consisting of humans, hamsters, mice, rats, deer, sheep, goats, elk, kudu, horses, dogs, cats, camels and pigs.

70. The method of claim 63, wherein the body fluid is selected from the group consisting of blood, urine, sweat, saliva, cerebrospinal fluid, sperm samples, serum, plasma and synovial fluid.

68. The method of 67, wherein the sample is a body fluid, a tissue or organ.

69. The method of claim 69, wherein the body fluid is selected from the group consisting of blood, urine, sweat, saliva, cerebrospinal fluid, sperm samples, serum, plasma and synovial fluid.

74. A method of detecting an isoform of a target polypeptide in a sample, comprising:

a) contacting a sample suspected of containing the target polypeptide with a reagent that specifically binds thereto as a monomer or dimer, wherein;

the target polypeptide is in a conformation that forms aggregates thereof;

the reagent is a hybrid polypeptide that comprises a scaffold and a polypeptide motif inserted therein;

the polypeptide motif when inserted into the scaffold binds to the target polypeptide; and

b) detecting the resulting complexes of the target polypeptide and reagent.

81. The method of claim 74, wherein the presence of the target polypeptide is indicative of a disease involving protein aggregation.

82. The method of claim 81, wherein the disease is selected from the group consisting of amyloid diseases.

83. The method of claim 81, wherein the disease is selected from the group consisting of Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy, Alzheimer'"'"'s Disease, Type II Diabetes, Huntington'"'"'s Disease, immunoglobulin amyloidosis, reactive amyloidosis associated with chronic inflammatory disease, hereditary systemic amyloidosis associated with autosomal dominant inheritance of variant transthyretin gene, ALS, Pick'"'"'s Disease, Parkinson'"'"'s disease, Frontotemporal dementia, Diabetes Type II, Multiple myeloma, Plasma cell dyscrasias, Familial amyloidotic polynueuropathy, Medullary carcinoma of thyroid;

chronic renal failure, congestive heart failure, senile cardiac and systemic amyloidosis, chronic inflammation, atherosclerosis and familial amyloidosis.

84. The method of claim 74, wherein the assay is a homogeneous assay.

85. The method of claim 84, wherein the reagent or hybrid polypeptide further comprises a second binding site and the method comprises capturing the complexes formed between the reagent and the target polypeptide on a solid support to thereby effect detection.

86. The method of claim 84, wherein the assay is a heterogeneous assay.

87. The method of claim 86, wherein the reagent or hybrid polypeptide is linked directly or indirectly to a solid support.

75. The method of 74, wherein the sample is a biological sample.

76. The method of 75, wherein the sample is a body fluid, tissue or organ.

77. The method of 75, wherein the sample is blood or blood-derived composition.

78. The method of 75, wherein the sample is a tissue or organ or is derived therefrom.

79. The method of 74, wherein the sample comprises a drug or other bio-active molecule prepared from the tissue or organ or is food.

80. The method of 79, wherein the drug or bioactive molecule is a hormone or growth factor.

88. A method of detecting PrP^Sc in a sample, comprising:

contacting a sample suspected of containing native PrP^Sc with a reagent that specifically binds as a monomer or dimer to native PrP^Sc in situ; and

detecting the resulting complexes.

89. the method of claim 88, wherein the reagent is a hybrid polypeptide comprising a sufficient portion of a PrP to specifically bind to PrP^Sc.

90. The method of claim 89, wherein the reagent comprises a hybrid polypeptide that comprises:

a scaffold; and

a polypeptide motif that specifically binds as a monomeric or dimeric unit to a disease-related form of a protein, wherein the disease is a disease of protein aggregation.

91. The method of claim 90, wherein the scaffold is selected from the group consisting of enzymes, chromogenic proteins, fluorescent proteins, antibodies and antibody fragments.

92. The method of claim 91, wherein the polypeptide motif comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.

93. The method of claim 91, wherein the polypeptide motif is inserted into in place of one or more amino acids of the scaffold.

106. A method for preparing a hybrid molecule that specifically interacts with one conformer of a protein that is involved in a disease of protein aggregation or conformation, comprising:

identifying a portion of a disease-related conformer that participates in the interaction of the conformer with a benign form of the conformer or in the aggregation reaction; and

inserting all or a portion of the identified portion into a scaffold, wherein the resulting hybrid molecule interacts with one conformer of a protein that is involved in a disease of protein aggregation or conformation with greater affinity than with a benign conformer.

107. An anti-idiotype antibody that specifically binds to an infectious form of a prion protein.

108. The anti-idiotype antibody of claim 107 that is produced by immunizing with Fab D13 or Fab D18 or with a hybrid polypeptide that comprises a motif from the replicative interface of cellular prion polypeptide inserted into a scaffold.