Nucleic acid molecules encoding serine protease 17, the encoded polypeptides and methods based thereon

• US 20030134794A1
• Filed: 11/20/2002
• Published: 07/17/2003
• Est. Priority Date: 11/20/2001
• Status: Abandoned Application

• Current Assignee: Dendreon Corporation (Bausch Health Cos., Inc.)
• Original Assignee: Dendreon Corporation (Bausch Health Cos., Inc.)
• Inventors: Madison, Edwin L., Ong, Edgar O.
• Application Number:
  US10/302,840
• Publication Number:
  US 20030134794A1
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)

Abstract

Provided herein are polypeptides designated CVSP17 polypeptides that exhibit protease activity as a single chain or as an activated two chain form. Methods using the polypeptides to identify compounds that modulate the protease activity thereof are provided. The polypeptides also serve as tumor markers.

Claims

1. A substantially purified single chain or two chain polypeptide, comprising the protease domain of serine protease 17 (CVSP17) or a catalytically active portion thereof, wherein:

a) the polypeptide also comprises at least 10 or more contiguous amino acids from residues 397-427 of SEQ ID No. 6 or comprises 10 or more contiguous amino acids encoded by a sequence of nucleotides that hybridizes under conditions of high stringency to a sequence of nucleotides that encodes residues 397-427 of SEQ ID No. 6;

or

b) the CVSP17 portion of the polypeptide consists essentially of the protease domain of the CVSP17 or a catalytically active portion thereof with the proviso that the protease domain does not include contiguous sequence Cys Arg Ser Thr Arg Ser (SEQ ID No.

     18);

c) the polypeptide consists essentially of residues 19-332 of SEQ ID No. 6;

d) the polypeptide comprises the sequence of amino acids set forth in SEQ ID No. 6;

e) the polypeptide is encoded by a sequence of nucleotides that hybridizes under conditions of high stringency along at least 70% of its full length to a sequence of nucleotides than encodes a polypeptide of any of a)-e); and

/or

f) the polypeptide has at least 60% sequence identity with a polypeptide of any of a)-e).

2. A purified polypeptide of claim 1, comprising a sequence of amino acids set forth as amino acids 105-332 in SEQ ID No. 6 or a catalytically active portion thereof.

3. The polypeptide of claim 1 that is a substantially purified activated two chain CVSP17 polypeptide or a catalytically active portion thereof.

4. A substantially purified polypeptide that has at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity with a polypeptide of claim 1.

5. A polypeptide of claim 1, wherein the CVSP17 portion thereof consists essentially of a protease domain or a catalytically active portion thereof.

6. A substantially purified polypeptide that has at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity with a polypeptide of claim 1 and has within at least 1% of the catalytic activity on the same substrate as a polypeptide of claim 1.

7. The substantially purified polypeptide of claim 1 that is a human polypeptide.

8. A polypeptide of claim 1 that comprises:

(a) the sequence of amino acids set forth in SEQ ID No. 6 or a catalytically active portion thereof, or that is encoded by a sequence of nucleotides that;

(b) hybridizes under conditions of moderate or high stringency to nucleic acid complementary to an mRNA transcript present in a mammalian cell that encodes a CVSP17 encoded by (a);

(c) encodes a splice variant of (a);

or

(d) comprises degenerate codons of the sequences of nucleotides of (a) or (b).

9. A polypeptide that is a mutein of the polypeptide of claim 1, wherein:

up to about 50% of the amino acids are replaced with another amino acid;

and the resulting polypeptide is a single chain or two chain polypeptide that has catalytic activity of at least 1% of the unmutated polypeptide.

10. The polypeptide of claim 9, wherein up to about 10% of the amino acids are replaced with another amino acid.

11. The polypeptide of claim 9, wherein the resulting polypeptide is a single chain or two chain polypeptide and has catalytic activity of at least 10% of the unmutated polypeptide.

12. The polypeptide of claim 9, wherein a free Cysteine in the protease domain is replaced with another amino acid.

13. The polypeptide of claim 9, wherein up to about 95% of the amino acids are conserved or are replaced by conservative amino acid substitutions.

14. The polypeptide of claim 12, wherein the replacing amino acid is a serine.

15. A nucleic acid molecule, comprising a sequence of nucleotides that encodes the polypeptide of any of claims 1-14.

16. The nucleic acid molecule of claim 15 that comprises a sequence of nucleotides selected from the group consisting of:

(a) a sequence of nucleotides set forth in SEQ ID No. 5 or a portion thereof;

(b) a sequence of nucleotides that hybridizes under high stringency along at least about 70% of its full length to the sequence of nucleotides set forth in SEQ ID No. 5 or a portion thereof;

(c) a sequence of nucleotides that has at least 60%, 70%, 80%, 90% or 95% sequence identity with (a); and

(d) a sequence of nucleotides comprising degenerate codon(s) of any of (a)-(c).

17. A vector comprising the nucleic acid molecule of claim 16.

18. The vector of claim 17 that is an expression vector.

19. The vector of claim 17 that is a eukaryotic vector.

20. The vector of claim 17 that is a prokaryotic vector.

21. The vector of claim 17 that includes a sequence of nucleotides that directs secretion of any polypeptide encoded by a sequence of nucleotides operatively linked thereto.

22. The vector of claim 17 that is a Pichia vector, a mammalian vector or an E. coli vector.

23. A cell, comprising a vector of claim 17.

24. The cell of claim 23 that is a prokaryotic cell.

25. The cell of claim 23 that is a eukaryotic cell.

26. The cell of claim 23 that is selected from among a bacterial cell, a yeast cell, a plant cell, an insect cell and an animal cell.

27. The cell of claim 26 that is a mammalian cell.

28. A recombinant non-human animal, wherein an endogenous gene that encodes a polypeptide of claim 1 has been deleted or inactivated by homologous recombination or insertional mutagenesis of the animal or an ancestor thereof.

29. A method for producing a polypeptide that contains a protease domain of a CVSP17 polypeptide, comprising:

culturing the cell of claim 23 under conditions whereby the encoded polypeptide is expressed by the cell; and

recovering the expressed polypeptide.

30. The method of claim 29, wherein the polypeptide is secreted into the culture medium.

31. The method of claim 29, wherein the polypeptide is expressed in the cytoplasm of the host cell.

32. A method for producing a polypeptide, comprising:

culturing of claim 23 under conditions whereby the encoded polypeptide is expressed by the cell; and

recovering the expressed polypeptide.

33. The method of claim 32, wherein the polypeptide is expressed in inclusion bodies, and the method further comprises

isolating the polypeptide from the inclusion bodies under conditions, whereby the polypeptide refolds into a proteolytically active form.

37. A double-stranded RNA (dsRNA) molecule that comprises at least 21 contiguous nucleotides or modified nucleotides from a sequence of nucleotides encoding a polypeptide of claim 1.

40. An antibody that specifically binds to the single chain form and/or two-chain form of a polypeptide of claim 1, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

41. The antibody of claim 40 that inhibits the enzymatic activity of the polypeptide.

42. An antibody that specifically binds to the leucine zipper portion of a polypeptide of claim 1, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

44. An antibody that specifically binds to activated two chain forms or active single chain protease domain of a CVSP17 polypeptide, wherein the antibody binds to the activated form or single chain form with at least 10-fold greater affinity than to an inactive form, wherein the CVSP17 is a polypeptide of claim 1.

45. A conjugate, comprising:

a) a polypeptide of claim 1, and

b) a targeting agent linked to the polypeptide directly or via a linker.

46. The conjugate of claim 45, wherein the targeting agent permits

i) affinity isolation or purification of the conjugate;

ii) attachment of the conjugate to a surface;

iii) detection of the conjugate;

or

iv) targeted delivery to a selected tissue or cell.

47. A combination, comprising:

a) an agent or treatment that modulates the catalytic activity of the polypeptide of claim 1;

and

b) another agent or treatment selected from anti-tumor and anti-angiogenic treatments and agents.

48. The combination of claim 47, wherein the modulator and the anti-tumor and/or anti-angiogenic agent are formulated in a single pharmaceutical composition or each is formulated in separate pharmaceutical compositions.

49. The combination of claim 47, wherein the modulator is an inhibitor.

50. The combination of claim 47, wherein the modulator is selected from among antibodies and antisense oligonucleotides and double-stranded RNA (dsRNA).

51. A solid support comprising two or more polypeptides of claim 1 linked thereto either directly or via a linker.

52. The support of claim 51, wherein the polypeptides comprise an array.

53. The support of claim 51, wherein the polypeptides comprise a plurality of different protease domains.

54. A solid support comprising two or more nucleic acid molecules of claim 15 or oligonucleotides portions thereof linked thereto either directly or via a linker, wherein the oligonucleotides contain at least 16 nucleotides.

55. The support of claim 54, wherein the nucleic acid molecules comprise an array.

56. The support of claim 54, wherein the nucleic acid molecules comprise a plurality of molecules that encode different protease domains.

58. A method for identifying compounds that modulate the protease activity of a CVSP17 polypeptide, comprising:

contacting a CVSP17 polypeptide or a catalytically active portion thereof with a substrate that is proteolytically cleaved by the polypeptide, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting compounds that change the amount of substrate cleaved compared to a control, whereby compounds that modulate the activity of the polypeptide are identified, wherein the CVSP17 polypeptide is a CVSP17 polypeptide of claim 1.

65. A method of identifying a compound that specifically binds to a single-chain and/or two-chain protease domain and/or to single or two-chain polypeptide and/or to a proteolytically active portion of the single or two chain form thereof of a CVSP17 polypeptide, comprising:

contacting a CVSP17 polypeptide or a proteolytically active portion thereof with a test compound or plurality thereof under conditions conducive to binding thereof; and

either;

a) identifying test compounds that specifically bind to the single chain and two chain form of the polypeptide or to a single chain or to a two chain form thereof or to a proteolytically active portion of the single and/or two chain forms thereof, or

b) identifying test compounds that inhibit binding of a compound known to bind a single chain and two chain form of the polypeptide or to a single or a two chain form thereof or to a proteolytically active portion of the single and/or two chain form thereof, wherein the known compound is contacted with the polypeptide before, simultaneously with or after the test compound, wherein the CVSP17 polypeptide is a CVSP17 polypeptide of claim 1.

71. A method for identifying activators of the zymogen form of a CVSP17, comprising:

contacting a zymogen form of a CVSP17 polypeptide or a proteolytically active portion thereof with a substrate of the activated form of the polypeptide;

adding a test compound, wherein the test compound is added before, after or simultaneously with the addition of the substrate; and

detecting cleavage of the substrate, thereby identifying compounds that activate the zymogen, wherein the CVSP17 polypeptide is a CVSP17 polypeptide of claim 1.

74. A method for treating or preventing a neoplastic disease, in a mammal, comprising administering to a mammal an effective amount of a modulator of the proteolytic activity of a polypeptide of claim 1.

75. The method of claim 74, wherein the modulator is an inhibitor.

76. The method of claim 74, wherein the modulator is an antibody that specifically binds to the polypeptide, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

80. A method of inhibiting tumor initiation, growth or progression or treating a malignant or pre-malignant condition, comprising administering an agent that inhibits activation of the zymogen form of a CVSP17 polypeptide or a potentially proteolytically active portion thereof or inhibits an activity of the activated form of CVSP17 or a potentially proteolytically active portion thereof, wherein the CVSP17 polypeptide is a CVSP17 polypeptide of claim 1.

83. A method of detecting neoplastic disease, comprising:

detecting a polypeptide that comprises a polypeptide of claim 1 or a portion of a polypeptide of claim 1 in a biological sample, wherein the amount detected differs from the amount of polypeptide detected from a subject who does not have neoplastic disease.

84. The method of claim 83, wherein the biological sample is selected from the group consisting of blood, urine, saliva, tears, synovial fluid, sweat, interstitial fluid, cerebrospinal fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells.

86. A method of diagnosing the presence of a pre-malignant lesion, a malignancy, or other pathologic condition in a subject, comprising:

obtaining a biological sample from the subject; and

exposing the biological sample to a detectable agent that binds to a two-chain and/or single-chain form of a CVSP17 polypeptide, wherein the pathological condition is characterized by the presence or absence of the two-chain or single-chain form, wherein the CVSP17 polypeptide is a CVSP17 polypeptide of claim 1.

88. A method of monitoring tumor progression and/or therapeutic effectiveness, comprising detecting and/or quantifying the level and/or activity CVSP17 polypeptide in a body tissue or fluid sample, wherein the CVSP17 polypeptide is a CVSP17 polypeptide of claim 1.

91. A transgenic non-human animal, comprising heterologous nucleic acid encoding a polypeptide of claim 1.

34. An antisense nucleic acid molecule that comprises at least 14, 16 or 30 contiguous nucleotides or modified nucleotides that are complementary to all or a portion of a contiguous sequence of nucleotides that encodes the sequence of amino acids set forth as 397-427 of SEQ ID No. 6.

35. A double-stranded RNA (dsRNA) molecule that comprises at acid molecule that comprises at least 21 contiguous nucleotides or modified nucleotides that are complementary to all or a portion of a contiguous sequence of nucleotides that encodes the sequence of amino acids set forth as 397-427 of SEQ ID No. 6.

36. A double-stranded RNA (dsRNA) molecule that comprises at acid molecule that comprises at least 21 contiguous nucleotides or modified nucleotides that are complementary to all or a portion of a contiguous sequence of nucleotides that encodes the sequence of amino set forth as SEQ ID No. 6.

38. The double-stranded dsRNA molecule of claim 36 that contains at least 8, 10, 12, 14, 15, 18, 21 contiguous nucleotides or modified nucleotides encoding all or a portion of amino acids 397-427 of SEQ ID No. 6.

39. A probe that comprises at least 14, 16 or 30 contiguous nucleotides or modified nucleotides that include all or a portion of a contiguous sequence of nucleotides that encodes the sequence of amino acids set forth as 397-427 of SEQ ID No. 6.

43. An antibody that specifically binds to activated two chain forms or active single chain protease domain of a CVSP17 polypeptide, wherein the antibody binds to the activated form or single chain form with at least 10-fold greater affinity than to an inactive form.

57. A method for identifying compounds that modulate the protease activity of a CVSP17 polypeptide, comprising:

contacting a CVSP17 polypeptide or a catalytically active portion thereof with a substrate that is proteolytically cleaved by the polypeptide, and, either simultaneously, before or after, adding a test compound or plurality thereof;

measuring the amount of substrate cleaved in the presence of the test compound; and

selecting compounds that change the amount of substrate cleaved compared to a control, whereby compounds that modulate the activity of the polypeptide are identified.

59. The method of claim 57, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof that modulate the activity of the polypeptide.

60. The method of claim 57, wherein a plurality of the test compounds are screened simultaneously.

61. The method of claim 57, wherein the change in the amount of substrate cleaved is assessed by comparing the amount of substrate cleaved in the presence of the test compound with the amount of substrate cleaved in the absence of the test compound.

62. The method of claim 60, wherein a plurality of the polypeptides are linked to a solid support, either directly or via a linker.

63. The method of claim 62, wherein the polypeptides comprise an array.

64. A method of identifying a compound that specifically binds to a single-chain and/or two-chain protease domain and/or to single or two-chain polypeptide and/or to a proteolytically active portion of the single or two chain form thereof of a CVSP17 polypeptide, comprising:

contacting a CVSP17 polypeptide or a proteolytically active portion thereof with a test compound or plurality thereof under conditions conducive to binding thereof; and

either;

a) identifying test compounds that specifically bind to the single chain and two chain form of the polypeptide or to a single chain or to a two chain form thereof or to a proteolytically active portion of the single and/or two chain forms thereof, or

b) identifying test compounds that inhibit binding of a compound known to bind a single chain and two chain form of the polypeptide or to a single or a two chain form thereof or to a proteolytically active portion of the single and/or two chain form thereof, wherein the known compound is contacted with the polypeptide before, simultaneously with or after the test compound.

66. The method of claim 64, wherein the polypeptide is linked either directly or indirectly via a linker to a solid support.

67. The method of claim 64, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

68. The method of claim 64, wherein a plurality of the test substances are screened simultaneously.

69. The method of claim 68, wherein a plurality of the polypeptides are linked to a solid support.

70. A method for identifying activators of the zymogen form of a CVSP17, comprising:

contacting a zymogen form of a CVSP17 polypeptide or a proteolytically active portion thereof with a substrate of the activated form of the polypeptide;

adding a test compound, wherein the test compound is added before, after or simultaneously with the addition of the substrate; and

detecting cleavage of the substrate, thereby identifying compounds that activate the zymogen.

72. The method of claim 70, wherein the substrate is a chromogenic substrate.

73. The method of claim 70, wherein the test compound is a small molecule, a nucleic acid or a polypeptide.

77. A method of inhibiting tumor initiation, growth or progression or treating a malignant or pre-malignant condition, comprising administering an agent that inhibits activation of the zymogen form of a CVSP17 polypeptide or a potentially proteolytically active portion thereof or inhibits an activity of the activated form of CVSP17 or a potentially proteolytically active portion thereof.

78. The method of claim 77, wherein the condition is a condition of the breast, cervix, prostate, lung, ovary or colon.

79. The method of claim 77, wherein the agent is an antisense oligonucleotide, double-stranded RNA (dsRNA) or an antibody.

81. The method of claim 77, further comprising administering another treatment or agent selected from anti-tumor and anti-angiogenic treatments or agents.

82. A method of identifying a compound that binds to the single-chain or two-chain form of a CVSP17 polypeptide or to a proteolytically active portion of a single-chain or two-chain form of a CVSP17 polypeptide, comprising:

contacting a test compound with both forms;

determining to which form the compound binds; and

if the compound binds to a form of polypeptide, further determining whether the compound has at least one of the following properties;

(i) inhibits activation of a single-chain zymogen form of the polypeptide;

(ii) inhibits activity of a two-chain and/or single-chain active form; and

(iii) inhibits dimerization of the polypeptide.

85. A method of diagnosing the presence of a pre-malignant lesion, a malignancy, or other pathologic condition in a subject, comprising:

obtaining a biological sample from the subject; and

exposing the biological sample to a detectable agent that binds to a two-chain and/or single-chain form of a CVSP17 polypeptide, wherein the pathological condition is characterized by the presence or absence of the two-chain or single-chain form.

87. A method of monitoring tumor progression and/or therapeutic effectiveness, comprising detecting and/or quantifying the level and/or activity CVSP17 polypeptide in a body tissue or fluid sample.

89. The, method of claim 87, wherein the tumor is a tumor of the breast, cervix, prostate, lung, ovary or colon.

90. The method of claim 87, wherein the body fluid is blood, urine, sweat, saliva, cerebrospinal fluid and synovial fluid.

92. A polypeptide comprising a portion of a CVSP17 polypeptide, wherein the CVSP17 portion of the polypeptide consists essentially of amino acids 1-19 of SEQ ID No. 6.

93. A nucleic acid molecule encoding a polypeptide of claim 92.