Trimerising Module

• US 20100022756A1
• Filed: 03/16/2009
• Published: 01/28/2010
• Est. Priority Date: 06/11/1997
• Status: Active Grant

• Current Assignee: Anaphore, Inc., Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Anaphore, Inc., Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Thogersen, Hans Christian, Holtet, Thor Las, Etzerodt, Michael, Graversen, Niels Jonas Heilskov, Nielsen, Bettina Bryde, Larsen, Ingrid Kjoller, Kastrup, Jette Sandholm
• Granted Patent: US 8,318,679 B2
• Time in Patent Office: Days
• US Class Current: 530/387.100
• 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 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/4726 Lectins
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/41 containing a Myc-tag
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/73 containing coiled-coiled motif (leucine zippers)
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

The present invention relates to the design of trimeric polypeptides using polypeptide structural elements derived from the tetranectin protein family, and their use in rational de novo design and production of multi-functional molecules including the application of the multi-functional molecules in protein library technology, such as phage display technology, diagnostic and therapeutic systems, such as human gene therapy and imaging. The trimeric polypeptides being constructed as a monomer polypeptide construct comprising at least one tetranectin trimerising structural element (TTSE) which is covalently linked to at least one heterologous moiety, said TTSE being capable of forming a stable complex with two other TTSEs; or as an oligomer which is comprised of two monomer polypeptide constructs as mentioned above, and which comprises three TTSEs or a multiplum of three TTSEs, or which is comprised of three monomer polypeptide constructs.

Claims

1-55. -55. (canceled)

56. An isolated polypeptide comprising:

(a) a first functional amino acid sequence that is capable of forming a trimeric complex with two other first functional amino acid sequences and that has up to five amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO;

40; and

(b) a second functional amino acid sequence that is heterologous to the first functional amino acid sequence;

wherein the isolated polypeptide does not consist of the amino acid sequence of SEQ ID NO;

27.

57. The isolated polypeptide of claim 56, further comprising a third functional amino acid sequence that is heterologous to the first amino acid sequence.

58. The isolated polypeptide of claim 56, wherein the first functional amino acid sequence is a tetranectin trimerising structural element (TTSE).

59. The isolated polypeptide of claim 56, wherein the trimeric complex is a stable triple alpha helical coiled coil complex.

60. The isolated polypeptide of claim 56, wherein the first functional amino acid has four amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

61. The isolated polypeptide of claim 56, wherein the first functional amino acid has three amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

62. The isolated polypeptide of claim 56, wherein the first functional amino acid has two amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

63. The isolated polypeptide of claim 56, wherein the first functional amino acid has one amino acid substitution at any one of positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

64. The isolated polypeptide of claim 56, wherein the first functional amino acid sequence comprises the consensus sequence of SEQ ID NO:

40.

65. The isolated polypeptide of claim 56, wherein the second functional amino acid sequence comprises an amino acid sequence encoding all or a part of:

(a) a ligand binding structure;

(b) a ligand;

(c) a receptor binding structure;

(d) a receptor;

(e) a detectable moiety;

(f) an in situ activatable substance;

(g) an enzyme;

or

(h) a cytokine.

66. The isolated polypeptide of claim 65, wherein the ligand binding structure is a receptor molecule, the ligand binding part of a receptor molecule, an antibody, an antigen binding antibody fragment, a monovalent scFv antibody fragment, a Fab antibody fragment, or a lectin.

67. The isolated polypeptide of claim 56, wherein the second functional amino acid sequence does not facilitate expression and/or purification of the polypeptide.

68. A trimeric polypeptide complex comprising three isolated polypeptides of claim 56.

69. The trimeric polypeptide complex of claim 68, wherein at least two of the second functional amino acid sequences are different from each other.

70. The trimeric polypeptide complex of claim 68, wherein all three of the second functional sequences are different from each other.

71. The trimeric polypeptide complex of claim 68, wherein the complex remains as a trimer at a temperature of at least 60°

C.

72. The trimeric polypeptide complex of claim 68, wherein the complex remains as a trimer at a temperature of about 70°

C.

73. A method for preparing the trimeric polypeptide complex of claim 68, comprising:

(a) mixing the polypeptides;

(b) effecting complex formation between the polypeptides; and

(c) isolating the resulting trimeric polypeptide complex.

74. An isolated polypeptide comprising:

(a) a tetranectin trimerising structural element (TTSE) that has up to five amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO;

40; and

(b) a heterologous amino acid sequence;

wherein the tetranectin trimerising structural element (TTSE) is capable of forming a stable triple alpha helical coiled coil complex with two other TTSEs.

75. The isolated polypeptide of claim 74, wherein the tetranectin trimerising structural element (TTSE) has four amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

76. The isolated polypeptide of claim 74, wherein the tetranectin trimerising structural element (TTSE) has three amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

77. The isolated polypeptide of claim 74, wherein the tetranectin trimerising structural element (TTSE) has two amino acid substitutions at positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

78. The isolated polypeptide of claim 74, wherein the tetranectin trimerising structural element (TTSE) has one amino acid substitution at any one of positions 10, 17, 20, 21, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, or 35 of the amino acid sequence of SEQ ID NO:

40.

79. The isolated polypeptide of claim 75, wherein the tetranectin trimerising structural element (TTSE) comprises the consensus sequence of SEQ ID NO:

40.

80. The isolated polypeptide of claim 75, wherein the heterologous amino acid sequence comprises the amino acid sequence encoding all or a part of:

(a) a ligand binding structure;

(b) a ligand;

(c) a receptor binding structure;

(d) a receptor;

(e) a detectable moiety;

(f) an in situ activatable substance;

(g) an enzyme;

or

(h) a cytokine.

81. The isolated polypeptide of claim 75, wherein the ligand binding structure is a receptor molecule, the ligand binding part of a receptor molecule, an antibody, an antigen binding antibody fragment, a monovalent scFv antibody fragment, a Fab antibody fragment, or a lectin.

82. The isolated polypeptide of claim 75, wherein the heterologous amino acid sequence does not facilitate expression and/or purification of the monomer polypeptide.

83. A trimeric polypeptide complex comprising three of the isolated polypeptides of claim 75, wherein the complex remains as a trimer at a temperature of at least 60°

C.

85. The trimeric polypeptide complex of claim 85, wherein at least two of the heterologous sequences are different from each other.

84. The trimeric polypeptide complex according to claim 85, wherein the complex remains as a trimer at a temperature of about 70°

C.

86. The trimeric polypeptide complex of claim 85, wherein all three of the heterologous sequences are different from each.