Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon

• US 20030119168A1
• Filed: 02/02/2001
• Published: 06/26/2003
• Est. Priority Date: 02/03/2000
• Status: Active Grant

• Current Assignee: Dendreon Pharmaceuticals, Inc. (Ya Fei Yuan,)
• Original Assignee: Corvas International, Inc. (Bausch Health Cos., Inc.)
• Inventors: Madison, Edwin L., Yeh, Jiunn-Chern, Ong, Edgar O.
• Granted Patent: US 7,700,341 B2
• Time in Patent Office: Days
• US Class Current: 435/226
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/14 Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)

Abstract

Provided herein is are polypeptides that include the protease domain of a type II transmembrane serine protease (MTSP) as a single chain. Methods using the polypeptides to identify compounds that modulate the protease activity of an MTSP are provided. Also provided are MTSPs designated MTSP3 and MTSP4 and a form of an MTSP designated MTSP6.

Claims

36-1. A combination, comprising:

a) an inhibitor of the catalytic activity of the polypeptide of claim 1;

and

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

55. A substantially purified membrane-type serine protease 3 (MTSP3).

69. A method for treating or preventing a neoplastic disease, in a mammal, comprising administering to a mammal an effective amount of an inhibitor of an MTSP3 of claim 55.

70. The method of claim 69, wherein the inhibitor is an antibody that specifically binds to the MTSP3, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

71. A substantially purified membrane-type serine protease 4 (MTSP4).

73. The MTSP4 of claim 71 that is selected from the group consisting of:

a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 7 or 9;

a polypeptide encoded by a sequence of nucleotides that hybridizes under conditions of high stringency to the sequence of nucleotides set forth in SEQ ID No. 7 or 9;

a polypeptide that comprises the sequence of amino acids set forth in SEQ ID No. 6, 8 or 10; and

a polypeptide encoded by a splice variant of the sequence of nucleotides set forth in SEQ ID No. 7 or 9.

74. The MTSP4 of claim 73 that is an MTSP4-L or an MTSP4-S.

75. A nucleic acid molecule, comprising a sequence of nucleotides that encodes the polypeptide of claim 73.

76. A vector comprising the nucleic acid molecule of claim 74.

77. A cell, comprising the vector of claim 76.

78. The cell of claim 77 that expresses the MTSP4 on its surface.

79. The cell of claim 77 that is a prokaryotic cell.

80. The cells of claim 77 that is a eukaryotic cell.

81. The cell of claim 77 that is selected from among a bacterial cell, a yeast cell, a yeast cell, a plant cell, an insect cell and an animal cell.

82. The cell of claim 77 that is a mammalian cell.

83. The cell of claim 77, wherein the MTSP4 comprises the sequence of amino acids set forth in SEQ ID No. 6, 8 or 10;

or a sequence of amino acids encoded by a sequence of nucleotides that hybridizes under conditions of high stringency to the sequence of nucleotides set forth in SEQ ID No. 7 or 9;

or a protein having at least about 90% sequence identity with the sequence of amino acids set forth in SEQ ID No. 8 or 10 and retaining protease activity.

84. A method for producing an MTSP4, comprising:

culturing the cell of claim 77 under conditions whereby the encoded protein is expressed by the cell; and

recovering the expressed protein.

85. An antisense nucleic acid molecule that comprises at least 14 contiguous nucleotides or modified nucleotides that are complementary to a contiguous sequence of nucleotides in the nucleic acid molecule of claim 75 or

comprises at least 16 contiguous nucleotides or modified nucleotides that are complementary to a contiguous sequence of nucleotides in the nucleic acid molecule of claim 75.

87. An antibody that specifically binds to the MTSP of claim 73, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

88. A method for treating or preventing a neoplastic disease, in a mammal, comprising administering to a mammal an effective amount of an inhibitor of an MTSP4 of claim 71.

89. The method of claim 88, wherein the inhibitor is an antibody that specifically binds to an MTSP4, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

72. The substantially purified MTSP4 that is an MTSP4-L or an MTSP4-S.

86. An antibody that specifically binds to the MTSP of claim 72, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

90. A substantially purified membrane-type serine protease 6 (MTSP6) selected from the group consisting of:

a polypeptide encoded by the sequence of nucleotides set forth in SEQ ID No. 11;

a polypeptide encoded by a sequence of nucleotides that hybridizes along the full length thereof under conditions of high stringency to the sequence of nucleotides set forth in SEQ ID No. 11;

a polypeptide that comprises the sequence of amino acids set forth as amino acids 217-443 of SEQ ID No. 12;

a polypeptide encoded by a splice variant of the sequence of nucleotides set forth in SEQ ID No. 11.

91. A nucleic acid molecule, comprising a sequence of nucleotides that encodes the polypeptide of claim 90.

92. A vector comprising the nucleic acid molecule of claim 91.

93. A cell, comprising the vector of claim 92.

94. The cell of claim 93 that expresses the MTSP6 on its surface.

95. The cell of claim 93 that is a prokaryotic cell.

96. The cells of claim 93 that is a eukaryotic cell.

97. The cell of claim 93 that is selected from among a bacterial cell, a yeast cell, a yeast cell, a plant cell, an insect cell and an animal cell.

98. The cell of claim 93 that is a mammalian cell.

99. The cell of claim 93, wherein the MTSP6 comprises the sequence of amino acids set forth in SEQ ID No. 12;

or a sequence of amino acids encoded by a sequence of nucleotides that hybridizes along the full length thereof under conditions of high stringency to the sequence of nucleotides set forth in SEQ ID No. 12;

or a protein having at least about 95% sequence identity with the sequence of amino acids set forth in SEQ ID No. 4 and retaining protease activity.

100. A method for producing an MTSP6, comprising:

culturing the cell of claim 93 under conditions whereby the encoded protein is expressed by the cell; and

recovering the expressed protein.

101. An antisense nucleic acid molecule that comprises at least 14 contiguous nucleotides or modified nucleotides that are complementary to a contiguous sequence of nucleotides in the nucleic acid molecule of claim 91;

or

comprises at least 16 contiguous nucleotides or modified nucleotides that are complementary to a contiguous sequence of nucleotides in the in the nucleic acid molecule of claim 91.

102. An antibody that specifically binds to the MTSP of claim 90, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

103. A method for treating or preventing a neoplastic disease, in a mammal, comprising administering to a mammal an effective amount of an inhibitor of an MTSP6 of claim 90.

104. The method of claim 103, wherein the inhibitor is an antibody that specifically binds to the MTSP6, or a fragment or derivative of the antibody containing a binding domain thereof, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

107. A conjugate, comprising:

a) an MTSP3 or MTSP4 or an MTSP6 of claim 90;

and

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

109. A combination, comprising:

a) an inhibitor of the catalytic activity of an MTSP3 or MTSP4 or MTSP6 of claim 90;

and

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

110. The combination of claim 109, wherein the inhibitor and the anti-tumor and/or anti-angiogenic agent are formulated in a single pharmaceutical composition or each is formulated in separate pharmaceutical compositions.

111. The combination of claim 109, wherein the inhibitor is selected from antibodies and antisense oligonucleotides.

112. A solid support comprising two or more MTSP3 polypeptides and/or MTSP4 polypeptides and/or MTSP6 polypeptides of claim 90 linked thereto either directly or via a linker.

113. The support of claim 112, wherein the polypeptides comprise an array.

114. A method for identifying compounds that modulate the protease activity of an MTSP selected from MTSP3, MTSP4 or MTSP6 of claim 90, comprising:

contacting the MTSP with a substrate proteolytically cleaved by the MTSP, 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 cleaved compared to a control, whereby compounds that modulate the activity of the MTSP are identified.

115. The method of claim 114, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

116. The method of claim 114, wherein a plurality of the test substances are screened for simultaneously.

117. The method of claim 114, wherein the change in the amount cleaved is assessed by comparing the amount cleaved in the presence of the test compound with the amount in the absence of the test compound.

118. The method of claim 114, wherein a plurality of the test substances are screened for simultaneously.

119. The method of claim 118, wherein a plurality of the polypeptides are linked to a solid support.

120. A method of identifying a compound that specifically binds to a MTSP selected from MTSP3, MTSP4 and the MTSP6 of claim 90, comprising:

contacting the MTSP polypeptide with a test compound or plurality thereof under conditions conducive to binding thereof; and

identifying compounds that specifically binds to the MTSP.

121. A method of identifying a compound that specifically binds to a MTSP selected from MTSP3, MTSP4 and the MTSP6 of claim 90, comprising:

contacting the MTSP polypeptide with a test compound or plurality thereof under conditions conducive to binding thereof; and

identifying compounds that specifically binds to the MTSP.

122. The method of any claims 121, wherein the polypeptide is linked either directly or indirectly via a linker to a solid support.

123. The method of claim 121, wherein the test compounds are small molecules, peptides, peptidomimetics, natural products, antibodies or fragments thereof.

124. The method of claim 121, wherein a plurality of the test substances are screened for simultaneously.

125. The method of claim 124, wherein a plurality of the polypeptides are linked to a solid support.

133. A method of detecting neoplastic disease, comprising:

detecting an MTSP3, MTSP4 or MTSP6 of claim 90 in a biological sample, wherein the amount detected differs from the amount is a subject who does not have neoplastic disease.

134. The method of claim 133, wherein the biological sample is selected from the group consisting of blood, urine, saliva, tears, interstitial fluid, cerebrospinal fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells.

135. The method of claim 133, wherein the extracellular domain of the MTSP3, MTSP4 or MTSP6 is in the sample.

105. A recombinant non-human animal, wherein an endogenous gene of an MTSP has been deleted or inactivated by homologous recombination or insertional mutagenesis of the animal or an ancestor thereof.

106. A recombinant non-human animal of claim 105, wherein the MTSP is an MTSP1, MTSP3, MTSP4 or MTSP6.

108. The conjugate of claim 106, 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.

126. An MTSP6 polypeptide, comprising amino acids set forth as amino acids 46-55 in SEQ ID No. 12 and/or amino acids 368-394 of SEQ ID No. 12, and that is encoded by a sequence of nucleic acids that hybridizes under moderate stringency to nucleic acid encoding the polypeptide set forth in SEQ ID No. 12.

127. The polypeptide of claim 126 that comprises the amino acids set forth as amino acids 46-55 in SEQ ID No. 12 and/or amino acids 368-394 of SEQ ID No. 12, and that is encoded by a sequence of nucleic acids that hybridizes under high stringency along its full length or full length of the protease domain to nucleic acid encoding the polypeptide set forth in SEQ ID No. 12.

128. The polypeptide of claim 126, comprising the sequence of amino acids set forth in SEQ ID No. 12.

129. An isolated nucleic acid molecule, comprising a sequence of nucleic acids that encodes the polypeptide of claim 126.

130. A method for treating tumors, comprising administering a prodrug that is specifically cleaved by an MTSP, whereby, upon contact with a cell that expresses MTSP activity, the prodrug is converted into an active drug.

131. The method of claim 130, wherein the MTSP is selected from among an MTSP3, MTSP4 and an MTSP6 of claim 90.

132. The method of claim 130, wherein the MTSP is selected from among corin, enterpeptidase, human airway trypsin-like protease (HAT), MTSP1, TMPRSS2, and TMPRSS4.