US2010015137A1PendingUtilityA1

Receptor that binds trail

59
Assignee: RAUCH CHARLESPriority: Feb 13, 1997Filed: Dec 23, 2008Published: Jan 21, 2010
Est. expiryFeb 13, 2017(expired)· nominal 20-yr term from priority
Inventors:Charles RauchHenning Walczak
C07K 14/70578A61K 38/00C07K 1/22C07K 14/715
59
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Claims

Abstract

A protein designated TRAIL receptor binds the protein known as TNF-Related Apoptosis-Inducing Ligand (TRAIL). The TRAIL receptor finds use in purifying TRAIL or inhibiting activities thereof. Isolated DNA sequences encoding TRAIL-R polypeptides are provided, along with expression vectors containing the DNA sequences, and host cells transformed with such recombinant expression vectors. Antibodies that are immunoreactive with TRAIL-R are also provided.

Claims

exact text as granted — not AI-modified
1 . A method of inducing apoptosis in mammalian cancel cells comprising contacting said mammalian cancer cells with an apoptosis inducing amount of an isolated TRAIL-R agonist monoclonal antibody which:
 (a) specifically binds to a TRAIL-R protein, wherein TRAIL-R is characterized by expression on cell membranes of Jurkat cells, binds TRAIL, comprises an amino acid sequence VPANEGD (amino acids 327-333 of SEQ ID NO: 2), and has a molecular weight of about 50 to 55 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis; and,   (b) induces apoptosis in said mammalian cancer cells.   
     
     
         2 . The method of  claim 1 , wherein said TRAIL-R protein consists of the contiguous amino acid residues 1 to 440 of SEQ ID NO: 2. 
     
     
         3 . The method of  claim 1 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2. 
     
     
         4 . The method of  claim 3 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         5 . The method of  claim 3 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         6 . The method of claim l, wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2. 
     
     
         7 . The method of  claim 6 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         8 . The method of  claim 6 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         9 . The method of  claim 1 , wherein said monoclonal antibody specifically binds to said TRAIL-R protein having an N-terminal amino acid residue at either position 52 or 54 of SEQ ID NO: 2. 
     
     
         10 . The method of  claim 9 , wherein said TRAIL-R protein is a soluble TRAIL-R protein. 
     
     
         11 . The method of  claim 10 , wherein said soluble TRAIL-R protein lacks a transmembrane region and a cytoplasmic domain. 
     
     
         12 . The method of  claim 11 , wherein said soluble TRAIL-R protein has an N-terminal amino acid residue at position 54 of SEQ ID NO: 2. 
     
     
         13 . The method of  claim 1 , wherein said TRAIL-R protein is a soluble TRAIL-R protein and lacks a transmembrane region and a cytoplasmic domain. 
     
     
         14 . A method of inducing apoptosis in mammalian cancer cells comprising contacting said mammalian cancer cells with an apoptosis inducing amount of an isolated TRAIL-R agonist monoclonal antibody which:
 (a) specifically binds to TRAIL-R, wherein an isolated and purified human TRAIL-R receptor protein is a product made by the process comprising the steps of:
 (i) isolating plasma membranes from Jurkat cells; 
 (ii) solubilizing and homogenizing said isolated plasma membranes of step (i); 
 (iii) centrifuging said solubilized and homogenized isolated plasma membranes of step (ii) to yield a plasma membrane extract and a pellet; 
 (iv) applying said plasma membrane extract of step (iii) to an anti-octapeptide monoclonal antibody affinity chromatography column, whereby said column of step (iv) adsorbs non-specifically bound material and wherein said octapeptide has the sequence presented in SEQ ID NO: 5; 
 (v) applying column flow-through from step (iv) to an octapeptide-TRAIL ligand affinity chromatography column, whereby said column of step (v) specifically binds said TRAIL-R receptor protein and wherein said octapeptide-TRAIL ligand is a fusion protein of said octapeptide having the sequence presented in SEQ ID NO:5 and TRAIL ligand; 
 (vi) eluting fractions with TRAIL ligand binding activity from said column of step (v); and, 
 (vii) purifying said fractions of step (vi) by reverse-phase HPLC to yield said isolated and purified TRAIL-R receptor protein, wherein said isolated and purified TRAIL-R receptor protein has a molecular weight of about 50 to 55 kilodaltons as determined by SDS polyacrylamide gel electrophoresis, and comprises the amino acid sequence VPANEGD (amino acids 327-333 of SEQ ID NO: 2); and, 
   (b) induces apoptosis in said mammalian cancer cells.   
     
     
         15 . The method of  claim 1   4 , wherein said TRAIL-R consists of the contiguous amino acid residues 1 to 440 of SEQ ID NO: 2. 
     
     
         16 . The method of  claim 14 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R, said extracellular domain sequence consisting of the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2. 
     
     
         17 . The method of  claim 16 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         18 . The method of  claim 16 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         19 . The method of  claim 14 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R, said extracellular domain sequence consisting of the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2. 
     
     
         20 . The method of  claim 19 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         21 . The method of  claim 19 , wherein said extracellular domain sequence is a membrane bound extracellular- domain sequence. 
     
     
         22 . The method of  claim 14 , wherein said monoclonal antibody specifically binds to said TRAIL-R having an N-terminal amino acid residue at either position 52 or 54 of SEQ ID NO: 2. 
     
     
         23 . The method of  claim 22 , wherein said I-RAIL-R is a soluble TRAIL-R. 
     
     
         24 . The method of  claim 23 , wherein said soluble TRAIL-R lacks a transmembrane region and a cytoplasmic domain. 
     
     
         25 . The method of  claim 24 , wherein said soluble TRAIL-R has an N-terminal amino acid residue at position 54 of SEQ ID NO: 2. 
     
     
         26 . The method of  claim 14 , wherein said TRAIL-R is a soluble TRAIL-R and lacks a transmembrane region and a cytoplasmic domain. 
     
     
         27 . A method of inducing apoptosis in mammalian cancer cells comprising contacting said mammalian cancer cells with an apoptosis inducing amount of an isolated TRAIL-R agonist monoclonal antibody which:
 (a) specifically binds to a TRAIL-R protein of SEQ ID NO:2: and   (b) induces apoptosis in said mammalian cancer cells.   
     
     
         28 . The method of  claim 27 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2. 
     
     
         29 . The method of  claim 28 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         30 . The method of  claim 28 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         31 . The method of  claim 27 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2. 
     
     
         32 . The method of  claim 31 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         33 . The method of  claim 31 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         34 . The method of  claim 27 , wherein said monoclonal antibody specifically binds to said TRAIL-R protein having an N-terminal amino acid residue at either position 52 or 54 of SEQ ID NO: 2. 
     
     
         35 . The method of  claim 34 , wherein said TRAIL-R protein is a soluble TRAIL-R receptor protein. 
     
     
         36 . The method of  claim 35 , wherein said soluble TRAIL-R protein lacks a transmembrane region and a cytoplasmic domain. 
     
     
         37 . The method of  claim 36 , wherein said soluble TRAIL-R protein has an N-terminal amino acid residue at position 54 of SEQ ID NO: 2. 
     
     
         38 . The method of  claim 27 , wherein said TRAIL-R protein is a soluble TRAIL-R receptor protein and lacks a transmembrane region and a cytoplasmic domain. 
     
     
         39 . A method of treating cancer comprising contacting mammalian cancer cells with an apoptosis inducing amount of an isolated TRAIL-R agonist monoclonal antibody which:
 (a) specifically binds to a TRAIL-R protein, wherein TRAIL-R is characterized by expression on cell membranes of Jurkat cells, binds TRAIL, comprises an amino acid sequence VPANEGD (amino acids 327-333 of SEQ ID NO: 2), and has a molecular weight of about 50 to 55 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis; and,   (b) induces apoptosis in said mammalian cancer cells.   
     
     
         40 . The method of  claim 39 , wherein said TRAIL-R protein consists of the contiguous amino acid residues 1 to 440 of SEQ ID NO: 2. 
     
     
         41 . The method of  claim 39 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2. 
     
     
         42 . The method of  claim 41 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         43 . The method of  claim 41 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         44 . The method of  claim 39 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2. 
     
     
         45 . The method of  claim 44 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         46 . The method of  claim 44 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         47 . The method of  claim 39 , wherein said monoclonal antibody specifically binds to said TRAIL-R protein having an N-terminal amino acid residue at either position 52 or 54 of SEQ ID NO: 2. 
     
     
         48 . The method of  claim 47 , wherein said TRAIL-R protein is a soluble TRAIL-R receptor protein. 
     
     
         49 . The method of  claim 48 , wherein said soluble TRAIL-R protein lacks a transmembrane region and a cytoplasmic domain. 
     
     
         50 . The method of  claim 49 , wherein said soluble TRAIL-R protein has an N-terminal amino acid residue at position 54 of SEQ ID NO: 2. 
     
     
         51 . The method of  claim 39 , wherein said TRAIL-R protein is a soluble TRAIL-R protein and lacks a transmembrane region and a cytoplasmic domain. 
     
     
         52 . A method of treating cancer comprising contacting mammalian cancer cells with an apoptosis inducing amount of an isolated TRAIL-R agonist monoclonal antibody which:
 (a) specifically binds to TRAIL-R, wherein an isolated and purified human TRAIL-R receptor protein is a product made by the process comprising the steps of:
 (i) isolating plasma membranes from Jurkat cells; 
 (ii) solubilizing and homogenizing said isolated plasma membranes of step (i); 
 (iii) centrifuging said solubilized and homogenized isolated plasma membranes of step (ii) to yield a plasma membrane extract and a pellet; 
 (iv) applying said plasma membrane extract of step (iii) to an anti-octapeptide monoclonal antibody affinity chromatography column, whereby said column of step (iv) adsorbs non-specifically bound material and wherein said octapeptide has the sequence presented in SEQ ID NO: 5; 
 (v) applying column flow-through from step (iv) to an octapeptide-TRAIL ligand affinity chromatography column, whereby said column of step (v) specifically binds said TRAIL-R receptor protein and wherein said octapeptide -TRAIL ligand is a fusion protein of said octapeptide having the sequence presented in SEQ ID NO:5 and TRAIL ligand; 
 (vi) eluting fractions with TRAIL ligand binding activity from said column of step (v); and, 
 (vii) purifying said fractions of step (vi) by reverse-phase HPLC to yield said isolated and purified TRAIL-R receptor protein, wherein said isolated and purified TRAIL-R receptor protein has a molecular weight of about 50 to 55 kilodaltons as determined by SDS polyacrylamide gel electrophoresis, and comprises the amino acid sequence VPANEGD (amino acids 327-333 of SEQ ID NO: 2); and, 
   (b) induces apoptosis in said mammalian cancer cells.   
     
     
         53 . The method of  claim 52 , wherein said TRAIL-R consists of the contiguous amino acid residues 1 to 440 of SEQ ID NO: 2. 
     
     
         54 . The method of  claim 52 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R, said extracellular domain sequence consisting of the contiguous amino acid residues 52 to 21 0 of SEQ ID NO: 2. 
     
     
         55 . The method of  claim 54 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         56 . The method of  claim 54 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         57 . The method of  claim 52 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R, said extracellular domain sequence consisting of the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2. 
     
     
         58 . The method of  claim 57 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         59 . The method of  claim 57 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         60 . The method of  claim 52 , wherein said monoclonal antibody specifically binds to said TRAIL-R having an N-terminal amino acid residue at either position 52 or 54 of SEQ ID NO: 2. 
     
     
         61 . The method of  claim 60 , wherein said TRAIL-R is a soluble TRAIL-R. 
     
     
         62 . The method of  claim 61 , wherein said soluble TRAIL-R lacks a transmembrane region and a cytoplasmic domain. 
     
     
         63 . The method of  claim 62 , wherein said soluble TRAIL-R has an N-terminal amino acid residue at position 54 of SEQ ID NO: 2. 
     
     
         64 . The method of  claim 52 , wherein said TRAIL-R is a soluble TRAIL-R and lacks a transmembrane region and a cytoplasmic domain. 
     
     
         65 . A method of treating cancer comprising contacting mammalian cancer cells with an apoptosis inducing amount of an isolated TRAIL-R agonist monoclonal antibody which:
   (a) specifically binds to a TRAIL-R protein of SEQ ID NO:2; and,   (b) induces apoptosis in said mammalian cancer cells.     
     
     
         66 . The method of  claim 65 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 52 to 210 of SEQ ID NO: 2. 
     
     
         67 . The method of  claim 66 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         68 . The method of  claim 66 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         69 . The method of  claim 65 , wherein said monoclonal antibody specifically binds to an extracellular domain sequence of said TRAIL-R protein, said extracellular domain sequence consisting of the contiguous amino acid residues 54 to 210 of SEQ ID NO: 2. 
     
     
         70 . The method of  claim 69 , wherein said extracellular domain sequence is a soluble extracellular domain sequence. 
     
     
         71 . The method of  claim 69 , wherein said extracellular domain sequence is a membrane bound extracellular domain sequence. 
     
     
         72 . The method of  claim 65 , wherein said monoclonal antibody specifically binds to said TRAIL-R protein having an N-terminal amino acid residue at either position 52 or 54 of SEQ ID NO: 2. 
     
     
         73 . The method of  claim 72 , wherein said TRAIL-R protein is a soluble TRAIL-R protein. 
     
     
         74 . The method of  claim 73 , wherein said soluble TRAIL-R protein lacks a transmembrane region and a cytoplasmic domain. 
     
     
         75 . The method of  claim 74 , wherein said soluble TRAIL-R protein has an N-terminal amino acid residue at position 54 of SEQ ID NO: 2. 
     
     
         76 . The method of  claim 65 , wherein said TRAIL-R protein is a soluble TRAIL-R protein and lacks a transmembrane region and a cytoplasmic domain. 
     
     
         77 . A method as in one of  claims 1 - 76 , wherein said cancer cells are further contacted with radiation or a chemotherapeutic agent. 
     
     
         78 . A method as in one of  claims 1 - 76 , wherein said cancer cells are lung, breast, ovary, prostate, kidney, liver, bladder, pancreas, or colon cancer cells. 
     
     
         79 . A method as in one of  claims 1 - 76 , wherein said cancer cells are leukemia, lymphoma, or melanoma cancel- cells. 
     
     
         80 . A method as in one of  claims 1 - 76 , wherein said monoclonal antibody is a humanized or chimeric monoclonal antibody. 
     
     
         81 . A method as in one of  claims 1 - 76 , wherein said monoclonal antibody comprises an Fab or F(ab′) 2  fragment.

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