Purified mammalian cytokines; related reagents and methods

Abstract

CTLA-8 related antigens from mammals, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding said antigens. Methods of using said reagents and diagnostic kits are also provided.

What is claimed is:  
     
         1 . An isolated or recombinant polynucleotide comprising sequence from: 
 a) a mammalian IL-173, which: 
 i) encodes at least 8 contiguous amino acids of mature SEQ ID NO: 6, 8, 10, or or 12;  
 ii) encodes at least two distinct segments of at least 5 contiguous amino acids of mature SEQ ID NO: 6, 8, 10, or 12; or  
 iii) comprises one or more segments at least 21 contiguous nucleotides of SEQ ID NO: 5, 7, 9, or 11;  
   b) a mammalian IL-174, which: 
 i) encodes at least 8 contiguous amino acids of mature SEQ ID NO: 14, 16, or 18;  
 ii) encodes at least two distinct segments of at least 5 contiguous amino acids of mature SEQ ID NO: 14, 16, or 18; or  
 iii) comprises one or more segments at least 21 contiguous nucleotides of SEQ ID NO: 14, 16, or 18;  
   c) a mammalian IL-176, which: 
 i) encodes at least 8 contiguous amino acids of mature SEQ ID NO: 28;  
   ii) encodes at least two distinct segments of at least 5 contiguous amino acids of mature SEQ ID NO: 28; or 
 iii) comprises one or more segments at least 21 contiguous nucleotides of SEQ ID NO: 27; or  
   d) a mammalian IL-177, which: 
 i) encodes at least 8 contiguous amino acids of mature SEQ ID NO: 30;  
 ii) encodes at least two distinct segments of at least 5 contiguous amino acids of mature SEQ ID NO: 30; or  
 iii) comprises one or more segments at least 21 contiguous nucleotides of SEQ ID NO: 29.  
   
     
     
         2 . The polynucleotide of  claim 1  in an expression vector, comprising sequence: 
 a) (IL-173) which: 
 i) encodes at least 12 contiguous amino acids of SEQ ID NO: 6, 8, 10, or 12;  
 ii) encodes at least two distinct segments of at least 7 and 10 contiguous amino acids of SEQ ID NO: 6, 8, 10, or 12; or  
 iii) comprises at least 27 contiguous nucleotides of SEQ ID NO: 5, 7, 9, or 11;  
 
 b) (IL-174) which: 
 i) encodes at least 12 contiguous amino acids of SEQ ID NO: 14, 16, or 18;  
 ii) encodes at least two distinct segments of at least 7 and 10 contiguous amino acids of SEQ ID NO: 14, 16, or 18; or  
 iii) comprises at least 27 contiguous nucleotides of SEQ ID NO: 13, 15, or 17;  
 
 c) (IL-176) which: 
 i) encodes at least 12 contiguous amino acids of SEQ ID NO: 28;  
 ii) encodes at least two distinct segments of at least 7 and 10 contiguous amino acids of SEQ ID NO: 28; or  
 iii) comprises at least 27 contiguous nucleotides of SEQ ID NO: 27; or  
 
 d) (IL-177) which: 
 i) encodes at least 12 contiguous amino acids of SEQ ID NO: 30;  
 ii) encodes at least two distinct segments of at least 7 and 10 contiguous amino acids of SEQ ID NO: 30; or  
 iii) comprises at least 27 contiguous nucleotides of SEQ ID NO: 29.  
 
 
     
     
         3 . The polynucleotide of  claim 2:   a) (IL-173) which: 
 i) encodes at least 16 contiguous amino acid residues of mature SEQ ID NO: 6, 8, 10, or 12;  
 ii) encodes at least two distinct segments of at least 10 and 13 contiguous amino acid residues of mature SEQ ID NO: 6, 8, 10, or 12;  
 iii) comprises at least 33 contiguous nucleotides of SEQ ID NO: 5, 7, 9, or 11; or  
 iv) comprises the entire mature coding portion of SEQ ID NO: 5, 7, 9, or 11;  
   b) (IL-174) which: 
 i) encodes at least 16 contiguous amino acid residues of mature SEQ ID NO: 14, 16, or 18;  
 ii) encodes at least two distinct segments of at least 10 and 13 contiguous amino acid residues of mature SEQ ID NO: 14, 16, or 18; or  
 iii) comprises at least 33 contiguous nucleotides of SEQ ID NO: 13, 15, or 17; or  
 iv) comprises the entire mature coding portion of SEQ ID NO: 13, 15, or 17;  
   c) (IL-176) which: 
 i) encodes at least 16 contiguous amino acids of mature SEQ ID NO: 28;  
 ii) encodes at least two distinct segments of at least 10 and 14 contiguous amino acid residues of mature SEQ ID NO: 28;  
 iii) comprises at least 33 contiguous nucleotides of SEQ ID NO: 27; or  
 iv) comprises the entire mature coding portion of SEQ ID NO: 27; or  
   d) (IL-177) which: 
 i) encodes at least 16 contiguous amino acids of mature SEQ ID NO: 30;  
 ii) encodes at least two distinct segments of at least 10 and 14 contiguous amino acid residues of mature SEQ ID NO: 30;  
 iii) comprises at least 33 contiguous nucleotides of SEQ ID NO: 29; or  
 iv) comprises the entire mature coding portion of SEQ ID NO: 29.  
   
     
     
         4 . A method of making: 
 a) a polypeptide comprising expressing said expression vector of  claim 2 , thereby producing said polypeptide;    b) a duplex nucleic acid comprising contacting a polynucleotide of  claim 2  with a complementary nucleic acid, thereby resulting in production of said duplex nucleic acid; or    c) a polynucleotide of  claim 2  comprising amplifying using a PCR method.    
     
     
         5 . An isolated or recombinant polynucleotide which hybridizes under stringent wash conditions of at least 55° C. and less than 400 mM salt to: 
 a) the (IL-173) polynucleotide of  claim 3  which consists of the mature coding portions of SEQ ID NO: 5, 7, 9, or 11;  
 b) the (IL-174) polynucleotide of  claim 3  which consists of the mature coding portions of SEQ ID NO: 13, 15, or 17;  
 c) the (IL-176) polynucleotide of  claim 3  which consists of the mature coding portions of SEQ ID NO: 27; or  
 d) the (IL-177) polynucleotide of  claim 3  which consists of the mature coding portions of SEQ ID NO: 29.  
 
     
     
         6 . A polynucleotide of  claim 5:   a) wherein said wash conditions are at least 65° C. and less than 300 mM salt; or    b) which comprises at least 50 contiguous nucleotides of the mature coding portion of: 
 i) SEQ ID NO: 5, 7, 9, or 11 (IL-173);  
 ii) SEQ ID NO: 13, 15, or 17 (IL-174);  
 iii) SEQ ID NO: 27 (IL-176); or  
 iv) SEQ ID NO: 29 (IL-177).  
   
     
     
         7 . A kit comprising said polynucleotide of  claim 6 , and 
 a) instructions for the use of said polynucleotide for detection;    b) instructions for the disposal of said polynucleotide or other reagents of said kit; or    c) both a and b.    
     
     
         8 . A cell containing said expression vector of  claim 3 , wherein said cell is: 
 a) a prokaryotic cell;    b) a eukaryotic cell;    c) a bacterial cell;    d) a yeast cell;    e) an insect cell;    f) a mammalian cell;    g) a mouse cell;    h) a primate cell; or    i) a human cell.    
     
     
         9 . An isolated or recombinant antigenic polypeptide: 
 a) (IL-173) comprising at least: 
 i) one segment of 8 identical contiguous amino acids from the mature coding portions of SEQ ID NO: 6, 8, 10, or 12; or  
 ii) two distinct segments of at least 5 contiguous amino acids from the mature coding portions of SEQ ID NO: 6, 8, 10, or 12;  
   b) (IL-174) comprising at least: 
 i) one segment of 8 identical contiguous amino acids from the mature coding portions of SEQ ID NO: 14, 16, or 18; or  
 ii) two distinct segments of at least 5 contiguous amino acids from the mature coding portions of SEQ ID NO: 14, 16, or 18;  
   c) (IL-176) comprising at least: 
 i) one segment of 8 identical contiguous amino acids from the mature coding portions of SEQ ID NO: 28; or  
 ii) two distinct segments of at least 5 contiguous amino acids from the mature coding portions of SEQ ID NO: 28; or  
   d) (IL-177) comprising at least: 
 i) one segment of 8 identical contiguous amino acids from the mature coding portions of SEQ ID NO: 30; or  
 ii) two distinct segments of at least 5 contiguous amino acids from the mature coding portions of SEQ ID NO: 30.  
   
     
     
         10 . The polypeptide of  claim 9 , wherein: 
 a) said segment of 8 identical contiguous amino acids is at least 14 contiguous amino acids; or    b) one of said segments of at least 5 contiguous amino acids comprises at least 7 contiguous amino acids.    
     
     
         11 . The polypeptide of  claim 9 , wherein: 
 A) (IL-173) said polypeptide: 
 a) comprises SEQ ID NO: 6, 8, 10, or 12;  
 b) binds with selectivity to a polyclonal antibody generated against an immunogen of the mature SEQ ID NO: 6, 8, 10, or 12;  
 c) comprises a plurality of distinct polypeptide segments of 10 contiguous amino acids of the mature SEQ ID NO: 6, 8, 10, or 12;  
 d) is a natural allelic variant of SEQ ID NO: 8 or 12;  
 e) has a length at least 30 amino acids; or  
 f) exhibits at least two non-overlapping epitopes which are selective for the mature SEQ ID NO: 6, 8, 10, or 12;  
   B) (IL-174) said polypeptide: 
 a) comprises the mature SEQ ID NO: 14, 16, or 18;  
 b) binds with selectivity to a polyclonal antibody generated against an immunogen of the mature SEQ ID NO: 14, 16, or 18;  
 c) comprises a plurality of distinct polypeptide segments of 10 contiguous amino acids of the mature SEQ ID NO: 14, 16, or 18;  
 d) is a natural allelic variant of SEQ ID NO: 14 or 18;  
 e) has a length at least 30 amino acids; or  
 f) exhibits at least two non-overlapping epitopes which are selective for primate protein of the mature SEQ ID NO: 14, 16, or 18;  
   C) (IL-176) said polypeptide: 
 a) comprises a mature sequence of SEQ ID NO: 28;  
 b) binds with selectivity to a polyclonal antibody generated against an immunogen of the mature SEQ ID NO: 28;  
 c) comprises a plurality of distinct polypeptide segments of 10 contiguous amino acids of the mature SEQ ID NO: 28;  
 d) is a natural allelic variant of SEQ ID NO: 28;  
 e) has a length at least 30 amino acids; or  
 f) exhibits at least two non-overlapping epitopes which are selective for the mature SEQ ID NO: 28; or  
   D) (IL-177) said polypeptide: 
 a) comprises a mature sequence of SEQ ID NO: 30;  
 b) binds with selectivity to a polyclonal antibody generated against an immunogen of the mature SEQ ID NO: 30;  
 c) comprises a plurality of distinct polypeptide segments of 10 contiguous amino acids of the mature SEQ ID NO: 30;  
 d) is a natural allelic variant of SEQ ID NO: 30;  
 e) has a length at least 30 amino acids; or  
 f) exhibits at least two non-overlapping epitopes which are selective for the mature SEQ ID NO: 30.  
   
     
     
         12 . The polypeptide of  claim 11 , which: 
 a) is in a sterile composition;    b) is not glycosylated;    c) is denatured;    d) is a synthetic polypeptide;    e) is attached to a solid substrate;    f) is a fusion protein with a detection or purification tag;    g) is a 5-fold or less substitution from a natural sequence; or    h) is a deletion or insertion variant from a natural sequence.    
     
     
         13 . A method using said polypeptide of  claim 9:   a) to label said polypeptide, comprising labeling said polypeptide with a radioactive label;    b) to separate said polypeptide from another polypeptide in a mixture, comprising running said mixture on a chromatography matrix, thereby separating said polypeptides;    c) to identify a compound that binds selectively to said polypeptide, comprising incubating said compound with said polypeptide under appropriate conditions; thereby causing said compound to bind to said polypeptide; or    d) to conjugate said polypeptide to a matrix, comprising derivatizing said polypeptide with a reactive reagent, and conjugating said polypeptide to said matrix.    
     
     
         14 . A binding compound comprising an antigen binding portion from an antibody which binds with selectivity to said polypeptide of  claim 11 , wherein said polypeptide: 
 a) (IL-173) comprises the mature SEQ ID NO 6, 8, 10, or 12; or    b) (IL-174) comprises the mature SEQ ID NO 14, 16, or 18;    c) (IL-176) comprises the mature SEQ ID No 28; or    d) (IL-177) comprises the mature SEQ ID NO 30.    
     
     
         15 . The binding compound of  claim 14 , wherein said antibody is a polyclonal antibody which is raised against: 
 a) (IL-173) SEQ ID NO: 6, 8, 10, or 12; or    b) (IL-174) SEQ ID NO: 14, 16, or 18;    c) (IL-176) SEQ ID NO: 28; or    d) (IL-177) SEQ ID NO: 30.    
     
     
         16 . The binding compound of  claim 14 , wherein said: 
 a) antibody: 
 i) is immunoselected;  
 ii) binds to a denatured protein; or  
 iii) exhibits a Kd to said polypeptide of at least 30 mM; or  
   b) said binding compound: 
 i) is attached to a solid substrate, including a bead or plastic membrane;  
 ii) is in a sterile composition; or  
 iii) is detectably labeled, including a radioactive or fluorescent label.  
   
     
     
         17 . A method of producing an antigen:antibody complex, comprising contacting-a polypeptide comprising sequence from SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 28, or 30 with a binding compound of  claim 14  under conditions which allow said complex to form.  
     
     
         18 . The method of  claim 17 , wherein said binding compound is an antibody, and said polypeptide is in a biological sample.  
     
     
         19 . A kit comprising said binding compound of  claim 14  and: 
 a) a polypeptide of the mature SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 28, or 30;  
 b) instructions for the use of said binding compound for detection; or  
 c) instructions for the disposal of said binding compound or other reagents of said kit.  
 
     
     
         20 . A method of evaluating the selectivity of binding of an antibody to a protein of the mature SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 28, or 30, comprising contacting said antibody to said protein and to another cytokine; and comparing binding of said antibody to said protein and said cytokine.