Btnl3 proteins, nucleic acids, and antibodies and uses thereof

Abstract

The invention provides novel BTNL3 proteins, including multimers, fragments, fusion proteins, and variants. In addition, antibodies that can bind to BTNL3 proteins and nucleic acids encoding BTNL3 proteins are provided. Methods of making BTNL3 proteins using such nucleic acids are also provided. Uses for BTNL3 proteins, and agonists or antagonists thereof, are described.

What is claimed is: 
     
         1 . An isolated multimeric BTNL3 protein comprising
 (a) a polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2, and   (b) a second polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2,   wherein the alignment window of the amino acid sequences of the polypeptides of (a) and (b) with amino acids 18-236 of SEQ ID NO:2 is at least 80 amino acids long,   wherein the BTNL3 protein is at least a dimer,   wherein the BTNL3 protein has been produced by a non-human host cell, and   wherein the multimeric BTNL3 protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         2 . An isolated multimeric BTNL3 protein comprising
 (a) a polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2, and   (b) a second polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2,   wherein the alignment window of the amino acid sequences of the polypeptides of (a) and (b) with amino acids 18-236 of SEQ ID NO:2 is at least 80 amino acids long,   wherein the BTNL3 protein has a molecular weight at least about two times as large as that of a polypeptide of (a),   wherein the BTNL3 protein has been produced by a non-human host cell, and   wherein the multimeric BTNL3 protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         3 . The multimeric BTNL3 protein of  claim 1  or  2 , wherein the amino acid sequences of the polypeptides of (a) and (b) are at least 95% identical to amino acids 18-236 of SEQ ID NO:2. 
     
     
         4 . The multimeric BTNL3 protein of  claim 3 , wherein the amino acid sequences of the polypeptides of (a) and (b) are at least 97% identical to amino acids 18-236 of SEQ ID NO:2. 
     
     
         5 . The multimeric BTNL3 protein of  claim 4 , wherein the polypeptides of (a) and (b) comprise the amino acid sequence of amino acids 18-236 of SEQ ID NO:2. 
     
     
         6 . The multimeric BNTL3 protein of any one of  claims 1  to  5 , wherein the amino acid sequences of (a) and (b) do not comprise amino acids 237 to 259 of SEQ ID NO:2. 
     
     
         7 . The multimeric BTNL3 protein of any one of  claims 1  to  6 , wherein the polypeptides of (a) and (b) each comprise an amino acid sequence additional to the amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2. 
     
     
         8 . The multimeric BTNL3 protein of  claim 7 , wherein the additional amino acid sequences of (a) and (b) are the amino acid sequences of Fc polypeptides. 
     
     
         9 . The multimeric BTNL3 protein of  claim 8 , wherein
 (i) the Fc polypeptides comprise the amino acid sequence of a native human Fc polypeptide or   (ii) the Fc polypeptides comprise amino acid sequences that have not more than 15 insertions, deletions, or substitutions of a single amino acid relative to the amino acid sequence of a native human Fc region.   
     
     
         10 . The multimeric BTNL3 protein of  claim 9 , wherein the amino acid sequences of the Fc polypeptides of (ii) have not more than 10 insertions, deletions, or substitutions of a single amino acid relative to the amino acid sequence of the native human Fc region. 
     
     
         11 . The multimeric BTNL3 protein of  claim 10 , wherein the Fc polypeptides of (ii) have not more than 5 insertions, deletions, or substitutions of a single amino acid relative to the amino acid sequence of the native human Fc region. 
     
     
         12 . The multimeric BTNL3 protein of any one of  claims 8  to  11 , wherein the multimeric BTNL3 protein can bind to a human neonatal Fc receptor (FcRn). 
     
     
         13 . The multimeric BTNL3 protein of  claim 12 , which comprises the amino acid sequence of the native human Fc region. 
     
     
         14 . The multimeric BTNL3 protein of any one of  claims 9  to  13 , wherein the native human Fc region is of the IgG1 isotype. 
     
     
         15 . The multimeric BTNL3 protein of any one of  claims 9  to  13 , wherein the native human Fc region is of the IgG2 isotype. 
     
     
         16 . The multimeric BTNL3 protein of any one of  claims 9  to  13 , wherein the native human Fc region is of the IgG4 isotype. 
     
     
         17 . The multimeric BTNL3 protein of any one of  claims 1  to  16 , which is a homotrimer or a higher order homomultimer. 
     
     
         18 . The multimeric BTNL3 protein of  claim 17 , which is a homotetramer or a higher order homomultimer. 
     
     
         19 . The multimeric BTNL3 protein of any one of  claims 1  to  16 , which is a heteromultimer. 
     
     
         20 . The multimeric BTNL3 protein of any one of  claims 1  to  19 , wherein the multimeric BTNL3 protein has a molecular weight that is:
 approximately 4 times as large as the molecular weight of the polypeptide of (a) or (b); 
 approximately the sum of two times the molecular weight of the polypeptide of (a) plus two times the molecular weight of the polypeptide of (b); 
 approximately the sum of three times the molecular weight of the polypeptide of (a) plus the molecular weight of the polypeptide of (b); or 
 approximately the sum of the molecular weight of the polypeptide of (a) or 2 plus three times the molecular weight of the polypeptide of (b). 
 
     
     
         21 . A BTNL3 fusion protein comprising
 (a) a first polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2, wherein the alignment window of the amino acid sequence of the BTNL3 fusion protein with amino acids 18-236 is SEQ ID NO:2 is at least 80 amino acids long, and   (b) a second polypeptide that has a different amino acid sequence from that of the first polypeptide and does not comprise a fragment of the sequence from amino acid 237 to 466 of SEQ ID NO:2 that is at least 20 amino acids long,   wherein the BTNL3 fusion protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         22 . The BTNL3 fusion protein of  claim 21 , wherein the second polypeptide is an IgG Fc polypeptide. 
     
     
         23 . The BTNL3 fusion protein of  claim 22 , wherein the Fc polypeptide has an amino acid sequence that contains not more than 15 insertions, deletions, or substitutions of a single amino acid relative to the amino acid sequence of a native human Fc region. 
     
     
         24 . The BTNL3 fusion protein of  claim 23 , wherein the Fc polypeptide has an amino acid sequence containing not more than 10 insertions, deletions, or substitutions of a single amino acid relative to the amino acid sequence of the native human Fc region. 
     
     
         25 . The BTNL3 fusion protein of  claim 24 , wherein the Fc polypeptide has an amino acid sequence containing not more than 5 insertions, deletions, or substitutions of a single amino acid relative to the amino acid sequence of the native human Fc region. 
     
     
         26 . The BTNL3 fusion protein of any one of  claims 23  to  25 , wherein the BTNL3 fusion protein can bind to FcRn. 
     
     
         27 . The BTNL3 fusion protein of  claim 26  comprising the amino acid sequence of the native human Fc region. 
     
     
         28 . The BTNL3 fusion protein of any one of  claims 23  to  27 , wherein the native human Fc region is of the IgG1 isotype. 
     
     
         29 . The BTNL3 fusion protein of any one of  claims 23  to  27 , wherein the native human Fc region is of the IgG2 isotype. 
     
     
         30 . The BTNL3 fusion protein of any one of  claims 23  to  27 , wherein the native human Fc region is of the IgG4 isotype. 
     
     
         31 . The BTNL3 fusion protein of any one of  claims 21  to  30 , wherein the amino acid sequence of the first polypeptide at least 95% identical to amino acids 18-236 of SEQ ID NO:2. 
     
     
         32 . The BTNL3 fusion protein of  claim 31 , wherein the amino acid sequence of the first polypeptide comprises amino acids 18-236 of SEQ ID NO:2. 
     
     
         33 . The BTNL3 fusion protein of any one of  claims 21  to  32 , which comprises an amino acid sequence that is substantially similar to SEQ ID NO:7, wherein the amino acid sequence comprises not more that 20 insertions, deletions, or substitutions of a single amino acid relative to SEQ ID NO:7. 
     
     
         34 . The BTNL3 fusion protein of  claim 33 , wherein the amino acid sequence contains no more than 15 insertions, deletions, or substitutions of a single amino acid relative to SEQ ID NO:7. 
     
     
         35 . The BTNL3 fusion protein of  claim 34 , wherein the amino acid sequence contains no more than 10 insertions, deletions, or substitutions of a single amino acid relative to SEQ ID NO:7. 
     
     
         36 . The BTNL3 fusion protein of  claim 35 , wherein the amino acid sequence contains no more than 5 insertions, deletions, or substitutions of a single amino acid relative to SEQ ID NO:7. 
     
     
         37 . The BTNL3 fusion protein of  claim 36 , wherein the amino acid sequence comprises the sequence of SEQ ID NO:7. 
     
     
         38 . The BTNL3 fusion protein of any one of  claims 21  to  37 , wherein the molecular weight of the BTNL3 fusion protein under non-reducing conditions is at least about four times the molecular weight of a monomer species of the BTNL3 fusion protein. 
     
     
         39 . The BTNL3 fusion protein of  claim 38 , wherein the molecular weight of the of the BTNL3 fusion protein under non-reducing conditions is about four times the molecular weight of a monomer species of the BTNL3 fusion protein. 
     
     
         40 . An isolated multimeric BTNL3 protein comprising
 (a) a polypeptide having an amino acid sequence at least 90% identical to amino acids 18-166 of SEQ ID NO:9, and   (b) a second polypeptide having an amino acid sequence at least 90% identical to amino acids 18-166 of SEQ ID NO:9,   wherein the alignment window of the amino acid sequences of the polypeptides of (a) and (b) with amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long,   wherein the multimeric BTNL3 protein is at least a dimer,   wherein the multimeric BTNL3 protein has been produced by a non-human host cell, and   wherein the multimeric BTNL3 protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         41 . An isolated multimeric BTNL3 protein comprising
 (a) a polypeptide having an amino acid sequence at least 90% identical to amino acids 18-166 of SEQ ID NO:9, and   (b) a second polypeptide having an amino acid sequence at least 90% identical to amino acids 18-166 of SEQ ID NO:9,   wherein the alignment window of the amino acid sequences of the polypeptides of (a) and (b) with amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long,   wherein the multimeric BTNL3 protein has a molecular weight greater than about three times as large as that of a polypeptide of (a),   wherein the multimeric BTNL3 protein has been produced by a non-human host cell, and   wherein the multimeric BTNL3 protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         42 . The multimeric BTNL3 protein of  claim 40  or  41 , wherein the amino acid sequences of the polypeptides of (a) and (b) contain no more than 10 insertions, deletions, or substitutions of a single amino acid relative to amino acids 18-166 of SEQ ID NO:9. 
     
     
         43 . A BTNL3 fusion protein encoded by a DNA, wherein the DNA comprises:
 (a) a polynucleotide, which encodes a polypeptide, wherein the polynucleotide
 (i) consists of the nucleotide sequence of nucleotides 52 to 708 of SEQ ID NO:1 or the nucleotide sequence of nucleotides 52 to 498 of SEQ ID NO:8; or 
 (ii) hybridizes under stringent conditions to the polynucleotide of (i); and 
   (b) another polynucleotide that does not hybridize to a polynucleotide consisting of the sequence of SEQ ID NO:1 or SEQ ID NO:8 and encodes a polypeptide in frame with the polypeptide encoded by the polynucleotide of (a);   wherein the fusion protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         44 . The multimeric BTNL3 protein or the BTNL3 fusion protein of any one of  claims 1  to  43 , comprising a linker sequence. 
     
     
         45 . The multimeric BTNL3 protein or the BTNL3 fusion protein of  claim 44 , wherein the molecular weight of the protein is about four times the molecular weight of a monomeric species of the protein. 
     
     
         46 . An isolated variant BTNL3 protein comprising a polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9,
 wherein the alignment window of the amino acid sequence with amino acids 18-236 of SEQ ID NO:2 or 18-166 of SEQ ID NO:9 is at least 80 amino acids long,   wherein the variant BTNL3 protein does not comprise the amino acid sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, and   wherein the variant BTNL3 protein can antagonize the inhibition by a BTNL3 protein comprising the amino acid sequence of SEQ IN NO:7 of proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         47 . The variant BTNL3 protein of  claim 46 , wherein the polypeptide comprises an amino acid sequence at least 95% identical to amino acids 18-236 of SEQ ID NO:2 or amino acid 18-166 of SEQ ID NO:9. 
     
     
         48 . An isolated DNA encoding the multimeric BTNL3 protein, the BTNL3 fusion protein, or the variant BTNL3 protein of any one of  claims 1  to  47 , wherein the DNA does not include exon sequences. 
     
     
         49 . An isolated DNA encoding a fusion protein comprising a BTNL3 protein and another polypeptide, wherein the DNA comprises:
 (a) a DNA, which encodes a polypeptide, wherein the DNA
 (i) consists of the nucleotide sequence of nucleotides 52 to 708 of SEQ ID NO:1 or the nucleotide sequence of nucleotides 52 to 498 of SEQ ID NO:8; or 
 (ii) hybridizes under stringent conditions to the DNA of (i); and 
   (b) another DNA that does not hybridize to a polynucleotide consisting of the sequence of SEQ ID NO:1 or SEQ ID NO:8 and encodes a polypeptide in frame with the polypeptide encoded by the polynucleotide of (a);   wherein the fusion protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         50 . A vector comprising the DNA of  claim 48  or  49 . 
     
     
         51 . A non-human host cell containing the DNA of  claim 48  or  49  or the vector of  claim 50 . 
     
     
         52 . A method of making a BTNL3 protein comprising
 culturing the host cell of  claim 51  in a medium under conditions suitable for expression of the nucleic acid and   recovering the expressed protein from the cells or the culture medium.   
     
     
         53 . A method of treating a patient having an autoimmune or inflammatory disease comprising administering to the patient a therapeutically effective dose of a BTNL3 protein comprising
 (a) the amino acid sequence of amino acids 18-236 of SEQ ID NO:2 or the amino acid sequence of amino acids 18-166 of SEQ ID NO:9,   (b) an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, wherein the alignment window of the amino acid sequence with amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long, or   (c) an amino acid sequence that has no more than 20 insertions, deletions, or substitutions of a single amino acid relative to the sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9,   wherein the BTNL3 protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         54 . The method of  claim 53 , wherein the autoimmune or inflammatory disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, an inflammatory bowel disease, a transplantation-related condition, Crohn's disease, ulcerative colitis, psoriasis, sarcoidosis, asthma, or a fibrotic disease. 
     
     
         55 . The method of  claim 54 , wherein the autoimmune or inflammatory disease is Crohn's disease. 
     
     
         56 . The method of  claim 54 , wherein the autoimmune or inflammatory disease is ulcerative colitis. 
     
     
         57 . The method of  claim 54 , wherein the autoimmune or inflammatory disease is a fibrotic disease. 
     
     
         58 . A method for inhibiting T cell proliferation comprising adding to the T cell a BTNL3 protein comprising
 (a) the amino acid sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9,   (b) an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, wherein the alignment window of the amino acid sequence with amino acids amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long, or   (c) an amino acid sequence that has no more than 20 insertions, deletions, or substitutions of a single amino acid relative to the sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9,   wherein the BTNL3 protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         59 . The method of  claim 58 , wherein the inhibition occurs in vitro or ex vivo. 
     
     
         60 . The method of  claim 58 , wherein the inhibition occurs in vivo. 
     
     
         61 . A method of treating a cancer patient comprising administering to the patient a therapeutically effective amount of an antibody that binds to a BTNL3 protein consisting of amino acids 18-236 of SEQ ID NO:2 and/or amino acids 18-166 of SEQ ID NO:9. 
     
     
         62 . The method of  claim 61 , wherein the antibody is an antagonistic antibody. 
     
     
         63 . The method of  claim 61  or  62 , wherein the cancer is an adenocarcinoma. 
     
     
         64 . A method of treating a cancer patient comprising administering to the patient a therapeutically effective amount of a variant BTNL3 protein comprising a polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9,
 wherein the alignment window of the amino acid sequence with amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long,   wherein the protein does not comprise the amino acid sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, and   wherein the protein can antagonize the inhibition by a BTNL3 protein comprising the amino acid sequence of SEQ IN NO:7 of proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         65 . The method of  claim 64 , wherein the cancer is an adenocarcinoma. 
     
     
         66 . A method of treating a patient infected with a pathogen comprising administering to the patient a therapeutically effective amount of an antibody that binds to a BTNL3 protein consisting of amino acids 18-236 of SEQ ID NO:2 and/or amino acids 18-166 of SEQ ID NO:9. 
     
     
         67 . The method of  claim 66 , wherein the antibody is an antagonistic antibody. 
     
     
         68 . A method of treating a patient infected with a pathogen comprising administering to the patient a therapeutically effective amount of a variant BTNL3 protein comprising a polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9,
 wherein the alignment window of the amino acid sequence with amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long,   wherein the variant BTNL3 protein does not comprise the amino acid sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, and   wherein the variant BTNL3 protein can antagonize the inhibition by a BTNL3 protein comprising the amino acid sequence of SEQ IN NO:7 of proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         69 . A method for vaccinating a patient against an antigen comprising administering to the patient the antigen and
 (a) an antagonistic antibody that binds to a protein consisting of amino acids 18-236 of SEQ ID NO:2 and/or amino acids 18-166 of SEQ ID NO:9 or   (b) a variant BTNL3 protein comprising a polypeptide comprising an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, wherein the alignment window of the amino acid sequence with amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long, wherein the variant BTNL3 protein does not comprise the amino acid sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, and wherein the variant BTNL3 protein can antagonize the inhibition by a BTNL3 protein comprising the amino acid sequence of SEQ IN NO:7 of proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         70 . The method of  claim 69 , wherein the antigen is a cancer antigen. 
     
     
         71 . The method of  claim 69 , wherein the antigen can elicit an immune response against a pathogen. 
     
     
         72 . The method of any one of  claims 69  to  71 , wherein the antigen can be administered before, concurrently with, or after the antagonistic antibody. 
     
     
         73 . A method for treating a patient having an autoimmune or inflammatory condition comprising the following steps:
 (a) removing T cells from the patient;   (b) stimulating the T cells with a combination of proteins comprising an anti-CD3 antibody and a BTNL3 protein, wherein the BTNL3 protein comprises
 (i) the amino acid sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, 
 (ii) an amino acid sequence at least 90% identical to amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, wherein the alignment window of the amino acid sequence with amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9 is at least 80 amino acids long, or 
 (iii) an amino acid sequence that has no more than 20 insertions, deletions, or substitutions of a single amino acid relative to the sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9, 
   (c) harvesting the stimulated T cells; and   (d) returning the harvested T cells to the patient,   wherein the BTNL3 protein can inhibit the proliferation of a T cell stimulated by an immobilized anti-CD3 antibody.   
     
     
         74 . The method of  claim 73 , wherein the BTNL3 protein is the BTNL3 protein of (b)(iii), wherein the amino acid sequence has no more than 10 insertions, deletions, or substitutions of a single amino acid relative to the sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9. 
     
     
         75 . The method of  claim 74 , wherein the amino acid sequence has no more than 5 insertions, deletions, or substitutions of a single amino acid relative to the sequence of amino acids 18-236 of SEQ ID NO:2 or amino acids 18-166 of SEQ ID NO:9. 
     
     
         76 . The method of  claim 73 , wherein the BTNL3 protein is the BTNL3 protein of (b)(i). 
     
     
         77 . The method of any one of  claims 73  to  76 , wherein the autoimmune or inflammatory condition is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, an inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriasis, sarcoidosis, asthma, a transplantation-related condition, types 1 or 2 diabetes, or a fibrotic disease.