Cytokine designated 4-1BB ligand and human receptor that binds thereto

Abstract

Novel 4-1BB ligand (4-1BB-L) polypeptides and a human cell surface receptor designated 4-1BB that binds 4-1BB-L are provided. Isolated 4-1BB-L-encoding and human 4-1BB-encoding DNA sequences, recombinant expression vectors comprising the isolated DNA sequences, and host cells transformed with the recombinant expression vectors are disclosed, along with methods for producing the novel polypeptides by cultivating such transformed host cells. Soluble forms of the 4-1BB-L or 4-1BB polypeptides are derived from the extracellular domains thereof.

1 . An isolated DNA encoding a 4-1BB-L polypeptide, wherein said DNA comprises a nucleotide sequence selected from the group consisting of nucleotides 53-979 of the murine 4-1BB-L sequence of SEQ ID NO:1 and nucleotides 4-765 of the human 4-1BB-L sequence of SEQ ID NO:3.  
     
     
         2 . An isolated DNA encoding a 4-1BB-L polypeptide, wherein said DNA is degenerate as a result of the genetic code to a nucleotide sequence of  claim 1 .  
     
     
         3 . An isolated DNA capable of hybridizing to a nucleotide sequence of  claim 1  under moderately stringent conditions, wherein said DNA encodes a 4-1BB-L polypeptide capable of binding to 4-1BB.  
     
     
         4 . An isolated DNA of  claim 3 , wherein said DNA encodes a soluble 4-1BB-L polypeptide.  
     
     
         5 . An isolated DNA of  claim 4 , wherein said DNA additionally encodes an antibody Fc polypeptide fused to the C-terminus of said soluble 4-1BB-L polypeptide.  
     
     
         6 . An isolated DNA of  claim 4  wherein the soluble 4-1BB-L polypeptide is a soluble human 4-1BB-L polypeptide comprising amino acids 49-254 of SEQ ID NO:3.  
     
     
         7 . A recombinant expression vector comprising a DNA according to  claim 1  operably linked to regulatory sequences suitable for expression of said DNA in a host cell.  
     
     
         8 . A recombinant expression vector comprising a DNA according to  claim 2  operably linked to regulatory sequences suitable for expression of said DNA in a host cell.  
     
     
         9 . A recombinant expression vector comprising a DNA according to  claim 3  operably linked to regulatory sequences suitable for expression of said DNA in a host cell.  
     
     
         10 . A recombinant expression vector comprising a DNA according to  claim 4  operably linked to regulatory sequences suitable for expression of said DNA in a host cell.  
     
     
         11 . A recombinant expression vector comprising a DNA according to  claim 5  operably linked to regulatory sequences suitable for expression of said DNA in a host cell.  
     
     
         12 . A process for preparing a 4-1BB-L polypeptide, comprising culturing a host cell comprising a vector according to  claim 7  under conditions that promote expression of the 4-1BB-L polypeptide, and purifying the 4-1BB-L polypeptide.  
     
     
         13 . A process for preparing a 4-1BB-L polypeptide, comprising culturing a host cell comprising a vector according to  claim 8  under conditions that promote expression of the 4-1BB-L polypeptide, and purifying the 4-1BB-L polypeptide.  
     
     
         14 . A process for preparing a 4-1BB-L polypeptide, comprising culturing a host cell comprising a vector according to  claim 9  under conditions that promote expression of the 4-1BB-L polypeptide, and purifying the 4-1BB-L polypeptide.  
     
     
         15 . A process for preparing a soluble 4-1BB-L polypeptide, comprising culturing a host cell comprising a vector according to  claim 10  under conditions that promote expression of the 4-1BB-L polypeptide, and purifying the 4-1BB-L polypeptide.  
     
     
         16 . A process for preparing a soluble 4-1BB-L/Fc fusion protein, comprising culturing a host cell comprising a vector according to  claim 11  under conditions that promote expression of the 4-1BB-L/Fc fusion protein, and purifying said fusion protein.  
     
     
         17 . A purified 4-1BB ligand polypeptide (4-1BB-L) comprising an N-terminal amino acid sequence selected from the group consisting of Met-Glu-Tyr-Ala-Ser-Asp-Ala-Ser-Leu-Asp-Pro-Glu-, Leu-Ala-Cys-Pro-Trp-Ala-Val-Ser-Gly-Ala-Arg-Ala-Ser-, Met-Asp-Gln-His-Thr-Leu-Asp-Val-Glu-Asp-Thr-Ala-, Arg-Thr-Glu-Pro-Arg-Pro-Ala-Leu-Thr-Ile-Thr-Thr-, Thr-Glu-Pro-Arg-Pro-Ala-Leu-Thr-Ile-Thr-Thr-, and Glu-Pro-Arg-Pro-Ala-Leu-Thr-Ile-Thr-Thr-.  
     
     
         18 . A purified 4-1BB-L according to  claim 17 , comprising an amino acid sequence selected from the group consisting of amino acids amino acids 1-254 of SEQ ID NO:3, amino acids 49-254 of SEQ D NO:3, amino acids 1-309 of SEQ ID NO:1, and amino acids/X-309 of SEQ ID NO:1, wherein x is selected from the group consisting of 104, 105, and 106.  
     
     
         19 . A purified 4-1BB-L according to  claim 17 , comprising an amino acid sequence that is identical to a sequence selected from the group consisting of amino acids amino acids 1-254 of SEQ ID NO:3, amino acids 49-254 of SEQ ID NO:3, amino acids 1-309 of SEQ ID NO:1, and amino acids x-309 of SEQ ID NO:1, wherein x is selected from the group consisting of 104, 105, and 106, except for conservative amino acid substitution(s).  
     
     
         20 . A purified 4-1BB-L polypeptide wherein said polypeptide is encoded by a DNA selected from the group consisting of: 
 (a) DNA comprising a nucleotide sequence selected from the group consisting of nucleotides 53-979 of the murine 4-1BB-L sequence of SEQ ID NO:1 and nucleotides 4-765 of the human 4-1BB-L sequence of SEQ ID NO:3;    (b) DNA capable of hybridizing to a DNA of (a) under moderately stringent conditions and which encodes a 4-1BB-L capable of binding to a 4-1BB; and    (c) DNA which, due to the degeneracy of the genetic code encodes a 4-1BB-L polypeptide encoded by a DNA of (a).    
     
     
         21 . A purified 4-1BB-L of  claim 20  wherein said 4-1BB-L is a soluble 4-1BB-L polypeptide.  
     
     
         22 . A purified soluble 4-1BB-L of  claim 21  which additionally comprises an antibody Fc polypeptide fused to the C-terminus of said soluble 4-1BB-L polypeptide.  
     
     
         23 . A purified soluble human 4-1BB-L comprising the amino acid sequence of amino acids 49-254 of SEQ ID NO:4.  
     
     
         24 . A pharmaceutical composition comprising an effective amount of a soluble human 4-1BB-L of  claim 21  in admixture with a suitable diluent, carrier, or excipient.  
     
     
         25 . A dimer comprising two soluble 4-1BB-L polypeptides of  claim 22 , joined via disulfide bonds between the Fc polypeptides fused to said soluble 4-1BB-L polypeptides.  
     
     
         26 . An antibody that is immunoreactive with a 4-1BB-L polypeptide of  claim 20 .  
     
     
         27 . An antibody of  claim 26 , wherein said antibody is a monoclonal antibody.  
     
     
         28 . An isolated nucleic acid molecule comprising a sequence of at least about 17 nucleotides of a DNA sequence according to  claim 1  or its DNA or RNA complement.  
     
     
         29 . An isolated DNA encoding a human 4-1BB polypeptide, wherein said DNA comprises a nucleotide sequence selected from the group consisting of: 
 a) nucleotides 120-884 of SEQ ID NO:7;    b) nucleotides 189-884 of SEQ ID NO:7; and    c) a nucleotide sequence that is degenerate as a result of the genetic code to a nucleotide sequence of (a) or (b).    
     
     
         30 . An isolated DNA encoding a soluble human 4-1BB polypeptide, wherein said polypeptide comprises the extracellular domain of human 4-1BB (amino acids 1-163 of SEQ ID NO:7) or a fragment thereof capable of binding a 4-1BB-L.  
     
     
         31 . An isolated DNA of  claim 30 , wherein said DNA additionally encodes an antibody Fc polypeptide fused to the C-terminus of said soluble human 4-1BB polypeptide.  
     
     
         32 . A recombinant expression vector comprising a DNA sequence according to  claim 29  operably linked to regulatory sequences suitable for expression of said DNA sequence in a host cell.  
     
     
         33 . A recombinant expression vector comprising a DNA sequence according to  claim 30  operably linked to regulatory sequences suitable for expression of said DNA sequence in a host cell.  
     
     
         34 . A recombinant expression vector comprising a DNA sequence according to  claim 31  operably linked to regulatory sequences suitable for expression of said DNA sequence in a host cell.  
     
     
         35 . A process for preparing a human 4-1BB polypeptide, comprising culturing a host cell comprising a vector according to  claim 32  under conditions that promote expression of the human 4-1BB polypeptide, and purifying said polypeptide.  
     
     
         36 . A process for preparing a soluble human 4-1BB polypeptide, comprising culturing a host cell comprising a vector according to  claim 33  under conditions that promote expression of the human 4-1BB polypeptide, and purifying said polypeptide.  
     
     
         37 . A process for preparing a fusion protein comprising an antibody Fc polypeptide fused to the C-terminus of a soluble human 4-1BB polypeptide, comprising culturing a host cell comprising a vector according to  claim 34  under conditions that promote expression of the human fusion protein, and purifying said fusion protein.  
     
     
         38 . A purified human 4-1BB polypeptide comprising the N-terminal amino acid sequence Leu-Gln-Asp-Pro-Cys-Ser-Asn-Cys-Pro-Ala-Gly-Thr-.  
     
     
         39 . A purified 4-1BB according to  claim 38 , comprising an amino acid sequence selected from the group consisting of amino acids 1-232 of SEQ ID-NO:7 and amino acids 1-163 of SEQ ID NO:7.  
     
     
         40 . A purified 4-1BB according to  claim 38 , comprising an amino acid sequence that is identical to a sequence selected from the group consisting of amino acids 1-232 of SEQ ID NO:7 and amino acids 1-163 of SEQ ID NO:7, except for conservative amino acid substitution(s).  
     
     
         41 . A purified soluble human 4-1BB polypeptide, wherein said polypeptide comprises the extracellular domain of human 4-1BB (amino acids 1-163 of SEQ ID NO:7) or a fragment thereof capable of binding a 4-1BB-L.  
     
     
         42 . A purified soluble human 4-1BB polypeptide of  claim 41 , additionally comprising an antibody Fc polypeptide fused to the C-terminus of said human 4-1BB polypeptide.  
     
     
         43 . A dimer comprising two soluble human 4-1BB polypeptides of  claim 42 , joined via disulfide bonds between the Fc polypeptides fused to said soluble 4-1BB polypeptides.  
     
     
         44 . A pharmaceutical composition comprising an effective amount of a soluble human 4-1BB of  claim 41  in admixture with a suitable diluent, carrier, or excipient.  
     
     
         45 . An antibody that is immunoreactive with a human 4-1BB polypeptide of  claim 38 .  
     
     
         46 . An antibody of  claim 45 , wherein said antibody is a monoclonal antibody.  
     
     
         47 . An isolated nucleic acid molecule comprising a sequence of at least about 17 nucleotides of a DNA sequence according to  claim 29  or its DNA or RNA complement.