Compositions and methods for producing bioactive fusion proteins

Abstract

Disclosed is a composition of matter involving a recombinant fusion protein comprising a a pharmacologically active protein partner, and a small pharmacologically inactive protein domain partner of human origin, such as but not limited to, a 10 th fibronectin III domain, a SH3 domain, a SH2 domain, a CH2 domain of IgG1, a PDZ domain, a thrombospondin repeat domain, an ubiquitin domain, a leucine-rich repeat domain, a villin headpiece HP35 domain, a villin headpiece HP76 domain, or a fragment or modification of any of these. Also disclosed are nucleic acids (e.g., DNA constructs) encoding the fusion protein, expression vectors and recombinant host cells for expression of the fusion protein, and pharmaceutical compositions containing the recombinant fusion protein and a pharmaceutically acceptable carrier, and method of producing a pharmacologically active recombinant fusion protein.

We claim: 
     
         1 . A composition of matter comprising a recombinant fusion protein, wherein said fusion protein comprises:
 (a) a pharmacologically inactive protein domain of human origin, wherein said pharmacologically inactive protein domain is a 10 th  fibronectin III domain that
 (i) has a mass of about 3 kDa to about 20 kDa, and 
 (ii) characteristically forms protein aggregates of less than about 10 percent of total mass of protein when suspended without other proteins in a pharmaceutically acceptable formulation buffer of interest; and 
   (b) at least one pharmacologically active protein about 5 to about 80 amino acid residues in length.   
     
     
         2 . The composition of matter of  claim 1  of the formula
   (F 1 ) a X 2    
 
       and multimers thereof, wherein:
 F 1  is a half-life extending moiety, and a is 0 or 1; 
 X 2  is D-(L) c -(P 5 ) d —(X 3 ) e , (X 4 ) f —(P 5 ) d -(L) e -D, or (X 4 ) f —(P 5 ) d -(L) e -D-(L) g -(P 6 ) h —(X 3 ) i , wherein 
 
       c and g are each independently 0 or 1, d and h are 1, and e, f, and i are each independently 0, 1, 2, 3, or 4; 
       X 3  is -(L) j -(P 7 ), j is 0 or 1; 
       X 4  is (P 8 )-(L) k -, k is 0 or 1; 
       D is the pharmacologically inactive protein domain of human origin, wherein the pharmacologically inactive protein domain is a 10 th  fibronectin III domain; 
       P 5 , P 6 , P 7  and P 8  are each independently pharmacologically active proteins; and 
       L is in each instance a peptidyl linker. 
     
     
         3 . The composition of matter of  claim 1 , wherein the pharmacologically inactive protein domain comprises an amino acid sequence selected from SEQ ID NOS: 2, 13, and 50. 
     
     
         4 . The composition of matter of  claim 2 , wherein a is 1, and F 1  is selected from peptide ligands or small molecule ligands that have binding affinity for a long half-life plasma protein. 
     
     
         5 . A pharmaceutical composition, comprising the composition of matter of  claim 1  or  claim 2 , and a pharmaceutically acceptable carrier. 
     
     
         6 . A nucleic acid comprising a polynucleotide sequence encoding a recombinant fusion protein comprising:
 (a) a pharmacologically inactive protein domain of human origin, wherein said pharmacologically inactive protein domain is a 10 th  fibronectin III domain that
 (i) has a mass from about 3 kDa to about 20 kDa, and 
 (ii) characteristically forms protein aggregates of less than about 10 percent of the total mass of protein when suspended without other proteins in a pharmaceutically acceptable formulation buffer of interest; and 
   (b) a pharmacologically active protein of about 5 to about 80 amino acid residues in length.   
     
     
         7 . The nucleic acid of  claim 6 , wherein the nucleic acid is a DNA. 
     
     
         8 . An expression vector comprising the nucleic acid of  claim 7 . 
     
     
         9 . A recombinant host cell comprising the expression vector of  claim 8 . 
     
     
         10 . A method of producing a pharmacologically active recombinant fusion protein, comprising:
 (a) placing the recombinant host cell of  claim 9  in a growth medium, such that the recombinant fusion protein is expressed; and   (b) isolating the fusion protein from the cell or growth medium.