Method for producing monomeric and multimeric molecules and uses thereof

Abstract

Herein is reported a method for the production of a polypeptide that is biologically active as n-mer comprising a nucleic acid encoding a fusion polypeptide according to the following formula (B n —CS o —I s —CS p —FC—CS q —I t —CS r —B m ) u , wherein B denotes a polypeptide that is biologically active as n-mer and forms non-defined aggregates/multimers upon expression in the absence of a fused Fc-region, FC denotes a heavy chain Fc-region polypeptide, CS denotes a cleavage site, and I denotes an intervening amino acid sequence, wherein FC does not substantially bind to an Fc-receptor, recovering the fusion polypeptide from the cell or the cultivation medium, optionally cleaving the fusion polypeptide with a protease, and thereby producing a polypeptide that is biologically active as n-mer and forms non-defined aggregates/multimers upon expression in the absence of a fused Fc-region.

1 . A method for the production of a polypeptide that is biologically active as n-mer comprising the following steps
 a) cultivating a cell comprising a nucleic acid encoding a fusion polypeptide according to formula I
   (B n —CS o —I s —CS p —FC—CS q —I t —CS r —B n ) u   (formula I)
 
 wherein 
 B denotes a polypeptide that is biologically active as n-mer, 
 FC denotes a heavy chain Fc-region polypeptide, 
 CS denotes a cleavage site, and 
 I denotes an intervening amino acid sequence, 
 wherein n=1 and m=0, or n=0 and m=1, 
 wherein if n=1 then o=0 or 1, and if o=0 then p=0 or 1, and if o=1 then p=0, and s=0 or 1, and q=0, and t=0, and r=0, 
 wherein if m=1 then q=0 or 1, and if q=0 then r=0 or 1, and if q=1 then r=0, and t=0 or 1, and o=0, and s=0, and p=0, 
 wherein u=1 or 2, 
 wherein FC does not substantially bind to an Fc-receptor, 
   b) recovering the fusion polypeptide from the cell or the cultivation medium,   c) optionally cleaving the fusion polypeptide with a protease,   and thereby producing a polypeptide that is biologically active as n-mer and forms non-defined aggregates/multimers upon expression in the absence of a fused Fc-region.   
     
     
         2 . A fusion polypeptide according to formula I
   (B n —CS o —I s —CS p —FC—CS q —I t —CS r —B m ) u   (formula I)
   wherein B denotes a polypeptide that is biologically active as n-mer,   FC denotes a heavy chain Fc-region polypeptide,   CS denotes a cleavage site, and   I denotes an intervening amino acid sequence,   wherein n=1 and m=0, or n=0 and m=1,   wherein if n=1 then o=0 or 1, and if o=0 then p=0 or 1, and if o=1 then p=0, and s=0 or 1, and q=0, and t=0, and r=0,   wherein if m=1 then q=0 or 1, and if q=0 then r=0 or 1, and if q=1 then r=0, and t=0 or 1, and o=0, and s=0, and p=0,   wherein u=1 or 2,   wherein FC does not substantially bind to an Fc-receptor.   
     
     
         3 . An n-mer polypeptide obtained by cleavage of a fusion polypeptide obtained by a method according to  claim 1 . 
     
     
         4 . The method according to  claim 1  or the fusion polypeptide according to  claim 2  or the n-mer polypeptide according to  claim 3 , characterized in that B is selected from the group of IL17, TWEAK, TNF and other TNF family members, TL1a, IL18, IL18R, IL33, and IL33R. 
     
     
         5 . The method according to  claim 1  or  4  or the fusion polypeptide according to  claim 2  or  4 , characterized in that FC is a variant of a heavy chain polypeptide selected from the group of human IgG1 heavy chain polypeptide, human IgG2 heavy chain polypeptide, human IgG3 heavy chain polypeptide, human IgG4 heavy chain polypeptide, murine IgG1 heavy chain polypeptide, murine IgG2 heavy chain polypeptide, murine IgG3 heavy chain polypeptide, rabbit IgG heavy chain polypeptide. 
     
     
         6 . The method according to any one of  claim 1 ,  4  or  5  or the fusion polypeptide according to any one of  claim 2 ,  4  or  5 , characterized in that FC is selected from human IgG1 heavy chain polypeptide with the mutations L234A, L235A and P329G, human IgG4 heavy chain polypeptide with the mutation S228P, L235E and P329G. 
     
     
         7 . The method according to any one of  claim 1 ,  4 ,  5  or  6  or the fusion polypeptide according to any one of  claim 2 ,  4 ,  5  or  6 , characterized in that u=2 and the first FC comprises the mutation T366W and optionally the mutation S354C and the second FC comprises the mutations T366S, L368A and Y407V and optionally the mutation Y349C. 
     
     
         8 . The method according to  claim 7  or the fusion polypeptide according to  claim 7 , characterized in that
 a) n=1 and m=0 and B fused to the first FC and B fused to the second FC are identical, 
 b) n=1 and m=0 and B fused to the first FC and B fused to the second FC are different, 
 c) n=0 and m=1 and B fused to the first FC and B fused to the second FC are identical, 
 d) n=0 and m=1 and B fused to the first FC and B fused to the second FC are different. 
 
     
     
         9 . Use of an immobilized fusion polypeptide according to any one of  claims 2  and  4  to  8  or of an n-mer polypeptide according to any one of  claims 3  to  4  as affinity chromatography ligand. 
     
     
         10 . Use of an immobilized fusion polypeptide according to any one of  claims 2  and  4  to  8  or of an n-mer polypeptide according to any one of  claims 3  to  4  in an immunoassay. 
     
     
         11 . The use according to any one of  claims 9  to  10 , characterized in that the fusion polypeptide or the n-mer polypeptide is bound to a solid phase. 
     
     
         12 . Pharmaceutical composition comprising a fusion polypeptide according to any one of  claims 2  and  4  to  8  or an n-mer polypeptide according to any one of  claims 3  to  4 . 
     
     
         13 . Use of a fusion polypeptide according to any one of  claims 2  and  4  to  8  or of an n-mer polypeptide according to any one of  claims 3  to  4  in the manufacture of a medicament. 
     
     
         14 . Use of a fusion polypeptide according to any one of  claims 2  and  4  to  8  or of an n-mer polypeptide according to any one of  claims 3  to  4  as immunogen. 
     
     
         15 . Use of a fusion polypeptide according to any one of  claims 2  and  4  to  8  or of a n-mer polypeptide according to any one of  claims 3  to  4  for obtaining an animal model of a disease by applying the fusion polypeptide according to any one of  claims 2  and  4  to  8  or the n-mer polypeptide according to any one of  claims 3  to  4  to an experimental animal. 
     
     
         16 . Use of a fusion polypeptide according to any one of  claims 2  and  4  to  8  or of a n-mer polypeptide according to any one of  claims 3  to  4  for selecting an antibody that specifically binds to B or an n-mer of B. 
     
     
         17 . Use of a fusion polypeptide according to any one of  claims 2  and  4  to  8  or of a n-mer polypeptide according to any one of  claims 3  to  4  for producing a disulfide-linked 2-mer of B. 
     
     
         18 . The method according to any one of  claims 1  and  4  to  8  for the production of a polypeptide that is biologically active as 2-mer and forms non-defined aggregates/multimers upon expression in the absence of a fused Fc-region comprising the following steps
 a) cultivating a cell comprising a nucleic acid encoding a fusion polypeptide according to formula II
   B n —CS o —I s —CS p —FC 1 —CS q —I t —CS r —B m :FC 2   (formula II)
 
 wherein 
 B denotes a polypeptide that is biologically active as 2-mer and forms non-defined aggregates/multimers upon expression in the absence of a fused Fc-region, 
 FC 1  denotes a first heavy chain Fc-region polypeptide, 
 FC 2  denotes a second heavy chain Fc-region polypeptide, 
 CS denotes a cleavage site, and 
 I denotes an intervening amino acid sequence, 
 wherein n=1 and m=0, or n=0 and m=1, 
 wherein if n=1 then o=0 or 1, and if o=0 then p=0 or 1, and if o=1 then p=0, and s=0 or 1, and q=0, and t=0, and r=0, 
 wherein if m=1 then q=0 or 1, and if q=0 then r=0 or 1, and if q=1 then r=0, and t=0 or 1, and o=0, and s=0, and p=0, 
 wherein the first FC and the second FC are covalently linked by one or more disulfide bond(s), 
 wherein FC 1  and FC 2  do not substantially bind to an Fc-receptor, 
 
 b) recovering the fusion polypeptide from the cell or the cultivation medium, 
 c) optionally cleaving the fusion polypeptide with a protease, 
 and thereby producing a polypeptide that is biologically active as 2-mer.