Polypeptides

Abstract

There is provided inter alia a polypeptide comprising an immunoglobulin chain variable domain comprising three complementarity determining regions (CDR1-CDR3) and four framework regions, wherein: (a) at least one lysine residue in CDR1, CDR2 and/or CDR3 has been substituted with at least one histidine residue, and/or (b) at least one arginine residue in CDR1, CDR2 and/or CDR3 has been substituted with at least one histidine residue; wherein the polypeptide has increased intestinal stability relative to a corresponding polypeptide not having said histidine substitutions.

1 . A method of increasing the intestinal stability of a polypeptide comprising an immunoglobulin chain variable domain, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions, wherein the method comprises the step of substituting:
 (a) at least one lysine residue in CDR1, CDR2 and/or CDR3 with at least one histidine residue, and/or   (b) at least one arginine residue in CDR1, CDR2 and/or CDR3 with at least one histidine residue.   
     
     
         2 . A method of making a polypeptide comprising an immunoglobulin chain variable domain, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions, wherein the method comprises the step of substituting:
 (a) at least one lysine residue in CDR1, CDR2 and/or CDR3 with at least one histidine residue, and/or   (b) at least one arginine residue in CDR1, CDR2 and/or CDR3 with at least one histidine residue   wherein the polypeptide has increased intestinal stability relative to a corresponding polypeptide not having said histidine substitutions.   
     
     
         3 . The method according to  claim 1 , wherein the substitutions increase the stability of the polypeptide in the intestinal tract, such as in the small and/or large intestine, such as in the duodenum, jejunum, ileum cecum, colon, rectum and/or anal canal, relative to a corresponding polypeptide not having said histidine substitutions. 
     
     
         4 . The method according to  claim 1 , wherein the substitutions increase the stability of the polypeptide in a model of the intestinal tract, such as in the small and/or large intestine, such as in the duodenum, jejunum, ileum cecum, colon, rectum and/or anal canal, relative to a corresponding polypeptide not having said histidine substitutions. 
     
     
         5 . The method according to  claim 4  wherein the model of the intestinal tract is the Standard Human Faecal Supernatant Intestinal Tract Model. 
     
     
         6 . The method according to  claim 5 , wherein the stability of the polypeptide is increased by at least 5%, more suitably 30%, more suitably 50%, relative to a corresponding polypeptide not having said histidine substitutions, after 1 hour incubation in the Standard Human Faecal Supernatant Intestinal Tract Model. 
     
     
         7 . The method according to  claim 1 , wherein the substitutions increase the stability of the polypeptide to one or more proteases produced in the small or large intestine, relative to a corresponding polypeptide not having said histidine substitutions. 
     
     
         8 . The method according to  claim 7 , wherein the one or more proteases are selected from the group consisting of trypsin, chymotrypsin, MMPs, cathepsin, enteropeptidase, host inflammatory proteases, proteases originating from gut commensal microflora and/or pathogenic bacteria actively secreted and/or released by lysis of microbial cells, and proteases originating from pathogenic bacteria, such as  C. difficile -specific proteases. 
     
     
         9 . The method according to  claim 1  wherein the potency of the polypeptide is substantially the same as the potency of a corresponding polypeptide not having said histidine substitutions. 
     
     
         10 . The method according to  claim 9  wherein the EC50 of the polypeptide is increased by no more than 400% relative to a corresponding polypeptide not having said histidine substitutions. 
     
     
         11 . The method according to  claim 1 , wherein the substitutions are introduced synthetically. 
     
     
         12 . The method according to  claim 11 , wherein the substitutions are introduced by a method selected from the group consisting of: error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multi mer creation, or a combination thereof. 
     
     
         13 . The method according to  claim 1 , wherein the substitutions are not introduced by V(D)J recombination or somatic mutation. 
     
     
         14 . The method according to  claim 1 , wherein the at least one lysine residue is present in a window defined as the second third of CDR1 and/or the second third of CDR2 and/or the second third of CDR3. 
     
     
         15 . The method according to  claim 1 , wherein the at least one arginine residue is present in a window defined as the second third of CDR1 and/or the second third of CDR2 and/or the second third of CDR3. 
     
     
         16 . The method according to  claim 1 , wherein each lysine and/or arginine residue in CDR1, CDR2 and/or CDR3 has been substituted with at least one histidine residue each. 
     
     
         17 . The method according to  claim 1 , wherein the polypeptide consists of an immunoglobulin chain variable domain. 
     
     
         18 . The method according to  claim 1 , wherein the polypeptide is selected from the group consisting of an antibody, a modified antibody containing additional antibody binding regions, an antibody fragment such as an scFv, a Fab fragment, a F(ab′)2 fragment and an immunoglobulin chain variable domain such as a VHH, a VH, a VL, a V-NAR. 
     
     
         19 . The method according to  claim 1 , wherein the polypeptide binds to a target accessible via the intestinal tract. 
     
     
         20 . The method according to  claim 19 , wherein the polypeptide binds to a target within the intestinal tract.