Method for the production of variable domains

Abstract

The present invention provides methods for the expression and/or production of variable domains with a C-terminal extension that can be used for coupling of the variable domain to one or more further groups, residues or moieties. In the method of the invention a yield of at least 80% of variable domains with a cysteine containing C-terminal extension is obtained. Also variable domains are provided and polypeptides comprising one or more variable domains obtainable by the methods of the present invention, as well as compounds that comprise such variable domains and/or polypeptides coupled to one or more groups, residues or moieties.

1 .- 31 . (canceled) 
     
     
         32 . Polypeptide comprising one or more single variable domains linked to a C-terminal extension of maximal 10 amino acid residues in which at least one amino acid residue is a cysteine residue, wherein the C-terminal extension is different from SEQ ID NO: 1 and/or wherein the C-terminal extension is different three glycine residues followed by a C-terminal cysteine residue. 
     
     
         33 . The polypeptide according to  claim 32 , wherein the at least one cysteine residue is positioned at the C-terminal end of the C-terminal extension. 
     
     
         34 . The polypeptide according to  claim 32 , wherein the at least one cysteine residue is positioned at a site in the C-terminal extension different from the C-terminal end. 
     
     
         35 . The polypeptide according to  claim 32 , wherein the C-terminal extension, in addition to the at least one cysteine residue, consists of glycine residues. 
     
     
         36 . The polypeptide according to  claim 32 , wherein the C-terminal extension, in addition to the at least one cysteine residue, consists of alanine residues. 
     
     
         37 . The polypeptide according to  claim 32 , wherein the C-terminal extension is selected from any of SEQ ID NO's: 2-7 or wherein the C-terminal amino acid of the one or more single variable domains is removed prior to addition of a C-terminal cysteine (e.g., as shown in SEQ ID NO: 8). 
     
     
         38 . The polypeptide according to  claim 32 , obtained by a method comprising the steps of:
 a) maintaining a host under conditions that are such that said host expresses and/or produces the polypeptide;   b) isolating and/or purifying the secreted polypeptide from the medium;   wherein at least 80% of the polypeptide isolated and/or purified in step b) contains the at least one cysteine residue in the C-terminal extension, as determined by mass spectrometry.   
     
     
         39 . The polypeptide according to  claim 38 , wherein said eukaryotic host is selected from insect cells, mammalian cells, and lower eukaryotic hosts comprising yeasts such as  Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis,  preferably  Pichia pastoris.    
     
     
         40 . The polypeptide according to  claim 32 , wherein the one or more single variable domains is a light chain variable domain sequence or a heavy chain variable domain sequence. 
     
     
         41 . The polypeptide according to  claim 40 , wherein the one or more single variable domains is a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or a heavy chain variable domain sequence that is derived from a heavy chain antibody. 
     
     
         42 . The polypeptide according to  claim 41 , wherein the one or more single variable domains is one or more (single) domain antibodies, one or more “dAb”s or one or more Nanobodies (including but not limited to a VHH sequence). 
     
     
         43 . Compound that comprises a polypeptide according to  claim 32  coupled to one or more groups, residues or moieties. 
     
     
         44 . Compound according to  claim 43 , wherein said one or more groups, residues or moieties are selected from polyethylene glycol (PEG) or derivatives thereof, a peptide, a small molecule-drug, a therapeutically active agent, a toxin or toxic residue, a label, part of a specific binding pair and/or a radiolabelled molecule. 
     
     
         45 . Compound according to  claim 44 , wherein said label is selected from fluorescent labels, phosphorescent labels, chemiluminescent labels, bioluminescent labels, radio-isotopes, metals, metal chelates, metallic cations, chromophores and enzymes. 
     
     
         46 . Compound according to  claim 44 , wherein said part of a specific binding pair is selected from biotin, avidin and streptavidin. 
     
     
         47 . Composition comprising at least a polypeptide according to  claim 32 . 
     
     
         48 . Composition comprising at least a compound according to  claim 44 . 
     
     
         49 . Composition according to  claim 47 , which is a pharmaceutical composition that further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and that optionally comprises one or more further pharmaceutically active polypeptides and/or compounds. 
     
     
         50 . Composition according to  claim 48 , which is a pharmaceutical composition that further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and that optionally comprises one or more further pharmaceutically active polypeptides and/or compounds. 
     
     
         51 . Diagnostic kit comprising a compound according to  claim 44 .