Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains

Abstract

This invention provides, and in certain specific but non-limiting aspects relates to:—assays that can be used to predict whether a given ISV will be subject to protein interference as described herein and/or give rise to an (aspecific) signal in such an assay (such as for example in an ADA immunoassay). Such predictive assays could for example be used to test whether a given ISV could have a tendency to give rise to such protein interference and/or such a signal; to select ISV's that are not or less prone to such protein interference or to giving such a signal; as an assay or test that can be used to test whether certain modification(s) to an ISV will (fully or partially) reduce its tendency to give rise to such interference or such a signal; and/or as an assay or test that can be used to guide modification or improvement of an ISV so as to reduce its tendency to give rise to such protein interference or signal;—methods for modifying and/or improving ISV's to as to remove or reduce their tendency to give rise to such protein interference or such a signal;—modifications that can be introduced into an ISV that remove or reduce its tendency to give rise to such protein interference or such a signal;—ISV's that have been specifically selected (for example, using the assay(s) described herein) to have no or low(er)/reduced tendency to give rise to such protein interference or such a signal;—modified and/or improved ISV's that have no or a low(er)/reduced tendency to give rise to such protein interference or such a signal.

1 . Protein or polypeptide, which protein or polypeptide contains an immunoglobulin single variable domain (ISV) at its C-terminal end, wherein said ISV is a serum albumin binding ISV, wherein said ISV is a VHH, humanized VHH or camelized VH, and wherein said ISV has a C-terminal end of the sequence VTVSS(X) n , in which:
 n=1, 2 or 3 in which each X=Ala or Gly; or   n=1, 2 or 3 in which each X=Ala; or   n=1, 2 or 3 in which each X=Gly; or   n=2 or 3 in which at least one X=Ala or Gly; or   n=2 or 3 in which all but one X=Ala or Gly.   
     
     
         2 . Protein or polypeptide according to  claim 1 , in which said ISV is Alb-1 or a humanized version of Alb-1. 
     
     
         3 . Protein or polypeptide according to  claim 1 , in which n=1 or n=2. 
     
     
         4 . Protein or polypeptide for use according to  claim 1 , in which n=2 or 3 and at least one X=Ala or Gly or n=2 or 3 and all but one X=Ala or Gly, with the remaining amino acid residue X being independently chosen from any naturally occurring amino acid. 
     
     
         5 . Protein or polypeptide according to  claim 4 , in which the remaining amino acid residue X is independently chosen from Val, Leu and/or Ile. 
     
     
         6 . Protein or polypeptide according to  claim 1 , in which:
 n=1, 2 or 3 in which each X=Ala or Gly; or   n=1, 2 or 3 in which each X=Ala; or   n=1, 2 or 3 in which each X=Gly.   
     
     
         7 . Protein or polypeptide according to  claim 1 , in which X is not cysteine. 
     
     
         8 . Protein or polypeptide which protein or polypeptide contains an ISV at its C-terminal end, wherein said ISV is a serum albumin binding ISV, wherein said ISV is a VHH, humanized VHH or camelized VH and wherein said ISV has a C-terminal end of the sequence VTVSS(X) n , in which n is 1 to 5, such as 1, 2, 3, 4 or 5, and in which each X is an amino acid residue that is independently chosen, with the proviso that X is not cysteine. 
     
     
         9 . Protein or polypeptide according to  claim 8 , in which n is 1, 2, or 3. 
     
     
         10 . Protein or polypeptide according to  claim 8 , in which each X is a naturally occurring amino acid. 
     
     
         11 . Protein or polypeptide according to  claim 8 , in which each X is chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). 
     
     
         12 . Protein or polypeptide according to  claim 8 , in which said ISV is Alb-1 or a humanized version of Alb-1. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . Pharmaceutical composition, that comprises a protein or polypeptide according to  claim 1 , and at least one suitable carrier, diluent or excipient. 
     
     
         16 .- 18 . (canceled) 
     
     
         19 . Pharmaceutical composition, that comprises a protein or polypeptide according to  claim 8 , and at least one suitable carrier, diluent or excipient. 
     
     
         20 .- 22 . (canceled) 
     
     
         23 . Protein or polypeptide according to  claim 2 , in which said humanized version of Alb-1 is Alb-8. 
     
     
         24 . Protein or polypeptide according to  claim 12 , in which said humanized version of Alb-1 is Alb-8.