Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains
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. A protein or polypeptide that comprises a VHH, a humanized VHH, or a camelized VH at its C-terminal end, wherein said VHH, humanized VHH, or camelized VH:
has a valine at position 11 according to Kabat numbering;
has a C-terminal sequence of VTVSS(X) n (SEQ ID NO:34), wherein: (i) n=1 or 2 in which each X is independently chosen from glycine (G), valine (V), leucine (L) or isoleucine (I); or (ii) n=3, 4 or 5 in which each X is independently chosen from alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I); such that said VHH, humanized VHH, or camelized VH has a substantially reduced tendency to give rise to protein interference compared to the same VHH, humanized VHH, or camelized VH without the modifications.
2. The protein or polypeptide according to claim 1 , wherein:
n=1 or 2 in which each X=Gly; or
n=3 in which each X=Ala or Gly; or
n=2 in which at least one X=Gly; or
n=2 in which all but one X=Gly;
n=3 in which at least one X=Ala or Gly; or
n=3 in which all but one X=Ala or Gly.
3. The protein or polypeptide according to claim 1 , wherein said VHH, humanized VHH, or camelized VH binds to a therapeutic target.
4. The protein or polypeptide according to claim 1 , comprising a serum albumin binding peptide or serum albumin binding domain.
5. The protein or polypeptide according to claim 4 , wherein the serum albumin binding domain is a serum albumin binding ISV, wherein said serum albumin binding ISV is a serum albumin binding VHH, humanized serum albumin binding VHH, or camelized serum albumin binding VH.
6. A pharmaceutical composition that comprises a protein or polypeptide according to claim 1 , and at least one suitable carrier, diluent or excipient.