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 polypeptide comprising a serum albumin binding immunoglobulin single variable domain (VH or VHH) at its C-terminal end, wherein the immunoglobulin single variable domain ends with the sequence VTVSS(X) n (SEQ ID NO: 34), in which n is 1 to 5, with each X being independently chosen from any amino acid, with the proviso that X is not cysteine (C); and wherein the immunoglobulin single variable domain:
(i) is not Alb-1; and
(ii) does not end with the sequence VTVSSA (SEQ ID NO: 90).
2. The polypeptide of claim 1 , in which:
(a) n is 2 or 3 in which each X is alanine (A) or glycine (G); or
(b) n is 2 or 3 in which each X is alanine (A); or
(c) n is 1, 2 or 3 in which each X is G glycine (G); or
(d) n is 2 or 3 in which at least one X is alanine (A) or glycine (G), with any remaining amino acid residue X being independently chosen from any naturally-occurring amino acid; or
(e) n is 2 or 3 in which all but one X is alanine (A) or glycine (G), with any remaining amino acid residue X being independently chosen from any naturally- occurring amino acid.
3. The polypeptide of claim 2 , in which in (d) or (e) the remaining amino acid residue X is independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) and isoleucine (I).
4. The polypeptide of claim 1 , wherein n is 1 or 2.
5. The polypeptide of claim 1 , wherein each X is a naturally-occurring amino acid.
6. The polypeptide of claim 1 , wherein each X is independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) and isoleucine (I).