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 .- 36 . (canceled)
37 . A method of modifying an immunoglobulin single variable domain (ISV) having a C-terminal end of the sequence VTVSS (SEQ ID NO: 33), or a protein or a polypeptide comprising the ISV at its C-terminus, the method comprising:
(i) extending the C-terminal end of the ISV, protein, or polypeptide by 1 to 10 amino acid residues to generate an extended protein ending with the sequence VTVSS(X)n (SEQ ID NO: 34), in which n is 1 to 10, with each X being independently chosen from any amino acid; and
(ii) determining whether the extended protein has reduced aspecific binding to its C-terminal end as compared to the ISV, protein, or polypeptide having the C-terminal end of the sequence VTVSS (SEQ ID NO: 33).
38 . The method of claim 37 , wherein the extended protein has reduced aspecific protein binding to its C-terminal end in a biological fluid of a human subject.
39 . The method of claim 38 , wherein the biological fluid is a whole blood sample, a serum sample, a plasma sample, an ocular fluid sample, a bronchoalveolar fluid sample, or a cerebrospinal fluid sample.
40 . The method of claim 37 , wherein n is 1 to 5, with each X being independently chosen from any naturally occurring amino acid.
41 . The method of claim 37 , wherein n is 1 to 5, with each X being independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) and isoleucine (I).
42 . The method of claim 41 , wherein the amino acid sequence of the ISV has valine at position 11 according to Kabat numbering.
43 . The method of claim 37 , wherein n is 1 or 2.
44 . The method of claim 37 , in which:
(a) n=1, 2 or 3 in which each X=Ala or Gly; or
(b) n=1, 2 or 3 in which each X=Ala; or
(c) n=1, 2 or 3 in which each X=Gly; or
(d) n=2 or 3 in which at least one X=Ala or Gly, with any remaining amino acid residue X being independently chosen from any naturally occurring amino acid; or
(e) n=2 or 3 in which all but one X=Ala or Gly, with any remaining amino acid residue X being independently chosen from any naturally occurring amino acid.
45 . The method of claim 44 , 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).
46 . The method of claim 44 , wherein n=1 or n=2.
47 . The method of claim 37 , wherein the ISV is a VHH, a humanized VHH, a VH, or a camelized VH.
48 . The method of claim 37 , wherein the ISV binds a therapeutic target.
49 . The method of claim 37 , wherein the ISV binds serum albumin.
50 . A method for reducing the tendency of an immunoglobulin single variable domain (ISV) having a C-terminal end of the sequence VTVSS (SEQ ID NO: 33), or a protein or a polypeptide comprising the ISV at its C-terminus, to give rise to protein interference in an anti-drug-antibody assay, the method comprising modifying the ISV, protein, or polypeptide by adding 1 to 10 amino acid residues to its C-terminal end, each amino acid independently chosen from any amino acid, to generate a modified ISV.
51 . The method of claim 50 , further comprising confirming that the modified ISV, protein or polypeptide has a reduced tendency to give rise to protein interference as compared to the ISV, protein, or polypeptide having the C-terminal end of the sequence VTVSS (SEQ ID NO: 33).
52 . The method of claim 50 , which comprises adding 1, 2, 3, 4 or 5 amino acid residues to the C-terminal end, each amino acid being independently chosen from any naturally occurring amino acid.
53 . The method of claim 50 , which comprises adding 1, 2, 3, 4 or 5 amino acid residues to the C-terminal end, each amino acid being independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) and isoleucine (I).
54 . The method of claim 50 , wherein the ISV is a VHH, a humanized VHH, a VH, or a camelized VH.
55 . The method of claim 50 , wherein the ISV binds a therapeutic target.
56 . The method of claim 50 , wherein the ISV binds serum albumin.