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 fusion protein comprising at least two immunoglobulin single variable domains (ISV), wherein one of the at least two ISVs is at the C-terminal end of the fusion protein, wherein the ISV at the C-terminal end of the fusion protein is a VHH, a humanized VHH, a VH, or a camelized VH that: does not bind to serum albumin; binds to a therapeutic target; and has a C-terminal end of the sequence VTVSS(X)n (SEQ ID NO: 34), in which n is 1, 2, 3, 4, or 5, and in which each X is an amino acid residue that is independently chosen.
38 . The fusion protein according to claim 37 , in which each X is chosen from any naturally occurring amino acid except cysteine.
39 . The fusion protein according to claim 37 , in which each X is chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), and isoleucine (I).
40 . The fusion protein of claim 37 , in which n is 1, 2, 4, or 5.
41 . The fusion protein according to claim 37 , 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, with the remaining amino acid residue X being independently chosen from any naturally occurring amino acid except cysteine; or
n=2 or 3 in which all but one X=Ala or Gly, with the remaining amino acid residue X being independently chosen from any naturally occurring amino acid except cysteine.
42 . The fusion protein according to claim 41 , in which n=1 or n=2.
43 . A pharmaceutical composition comprising a fusion protein according to claim 37 and at least one suitable carrier, diluent or excipient.
44 . A fusion protein comprising at least two immunoglobulin single variable domains (ISV), wherein one of the at least two ISVs is at the C-terminal end of the fusion protein, wherein the ISV at the C-terminal end of the fusion protein is a VHH, a humanized VHH, a VH, or a camelized VH that: does not bind to serum albumin; has a valine at Kabat position 11; and has a C-terminal end of the sequence VTVSS(X)n (SEQ ID NO: 34), in which n is 1, 2, 3, 4, or 5, and in which each X is an amino acid residue that is independently chosen.
45 . The fusion protein according to claim 44 , in which each X is chosen from any naturally occurring amino acid except cysteine.
46 . The fusion protein according to claim 44 , in which each X is chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), and isoleucine (I).
47 . The fusion protein of claim 44 , in which n is 1, 2, 4, or 5.
48 . The fusion protein according to claim 44 , 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, with the remaining amino acid residue X being independently chosen from any naturally occurring amino acid except cysteine; or
n=2 or 3 in which all but one X=Ala or Gly, with the remaining amino acid residue X being independently chosen from any naturally occurring amino acid except cysteine.
49 . The fusion protein according to claim 48 , in which n=1 or n=2.
50 . A pharmaceutical composition comprising a fusion protein according to claim 44 and at least one suitable carrier, diluent or excipient.
51 . A fusion protein comprising at least two immunoglobulin single variable domains (ISV), wherein one of the at least two ISVs is at the C-terminal end of the fusion protein, wherein the ISV at the C-terminal end of the fusion protein is a VHH, a humanized VHH, a VH, or a camelized VH that: does not bind to serum albumin; and has a C-terminal end of the sequence VTVSS(X)n (SEQ ID NO: 34), in which n is 1, 2, 3, 4, or 5, and in which each X is an amino acid residue that is independently chosen, except with the proviso that when n is 3, each X is chosen from the group consisting of glycine (G), valine (V), leucine (L), and isoleucine (I).
52 . The fusion protein according to claim 51 , in which when n is 1, 2, 4, or 5, each X is chosen from any naturally occurring amino acid except cysteine.
53 . The fusion protein according to claim 51 , in which each X is chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), and isoleucine (I).
54 . The fusion protein according to claim 51 , in which:
n=1 or 2 in which each X=Ala or Gly; or
n=1 or 2 in which each X=Ala; or
n=1, 2 or 3 in which each X=Gly; or
n=2 in which at least one X=Ala or Gly, with the remaining amino acid residue X being independently chosen from any naturally occurring amino acid except cysteine; or
n=2 in which all but one X=Ala or Gly, with the remaining amino acid residue X being independently chosen from any naturally occurring amino acid except cysteine.
55 . The fusion protein according to claim 51 , in which n=1 or n=2.
56 . A pharmaceutical composition comprising a fusion protein according to claim 51 and at least one suitable carrier, diluent or excipient.