Note: Descriptions are shown in the official language in which they were submitted.
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PEPTIDES CAPABLE OF BINDING TO SERUM ALBUMIN AND COMPOUNDS,
CONSTRUCTS AND POLYPEPTIDES COMPRISING THE SAME
Field of the Invention
The present invention relates to amino acid sequences that are capable of
binding to
serum proteins; to peptides that comprise or essentially consist of such amino
acid sequences;
to compounds and constructs (such as fusion proteins and polypeptides) that
comprise such
amino acid sequences; to nucleic acids that encode such amino acid sequences,
peptides,
fusion proteins or polypeptides; to compositions, and in particular
pharmaceutical
compositions, that comprise such amino acid sequences, peptides constructs,
compounds,
fusion proteins or polypeptides; and to uses of such amino acid sequences,
peptides
constructs, compounds, fusion proteins or polypeptides.
Other aspects, embodiments, advantages and applications of the invention will
become clear from the further description herein.
Background of the Invention
The International application WO 09/127691 entitled "Peptides capable of
binding to
serum proteins and compounds, constructs and polypeptides comprising the same"
describes
a class of peptides that are capable of binding to serum albumin, which
peptides can be linked
or fused to a therapeutic moiety, compound, protein or other therapeutic
entity in order to
increase the half-life thereof.
For a description of the peptides disclosed in WO 09/127691, reference is made
to the
specification, claims, figures and sequence listing of WO 09/127691, which are
incorporated
herein by reference. By means of illustration (and not by means of limitation
either of WO
09/127691 or the present application), some of the exemplary peptides
described in WO
09/127691 may for example contain one or more of the following features:
(i) an Arg (R) residue, in particular an Arg (R) residue that is capable of
forming a
hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135 of human
serum albumin and/or capable of forming electrostatic interactions with the
main-chain
oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum
albumin;
and/or
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(ii) a Trp (W) residue, in particular a Trp (W) residue that is capable of
forming
electrostatic interactions with the Arg (R) 138 residue of human serum
albumin; and/or
(iii) the sequence motif GGG;
and preferably at least any two and more preferably all three of (i), (ii) and
(iii)..
In particular, some of the exemplary peptides described in WO 09/127691 may
for
example contain one or more of the following features-
(i) the sequence motif RXWD, in which X may be any amino acid sequence but is
preferably W, Y, F, S or D; and/or
(ii) the sequence motif GGG, preferably the sequence motif FGGG (SEQ ID NO:6),
more
preferably the sequence motif DVFGGG (SEQ ID NO: 15), and in particular the
sequence motif DVFGGGT (SEQ ID NO:19);
and most preferably both these sequence motifs (i) and (ii).
Also, although it is stated in WO 09/127691 that this application is not
limited to any
specific (or complete) explanation or hypothesis on where (i.e. to which
epitope) and how
(i.e. via which amino acid residues) the amino acid sequences from WO
09/127691 bind to
human serum albumin, it is mentioned that, from the crystal structure and
modeling data, a
number of observations have been made regarding the binding interaction of one
of the
peptides from WO 09/127691 (SEQ ID NO, 143 in WO 09/127691) and human serum
albumin. These are described in Example 8 on pages 84-89 of WO 09/127691, and
again
incorporated herein by reference.
The International application WO 08/068280 entitled "Peptides capable of
binding to
serum proteins" describes methods for generating peptides that are capable of
binding to
serum proteins, which peptides can be liniked or fused to a therapeutic
moiety, compound,
protein or other therapeutic entity in order to increase the half-life
thereof. SEQ ID NO:1 is
an example of a peptide that has been generated using the methodology
described in WO
08/068280 (mentioned herein as a reference).
Summary of the Invention
It is an object of the present invention to provide amino acid sequences with
improved
properties compared to the amino acid sequences described in WO 08/068280 and
in WO
09/12769 1. In particular, it is an object of the invention to provide amino
acid sequences that:
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- bind better (as defined herein) to human serum albumin than the amino acid
sequences
described in WO 08/068280 (and in particular, better than the amino acid
sequence from
SEQ ID NO:1, which is taken from WO 08/068280) and in WO 09/127691 (and in
particular, better than 59F2 (WO 09/127691: SEQ ID NO: 149/ SEQ ID NO, 76
herein);
59H12 (WO 09/1.27691: SEQ ID NO: 1551 SEQ ID NO: 77 herein); and/or 59C2 (WO
09/127691: SEQ ID NO: 156/SEQ ID NO: 75 herein);
and/or
- can specifically bind (as defined herein) to human serum albumin and that
can also
specifically bind serum albumin from at least one other species of mammal
(such as
serum albumin from a mouse, rat, rabbit, dog or a species of primate such as
baboon or
rhesus monkey), and in particular can specifically bind to human serum albumin
and to
serum albumin from cynom.olgus monkey;
and/or
- can bind to (human) serum albumin and that have other improved properties
for
pharmaceutical use compared to the described in WO 08/068280 and in WO
09/127691,
such as improved stability, improved protease resistance, etc.;
and/or
- when linked or fused to a therapeutic moiety, compound, protein or other
therapeutic
entity provide a greater increase of the serum half-life or other
pharmacologically relevant
properties than the amino acid sequences described in WO 08/068280 and in WO
09/127691 (when linked or fused to the same therapeutic).
It is also an object of the invention to provide amino acid sequences that can
be linked
or fused to a therapeutic moiety, compound, protein or other therapeutic
entity, such that the
resulting compound or construct has an improved half-life compared to a
corresponding
compound or construct that contains one of the amino acid sequences described
in WO
08/068280 and in WO 09/127691.
It is an object of the present invention to provide amino acid sequences that
are an
alternative, and in particular an improved alternative, to the serum protein-
binding amino acid
sequences described in WO 08/068280 and in WO 09/127691.
Generally, the invention achieves this objective by providing the amino acid
sequences described herein. These amino acid sequences can bind to (and in
particular,
specifically bind to, as defined herein) serum albumin (and in particular to
human serum
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albumin) and can be used as small peptides or as peptide moieties for linking
or fusing to a
therapeutic compound (such as a therapeutic protein or polypeptide) in order
to increase the
half-life thereof These amino acid sequences (which are also referred to
herein as "amino
acid sequences of the invention") are as further defined herein.
In particular, according to the present invention, it has been found that the
serum-
albumin binding peptides described in WO 08/068280 and in WO 09/127691 can be
further
improved by providing (a stretch of) one or more amino acid residues (or
combination of one
or more amino acid residues) towards the N-terminal end of the peptides
described in WO
09/127691. These one or more amino acid residues (or combination of one or
more amino
acid residues) towards the N-terminal end of the peptides may be as further
described herein.
Thus, the amino acid sequences of the invention are generally based on the
peptides
described in WO 09/127691, but with, in addition to the amino acid residues
and/or sequence
motif described in WO 09/127691, (a stretch of) said one or more amino acid
residues (or
combination of one or more amino acid residues) towards the N-terminal end of
the peptide,
as further described herein.
In particular, in the amino acid sequences of the invention, (this stretch of)
these one
or more amino acid residues (or combination of one or more amino acid
residues) may be
provided towards the N-terminal end of the peptide, relative to position 3
(numbering as
described in WO 09/127691 and further herein) of the peptides described in WO
09/127691.
This is also referred to herein as being "upstream" of said position 3.
For example, (this stretch of) these amino acids may be provided towards the N-
terminal end of the peptide relative to the position of the Arg (R) residue
that is (preferably)
present at position 3 in the peptides described in WO 09/127691 (which, as
described in WO
09/127691, may in particular be an Arg (R) residue that is capable of forming
a hydrogen
bond with the amino acid residues Asn (N) 133 & Asn (N) 135 of human serum
albumin
and/or capable of forming electrostatic interactions with the main-chain
oxygen atoms of the
Pro (P) 134 and Leu (L) 136 residues of human serum albumin). If such an Arg
residue is
present, in the present specification and claims, said one or more amino acid
residues (or
combination of one or more amino acid residues) are also said to be "upstream"
of said Arg
(R) residue.
Where the amino acid sequences of the invention contain an RXWD motif (which
is
preferred for the amino acid sequences of the invention, as it is for the
peptides described in
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09/127691), said one or more amino acid residues (or combination of one or
more amino acid
residues) may again be (and is preferably) provided towards the N-terminal end
of the
peptide, relative to said RXWD motif. Again, if such a motif is present, in
the present
specification and claims, said one or more amino acid residues (or combination
of one or
more amino acid residues) are also said to be "upstream" of said RXWD motif.
Also, when a stretch of amino acid residues is said herein to be "upstream" of
a
residue (such as the R at position 3), a position (such as position 3) or a
motif (such as the
RXWD motif), said stretch of amino acids is preferably directly precedes (i.e.
is immediately
linked to and essentially contiguous with) said residue, position or motif
(although it is not
excluded that there are 1, 2 or 3 amino acid residues in between the stretch
and the
residue/position/motif). Preferably, the last amino acid of the stretch of
amino acids
"upstream of position 3" is at position 0, 1 or 2 (numbering as below), and
preferably at
position 2.
Thus, generally, the stretch of amino acids described herein that provides the
improved properties to the amino acid sequences of the invention (compared to
the serum-
albumin binding peptides described in WO 08/068280 and in WO 09/127691) is
generally
upstream of position 3 (numbering as shown below, which corresponds to the
numbering
used in WO 09/127691).
By means of illustration, the terms "towards the N-terminal end" and
"upstream" are
explained by means of the following amino acid sequence of the invention
(clone EXPGMP
89D03, SEQ ID NO:72)
WWEQDRDWDFDVFGGGTP
As can be seen, this amino acid sequence of the invention contains an RXWD
motif
(underlined), as well as a DVFGGG motif (SEQ ID NO: 15; also underlined),
which is located
"towards the C-terminal end" or "downstream" of the RXWD motif. This amino
acid
sequence also the invention also contains (by means of illustration) an
example of some of
the one or more amino acid residues that provide the amino acid sequences of
the invention
with improved properties (as described herein) compared to the peptides
described in WO
09/127691. These exemplary amino acid residues (WWEQD; SEQ TD NO:96) are said
to be
"towards the N-terminal end" or "upstream" of the RXWD motif (it should also
be noted that,
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as described in WO 09/127691) the Arg (R) residue referred to in the preceding
paragraphs
is usually the Arg (R) residue that is present in the sequence motif RXWD).
As can also be seen from this example, one or more of the amino acid residues
upstream of the RXWD motif that, in the amino acid sequence of the invention,
provides for
the improved properties (i.e. compared to the peptides described in WO
09/127691.) may also
replace one or more of the amino acid residues that, in the peptides described
in WO
09/127691, are upstream of the RXWD motif (which, for example, in the sequence
of SEQ
ID NO: 143 from WO 09/127691 are three alanine residues, AAA).
In the present invention, the same numbering for the amino acid residues is
used as in
WO 09/127691 and in WO 08/068280. This numbering is further described in WO
09/127691 and in WO 08/068280, and is further used and illustrated throughout
the present
description. According to this numbering, the Arg (R) residue is at position
3, and the amino
acid residues upstream of the Arg (R) residues are at positions I and 2.
Reference is for
example made to Table 1 on page 15 of page WO 09/127691 (As mentioned there,
instead of
the preferred Arg residue at position 3, the peptides of WO 09/127691 may
instead contain L,
F, Y, W, P, T. S, M, A, D, 1, K, Q or V at position 3, and the same applies to
the amino acid
sequences of the invention).
It should also be noted that the amino acid sequences of the invention usually
contain
more than two amino acid residues upstream of position 3. With reference to
the exemplary
sequence of SEQ ID NO: 1 above, these amino acid residues are numbered as
follows.
Amino acid W W E Q D R D W D F D V ...
residue
Position [etc.] -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 [etc.].
"Upstream" of position 3 << 3 >> "Downstream" of position 3
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In order to further illustrate and clarify this numbering of amino acid
residues in the
present specification and the amino acid sequences of the invention, Figure
III is a table
showing an alignment of the following amino acid sequences: SEQ ID NO:I
(reference),
59C2 (SEQ ID NO: 75, reference), 80B10 (SEQ ID NO: 56, invention); 89D03 (SEQ
ID
NO:72, invention), 89D03V 1 (SEQ ID NO: 103, invention), 89D03 V 1 VG (SEQ ID
NO:106,
invention) and 89D03V3 (SEQ ID NO: 105, invention). Where a box is left open,
no amino
acid residue is present in the sequences that are exemplified in this table.
Generally, the amino acid sequences of the invention may contain, upstream of
position 3, a stretch of amino acid residues of between 2 and 10 amino acid
residues, which
comprises at least one hydrophobic and/or aromatic amino acid residue (and for
the
remainder one or more further suitable amino acid residues, as for example
exemplified
herein).
In particular, said at least one hydrophobic amino acid residue may be chosen
from L,
I, V and/or M and/or said at least one aromatic amino acid residue may be
chosen from W, Y
and/or F. For the remainder (i.e. at position 3 and further downstream from
position 3), the
amino acid sequences of the invention may be as described in WO 09/127691
(with the same
preferred aspects and features applying).
In particular, the amino acid sequences of the invention may contain, upstream
of
position 3, a stretch of amino acid residues of between 2 and 10 amino acid
residues, which
comprises at least one hydrophobic and/or aromatic amino acid residue such
that at least one
of said hydrophobic and/or aromatic amino acid residues can bind (in)to a
subpocket in
(human) serum albumin that comprises (at least) one or more of the following
amino acid
residues of human serum albumin: V442, S443, T446, L484, L487, H488, K490,
T491 and/or
V493 (numbering as described in Example 8 of WO 09/127691 ). For the remainder
(i.e. at
position 3 and further downstream from position 3), the amino acid sequences
of the
invention may be as described in WO 09/127691 (with the same preferred aspects
and
features applying).
In one non-limiting aspect, the amino acid sequences of the invention may
contain,
upstream of position 3, a stretch of amino acid residues of between 2 and 10
amino acid
residues, which comprises (i) at least two hydrophobic amino acid residues;
(ii) at least two
aromatic amino acid residues; and/or (iii) at least one hydrophobic amino acid
residue and at
least aromatic amino acid residue. Again, said hydrophobic amino acid
residue(s) may be
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chosen from L, I, V and/or M and/or said aromatic amino acid residue(s) may be
chosen from
W. Y and/or F. For the remainder (i.e. at position 3 and further downstream
from position 3),
the amino acid sequences of the invention may be as described in WO 09/127691
(with the
sate preferred aspects and features applying).
In particular, the amino acid sequences of the invention may contain, upstream
of
position 3, a stretch of amino acid residues of between 2 and 10 amino acid
residues, which
comprises (i) at least two hydrophobic an-iino acid residues; (ii) at least
two aromatic amino
acid residues; and/or (iii) at least one hydrophobic amino acid residue and at
least aromatic
amino acid residue, such that at least one of said hydrophobic and/or aromatic
amino acid
residues can bind (in)to a subpocket in (human) serum albumin that comprises
(at least) one
or more of the following amino acid residues of human serum albumin: V442,
5443, T446,
L484, L487,11488, K490, T491 and/or V493. Again, said hydrophobic amino acid
residue(s)
may be chosen from L, I, V and/or M and/or said aromatic amino acid residue(s)
may be
chosen from W, Y and/or F. For the remainder (i.e. at position 3 and further
downstream
from position 3), the amino acid sequences of the invention may be as
described in WO
09/127691 (with the same preferred aspects and features applying).
The above amino acid sequences preferably comprise at least one hydrophobic
amino
acid residue or aromatic amino acid residue at position -2, -1, 0, 1 and/or 2
(numbering as
described above), and in particular at least one hydrophobic amino acid
residue or aromatic
amino acid residue at position -2, -1 and/or 0.
The amino acid sequences of the invention are further preferably such that
they bind
to human serum albumin (as determined using Biacore) with an affinity
(expressed as Ko)
better than 100 nM, preferably better than 5OnM, more preferably better than
30 nM, such as
equal to or better than 20nM (measured either using the amino acid sequence of
the invention
per se or measured using a fusion of the amino acid sequence of the invention
to another
protein or peptide, such as the Nanobody 2D3 used as an example herein.
Reference is for
example made to Example 2 below).
In one specific, but non-limiting aspect, the amino acid sequences of the
invention
may contain, upstream of position 3, a stretch of amino acid residues of
between 2 and 10
amino acid residues, which comprises at least one W residue and/or at least
one Y residue
(and again, for the remainder one or more further suitable amino acid
residues, as for
example exemplified herein). For the remainder (i.e. at position 3 and further
downstream
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from position 3), the amino acid sequences of the invention may be as
described in WO
09/127691 (with the same preferred aspects and features applying).
In particular, the amino acid sequences of the invention may contain, upstream
of
position 3, a stretch of amino acid residues of between 2 and 10 amino acid
residues, which
comprises at least one W residue and/or at least one Y residue, such that at
least one of said
W or Y residues can bind (in)to a subpocket in (human) serum albumin that
comprises (at
least) one or more of the following amino acid residues of human serum
albumin: V442,
S443, T446, L484, L487,11488, K490, T491 and/or V493.
In another specific, but non-limiting aspect, the amino acid sequences of the
invention
may contain, upstream of position 3, a stretch of amino acid residues of
between 2 and 10
amino acid residues, which comprises (i) at least two W residues; (ii) at
least two Y residues;
and/or (iii) at least one W residue and at least one Y residue (and again, for
the remainder one
or more further suitable amino acid residues, as for example exemplified
herein). For the
remainder (i.e. at position 3 and further downstream from position 3), the
amino acid
sequences of the invention may be as described in WO 09/127691 (with the same
preferred
aspects and features applying).
In particular, the amino acid sequences of the invention may contain, upstream
of
position 3, a stretch of amino acid residues of between 2 and 10 amino acid
residues, which
comprises (i) at least two W residues; (ii) at least two Y residues; and/or
(iii) at least one W
residue and at least one Y residue, such that at least one of said W or Y
residues can bind
(in)to a subpocket in (human) serum albumin that comprises (at least) one or
more of the
following amino acid residues of human serum albumin: V442, S443, T446, L484,
L487,
H488, K490, T491 and/or V493.
Preferably, the amino acid sequences of the invention may contain, upstream of
position 3, a stretch of amino acid residues of between 2 and 10 amino acid
residues which
comprises 1, 2 or 3 W residues. In particular, the amino acid sequences of the
invention may
contain, upstream of position 3, a stretch of amino acid residues of between 2
and 10 amino
acid residues, which comprises 1, 2 or 3 W residues such that at least one of
said W residues
can bind (in)to a subpocket in (human) serum albumin that comprises (at least)
one or more
of the following amino acid residues of human serum albumin: V442, S443, T446.
L484,
L487, H488, K490, T491 and/or V493.
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Generally, the amino acid sequences of the invention may contain, upstream of
position 3, a stretch of amino acid residues comprising 3, 4, 5, 6 or 7 amino
acid residues,
which comprises at least one hydrophobic and/or aromatic amino acid residue
(and for the
remainder one or more further suitable amino acid residues, as for example
exemplified
herein).
In particular, said at least one hydrophobic amino acid residue may be chosen
from L,
I, V and/or M and/or said at least one aromatic amino acid residue may be
chosen from W, Y
and/or F. For the remainder (i.e. at position 3 and further downstream from
position 3), the
amino acid sequences of the invention may be as described in WO 09/127691
(with the same
10 preferred aspects and features applying).
In particular, the amino acid sequences of the invention may contain,
upstream. of
position 3, a stretch of amino acid residues comprising 3, 4, 5, 6 or 7 amino
acid residues,
which comprises at least one hydrophobic and/or aromatic amino acid residue at
least one
hydrophobic and/or aromatic amino acid residue such that at least one of said
hydrophobic
and/or aromatic amino acid residues can bind (in)to a subpocket in (human)
serum albumin
that comprises (at least) one or more of the following amino acid residues of
human serum
albumin: V442, 5443, T446, L484, L487, H488, K490, T491 and/or V493. For the
remainder
(i.e. at position 3 and further downstream from position 3), the amino acid
sequences of the
invention may be as described in WO 09/127691 (with the same preferred aspects
and
features applying).
In one non-limiting aspect, the amino acid sequences of the invention may
contain,
upstream of position 3, a stretch of amino acid residues comprising 3, 4, 5, 6
or 7 amino acid
residues, which comprises (i) at least two hydrophobic amino acid residues;
(ii) at least two
aromatic amino acid residues; and/or (iii) at least one hydrophobic amino acid
residue and at
least aromatic amino acid residue. Again, said hydrophobic amino acid
residue(s) may be
chosen from L, I, V and/or M and/or said aromatic amino acid residue(s) may be
chosen from
W, Y and/or F. For the remainder (i.e. at position 3 and further downstream
fTom position 3),
the amino acid sequences of the invention may be as described in WO 09/127691
(with the
same preferred aspects and features applying).
In particular, the amino acid sequences of the invention may contain, upstream
of
position 3, a stretch of amino acid residues comprising 3, 4, 5, 6 or 7 amino
acid residues,
which comprises (i) at least two hydrophobic amino acid residues; (ii) at
least two aromatic
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amino acid residues; and/or (iii) at least one hydrophobic amino acid residue
and at least
aromatic amino acid residue, such that at least one of said hydrophobic and/or
aromatic
amino acid residues can bind (in)to a subpocket in (human) serum albumin that
comprises (at
least) one or more of the following amino acid residues of human serum
albumin: V442,
S443, T446, L484, L487, H488, K490, T491 and/or V493. Again, said hydrophobic
amino
acid residue(s) may be chosen from L, 1, V and/or M and/or said aromatic amino
acid
residue(s) may be chosen from W, Y and/or F. For the remainder (i.e. at
position 3 and
further downstream from position 3), the amino acid sequences of the invention
may be as
described in WO 09/127691 (with the same preferred aspects and features
applying).
All the amino acid sequences of the invention described herein are preferably
such
that they bind to human serum albumin (as determined using Biacore) with an
affinity
(expressed as KD) better than 100 nM, preferably better than 50 nM, more
preferably better
than 30 nM, such as equal to or better than 20 nM, measured either using the
amino acid
sequence of the invention per se or (preferably) measured using a fusion of
the amino acid
sequence of the invention to another protein or peptide, for example as a
fusion with a
NANOBODY (such as the Nanobody 2D3 used as an example herein).
In one specific, but non-limiting aspect, the amino acid sequences of the
invention
may contain, upstream of position 3, a stretch of amino acid residues
comprising 3, 4, 5, 6 or
7 amino acid residues, which comprises at least one W residue and/or at least
one Y residue
(and again, for the remainder one or more further suitable amino acid
residues, as for
example exemplified herein). For the remainder (i.e. at position 3 and further
downstream
from position 3), the amino acid sequences of the invention may be as
described in WO
09/127691 (with the same preferred. aspects and features applying).
In particular, the amino acid sequences of the invention may contain, upstream
of
position 3, a stretch of amino acid residues comprising 3, 4, 5, 6 or 7 amino
acid residues,
which comprises at least one W residue and/or at least one Y residue, such
that at least one of
said W or Y residues can bind (in)to a subpocket in (human) serum albumin that
comprises
(at least) one or more of the following amino acid residues of human serum
albumin: V442,
S443, T446, L484, L487, H488, K490, T491 and/or V493.
In another specific, but non-limiting aspect, the amino acid sequences of the
invention
may contain, upstream of position 3, a stretch of amino acid residues
comprising 3, 4, 5, 6 or
7 amino acid residues, which comprises (i) at least two W residues; (ii) at
least two Y
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residues; and/or (iii) at least one W residue and at least one Y residue (and
again, for the
remainder one or more further suitable amino acid residues, as for example
exemplified
herein). For the remainder (i.e. at position 3 and further downstream from
position 3), the
amino acid sequences of the invention may be as described in WO 09/127691
(with the same
preferred aspects and features applying).
In particular, the amino acid sequences of the invention may contain, upstream
of
position 3, a stretch of amino acid residues comprising 3, 4, 5, 6 or 7 amino
acid residues,
which comprises (i) at least two W residues; (ii) at least two Y residues;
and/or (iii) at least
one W residue and at least one Y residue, such that at least one of said W or
Y residues can
bind (in)to a subpocket in (human) serum albumin that comprises (at least) one
or more of the
following amino acid residues of human serum albumin: V442, 5443, T446, L484,
L487,
11488, K490, T491 and/or V493.
Preferably, the amino acid sequences of the invention may contain, upstream of
position 3, a stretch of amino acid residues comprising 3, 4, 5, 6 or 7 amino
acid residues
which comprises 1, 2 or 3 W residues. In particular, the amino acid sequences
of the
invention may contain, upstream of position 3, a stretch of amino acid
residues comprising 3,
4, 5, 6 or 7 amino acid residues, which comprises 1, 2 or 3 W residues such
that at least one
of said W residues can bind (in)to a subpocket in (human) serum albumin that
comprises (at
least) one or more of the following amino acid residues of human serum
albumin: V442,
S443, T446, L484, L487, H488, K490, T491 and/or V493.
Again, the above amino acid sequences preferably comprise at least one W or Y
residue at position -2, -1, 0, 1 and/or 2 (numbering as described above), and
in particular at
least one W or Y residue at position -2, -1 and/or 0.
Generally, it should be noted that, in addition to the stretch of amino acid
residues
upstream of position 3 referred to in the above paragraphs, the amino acid
sequences of the
invention may comprise one or more further amino acid residues (further)
upstream of said
stretch of between 2 and 10 (preferably 3, 4, 5, 6 or 7) amino acid residues.
However,
although not excluded from the scope of the invention, this is neither
required nor essential.
Some preferred, but non-limiting examples of suitable stretches of amino acids
upstream of position 3 that can be present in the amino acid sequences of the
invention can be
seen in the peptides listed in Table II below. For example, the part of the
amino acid
sequences upstream from position 3 may be one of the sequences LWYML, LWYLY,
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YWWER, AWYDY, WWNWR, EWWWR, VDWFY, RDWFL, DWWNR, YGDWF,
WWTWG, PIDFW, WWTSD. QKLYW, KWWEI, WWSTP, LFWWE, WEE,
Wes, N LIV, WWELD (see SEQ ID NO's 78 to 98, respectively); or an amino acid
sequence that has 3, 2 or 1 amino acid differences (as defined herein) with
one of these
sequences (provided it still meets the features set out above). The presence
of one of the
underlined sequences, or an amino acid sequence that has 2 or I amino acid
differences (as
defined herein) with one of these sequences (provided it still meets the
features set out
above), is particularly preferred.
The other part of the peptide (at position 3 and further downstream) can in
particular
be as described in WO 09/127691 (with the same preferred aspects and features
applying).
For example, the part of the peptide at position 3 and further downstream may
for example be
the same as, or essentially be the same as, the part of the peptide at
position 3 and further
downstream from one of the sequences of SEQ ID NOs: 2-115 and 147-157 from WO
09/127691 (see Table II in WO 09/127691).
Thus, according to a first aspect, the invention relates to an amino acid
sequence that:
a) has at least 50%, preferably at least 65 %, more preferably at least 70%,
even more
preferably at least 75%, such as at least 80%, such as at least 90%, but not
100%,
sequence identity (as defined herein) with the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
and that:
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
and that:
c) contains a stretch of amino acid residues upstream of position 3 that is as
described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein).
Another aspect of the invention relates to an amino acid sequence that:
a) that has no more than 9, preferably no more than 8, in particular no more
than 7, such
as 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the
amino acid
sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:l);
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: 1);
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and that:
c) contains a stretch of amino acid residues upstream of position 3 that is as
described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein).
In yet another aspect, the invention relates to an amino acid sequence that
a) contains one or more of the following sequence motifs: DYDV (SEQ JD NO:2),
YDVF
(SEQ ID NO:3), DVFG (SEQ ID NO:4), VFGG (SEQ ID NO:5), FGGG (SEQ ID
NO:6) and/or GGGT (SEQ ID NO:7);
b) has a total length of between 5 and 50, preferably between 7 and 40, more
preferably
between 10 and 35, such as about 15, 20, 25 or 30 amino acid residues;
c) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:l);
and that:
d) contains a stretch of amino acid residues upstream of position 3 that is as
described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein);
which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO:1).
In yet another aspect, the invention relates to an amino acid sequence that
a) contains one or more of the following sequence motifs: DYDVF (SEQ ID NO:8),
YDVFG (SEQ ID NO:9), DVFGG (SEQ ID NO:10), VFGGG (SEQ ID NO.- 11) and/or
FGGGT (SEQ ID NO: 12);
b) has a total length of between 5 and 50, preferably between 7 and 40, more
preferably
between 10 and 35, such as about 15, 20, 25 or 30 amino acid residues;
c) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
and that:
d) contains a stretch of amino acid residues upstream of position 3 that is as
described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein);
which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO: 1). Preferably, such an amino acid sequence is as further described
herein. For example,
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it preferably also comprises a sequence motif RXWD (i.e, upstream of the
aforementioned
sequence motif, e.g. at positions 3 to 6), as further described herein.
As further described herein, it may be possible in the above amino acid
sequences of
the invention to replace the aspartate (D) residue at position 8 by a
threonine (T) residue (for
example, but without limitation, in amino acid sequences of the invention that
do not contain
a threonine residue at position 14). In such a case, such an amino acid
sequence may be as
described above, but may instead of the sequence motif DYDVF (SEQ ID NO:8),
YDVFG
(SEQ ID NO:9), DVFGG (SEQ ID NO, 10) contain the sequence motif DYTVF (SEQ ID
NO:126), YTVFG (SEQ ID NO:127) or TVFGG (SEQ ID NO:128), respectively. Such
10 amino acid sequences may be as further described herein, and may according
to a specific but
non-limiting aspect contain an amino acid residue at position 14 other than
threonine (for
example, A, N and in particular D).
In yet another aspect, the invention relates to an amino acid sequence that
a) contains one or more of the following sequence motifs: DYDVFG (SEQ ID NO:
13),
YDVFGG (SEQ ID NO:14), DVFGGG (SEQ ID NO:15) and/or VFGGGT (SEQ ID
NO:16);
b) has a total length of between 6 and 50, preferably between 7 and 40, more
preferably
between 10 and 35, such as about 15, 20, 25 or 30 amino acid residues;
c) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: I);
and that:
d) contains a stretch of amino acid residues upstream. of position 3 that is
as described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein);
which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO:1). Preferably, such an amino acid sequence is as further described herein.
For example,
it preferably also comprises a sequence motif RXWD (i.e. upstream of the
aforementioned
sequence motif, e.g. at positions 3 to 6), as further described herein.
As further described herein, it may be possible in the above amino acid
sequences of
the invention. to replace the aspartate (D) residue at position 8 by a
threonine (T) residue (for
example, but without limitation, in amino acid sequences of the invention that
do not contain
a threonine residue at position 14). In such a case, such an amino acid
sequence may be as
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described above, but may instead of the sequence motif DYDVFG (SEQ ID NO: 13),
YDVFGG (SEQ ID NO: 14) or DVFGGG (SEQ ID NO: 15) contain the sequence motif
DYDVFG (SEQ ID NO:129), YDVFGG (SEQ ID NO: 1.30) or TVFGGG (SEQ ID NO:131),
respectively. Such amino acid sequences may be as further described herein,
and may
according to a specific but non-limiting aspect contain an amino acid residue
at position 14
other than threonine (for example, A, N and in particular D).
In yet another aspect, the invention relates to an amino acid sequence that
a) contains one or more of the following sequence motifs: DYDVFGG (SEQ ID
NO:17),
YDVFGGG (SEQ ID NO:18) and/or DVFGGGT (SEQ ID NO:19);
b) has a total length of between 7 and 50, preferably between 8 and 40, more
preferably
between 10 and 35. such as about 15, 20, 25 or 30 amino acid residues;
c) binds better to human. serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
and that:
d) contains a stretch of amino acid residues upstream of position 3 that is as
described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein);
which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO:I). Preferably, such an amino acid sequence is as further described herein.
For example,
it preferably also comprises a sequence motif RXWD (i.e. upstream of the
aforementioned
sequence motif. e.g. at positions 3 to 6), as further described herein.
As further described herein, it may be possible in the above amino acid
sequences of
the invention to replace the aspartate (D) residue at position 8 by a
threonine (T) residue (for
example, but without limitation, in amino acid sequences of the invention that
do not contain
a threonine residue at position 14). In such a case, such an amino acid
sequence may be as
described above, but may instead of the sequence motif DYDVFGG (SEQ ID NO:
17),
YDVFGGG (SEQ ID NO: 18) or DVFGGGT (SEQ ID NO. 19) contain the sequence motif
DYTVFGG (SEQ ID NO:132), YTVFGGG (SEQ ID NO:133) or TVFGGGT, (SEQ ID
NO: 134), respectively. Such amino acid sequences may be as further described
herein.
Also, as further described herein, it may be possible in the above amino acid
sequences of the invention to replace the threonine (T) residue at position 14
with another
amino acid residue, such as (for example and without limitation) A, N or D. In
such a case,
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such an amino acid sequence may be as described above, but may for example
instead of the
sequence motif DVFGGGT (SEQ ID NO: 19) contain the sequence motif DVFGGGA (SEQ
ID NO:135), DVFGGGN (SEQ ID NO: 136) or DVFGGGD (SEQ ID NO:137).
It is also possible that both the aspartate (D) residue at position 8 is
replaced by a
threonine (T) residue, and that the threonine residue at position 14 is
replaced by another
amino acid (for example, but without limitation, A, N and in particular D). In
such a case,
such an amino acid sequence may be as described above, but may for example
instead of the
sequence motif DVFGGGT (SEQ ID NO: 19) contain a sequence motif TVFGGGA (SEQ
ID
NO: 138), TVFGGGN (SEQ ID NO:139) or TVFGGGD (SEQ ID NO: 140).
In yet another aspect, the invention relates to an amino acid sequence that
a) contains one or more of the following sequence motifs: DYDVFGGG (SEQ ID
NO:20)
and/or YDVFGGGT (SEQ ID NO:21);
b) has a total length of between 8 and 50, preferably between 9 and 40, more
preferably
between 10 and 35, such as about 15, 20, 25 or 30 amino acid residues;
c) binds better to human serum albumin than. the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
and that:
d) contains a stretch of amino acid residues upstream of position 3 that is as
described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein);
which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO: 1). Preferably, such an amino acid sequence is as further described
herein.
In yet another aspect, the invention relates to an amino acid sequence that
a) contains the following sequence motif: DYDVFGGGT (SEQ ID NO:23);
b) has a total length of between 9 and 50, preferably between 9 and 40, more
preferably
between 10 and 35, such as about 15, 20, 25 or 30 amino acid residues;
c) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: I);
and that:
d) contains a stretch. of amino acid residues upstream of position 3 that is
as described
herein (including the preferred aspects of such a stretch of amino acid
residues, also as
described herein);
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which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO: 1). Preferably, such an amino acid sequence is as further described
herein. For example,
it preferably also comprises a sequence motif RXWD (i.e. upstream of the
aforementioned
sequence motif e.g. at positions 3 to 6), as further described herein.
As further described herein, it may be possible in the above amino acid
sequences of
the invention to replace the aspartate (D) residue at position 8 to a
threonine (T) residue (for
example, but without limitation, in amino acid sequences of the invention that
do not contain
a threonine residue at position 14). In such a case, such an amino acid
sequence may be as
described above, but may instead of the sequence motif DYTVFGGG (SEQ ID NO:20)
and/or YDVFGGGT (SEQ ID NO:21) contain the sequence motif DYTVFGGG (SEQ ID
NO:141) or YTVFGGGT (SEQ ID NO:142), respectively.
Also, as further described herein, it may be possible in the above amino acid
sequences of the invention to replace the threonine (T) residue at position 14
with another
amino acid residue, such as (for example and without limitation) A, N or D. In
such a case,
such an amino acid sequence may be as described above, but may for example
instead of the
sequence motif YDVFGGGT (SEQ ID NO:21) contain the sequence motif YDVFGGGA
(SEQ ID NO:143), YDVFGGGN (SEQ ID NO:144) or YDVFGGGD (SEQ ID NO:145).
It is also possible that both the aspartate (D) residue at position 8 is
replaced by a
threonine (T) residue, and that the threonine residue at position 14 is
replaced by another
amino acid (for example, but without limitation, A, N and in particular D). In
such a case,
such an amino acid sequence may be as described above, but may for example
instead of the
sequence motif YDVFGGGT (SEQ ID NO:21) contain a sequence motif YTVFGGGA (SEQ
ID NO, 146), YTVFGGGN (SEQ ID NO:147) or YTVFGGGD (SEQ ID NO:148).
The amino acid sequences of the invention (as further described herein)
preferably (at
least) contain:
(i) an Arg (R) residue, in particular an Arg (R) residue that is capable of
forming a
hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135 of human
serum albumin and/or capable of forming electrostatic interactions with the
main-chain
oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum
albumin;
and/or
(ii) a Trp (W) residue, in particular a Trp (W) residue that is capable of
forming
electrostatic interactions with the Arg (R) 138 residue of human serum
albumin; and/or
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(iii) the sequence motif GGG;
and preferably at least any two and more preferably all three of (i), (ii) and
(iii); and in
addition contain, upstream of said Arg residue, at least one hydrophobic
and/or aromatic
amino acid residue such that said at least one of said hydrophobic and/or
aromatic amino acid
residues can bind (in)to a subpocket in (human) serum albumin that comprises
(at least) one
or more of the following amino acid residues of human serum albumin: V442,
S443, T446,
L484, L487, H488, K490, 7491 and/or V493 (numbering as described in Example 8
of WO
09/127691 ). In particular, the amino acid sequences of the invention may
contain a stretch of
amino acid residues upstream of said Arg residue that is as described herein
(including the
preferred aspects of such a stretch of amino acid residues, also as described
herein).
The amino acid sequences of the invention (as further described herein)
preferably (at
least) contain:
(iii) the sequence motif RXWD, in which X may be any amino acid sequence but
is
preferably W, Y, F, S or D; and/or
(iv) the sequence motif GGG, preferably the sequence motif FGGG (SEQ ID NO:6),
more
preferably the sequence motif DVFGGG (SEQ ID NO:15) (or alternatively, the
sequence motif TVFGGG (SEQ ID NO: 131), and in particular the sequence motif
DVFGGGT (SEQ ID NO: 19) (or alternatively one of the sequence motifs of SEQ ID
NO's: 134 to 139);
and most preferably both these sequence motifs (i) and (ii); upstream of said
RXWD motif,
at least one hydrophobic and/or aromatic amino acid residue such that said at
least one of said
hydrophobic and/or aromatic amino acid residues can bind (in)to a subpocket in
(human)
serum albumin that comprises (at least) one or more of the following amino
acid residues of
human serum albumin: V442. 5443. T446, L484, L487, H488, K490, T491 and/or
V493
(numbering as described in Example 8 of WO 09/127691 ). In particular, the
amino acid
sequences of the invention may contain a stretch of amino acid residues
upstream of said
RXWD motif that is as described herein (including the preferred aspects of
such a stretch of
amino acid residues, also as described herein).
Again, all the amino acid sequences of the invention as described herein are
preferably such that they bind to human serum albumin (as determined using
Biacore) with
an affinity (expressed as KD) better than 100 nM, preferably better than 5OnM,
more
preferably better than 3 0 nM, such as equal to or better than 20nM, measured
either using the
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amino acid sequence of the invention per se or (preferably) measured using a
fusion of the
amino acid sequence of the invention to another protein or peptide, for
example as a fusion
with a NANOBODY (such as the Nanobody 2D3 used as an example herein).
Instead of the sequence motif DVFGGG (SEQ ID NO:15), an preferred amino acid
sequence of the invention may for example also contain the sequence motif
DAFGGG (SEQ
ID NO:44). Similarly, in sequence motifs with a threonine (T) on position 8
(for example,
one of the sequence motifs of SEQ ID NO's: 126 to 148), the valine (V) at
position 9 may be
replaced by an alanine (A), to provide - as a non-limiting example - the
sequence motif
TAFGGG (SEQ ID NO: 149) i.e. instead of either the sequence motif DVFGGG (SEQ
ID
10 NO: 15) or the sequence motif TVFGGG (SEQ ID NO: 131). Also, instead of the
sequence
motif DVFGGGT (SEQ ID NO:19). a preferred amino acid sequence of the invention
may
for example also contain the sequence motifs DVFGGGS (SEQ ID NO:45) or DAFGGGT
(SEQ ID NO:46). Other similar sequence motifs that may be present in the amino
acid
sequences of the invention will be clear to the skilled person based on the
disclosure herein
(such as the sequences mentioned in Table II ).
Thus, in another aspect, the invention relates to an amino acid sequence that:
a) has at least 50%, preferably at least 65 %, more preferably at least 70%,
even more
preferably at least 75%, such as at least 80%, such as at least 90%, but not
100%,
sequence identity (as defined herein) with the amino acid sequence
20 AASYSDYDVFGGGTDFGP (SEQ ID NO:I);
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
c) comprises an Arg (R) residue, in particular an Arg (R) residue that is
capable of
forming a hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135
of
human serum albumin and/or capable of forming electrostatic interactions with
the
main-chain oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human
serum
albumin;
and that
d) contains, upstream of said Arg residue, at least one hydrophobic and/or
aromatic
amino acid residue such that said at least one of said hydrophobic and/or
aromatic
amino acid residues can bind (in)to a subpocket in (human) serum albumin that
comprises (at least) one or more of the following amino acid residues of human
serum
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albumin: V442. S443, T446, L484, L487, H488, K490, T491 and/or V493 (numbering
as described in Example 8 of WO 09/127691 ).
In particular, such an amino acid sequences of the invention may contain a
stretch of
amino acid residues upstream of said Arg residue that is as described herein
(including the
preferred aspects of such a stretch of amino acid residues, also as described
herein).
This amino acid sequence preferably also comprises (i) the sequence motif
RXWD, in
which X may be any amino acid sequence but is preferably W, Y, F, S or D;
and/or (ii) the
sequence motif GGG, preferably the sequence motif FGGG (SEQ ID NO:6), more
preferably
the sequence motif DVFGGG (SEQ ID NO: IS) (or alternatively, the sequence
motif
TVFGGG (SEQ ID NO: 131), and in particular the sequence motif DVFGGGT (SEQ ID
NO: 19) (or alternatively one of the sequence motifs of SEQ ID NO's: 134 to
139); and most
preferably both these sequence motifs.
The above amino acid sequence is also preferably as further described herein.
In another aspect, the invention relates to an amino acid sequence that:
a) has at least 50%, preferably at least 65 %, more preferably at least 70%,
even more
preferably at least 75%, such as at least 80%, such as at least 90%, but not
100%,
sequence identity (as defined herein) with the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: 1);
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
c) comprises a Trp (W) residue, in particular a Trp (W) residue that is
capable of forming
electrostatic interactions with the Arg (R) 138 residue of human serum
albumin;
and that
d) contains a stretch of amino acid residues upstream of position 3 (and/or
said W residue)
that is as described herein (including the preferred aspects of such a
stretch. of amino
acid residues, also as described herein).
This amino acid sequence preferably also comprises (i) the sequence motif
RXWD, in
which X may be any amino acid sequence but is preferably W, Y, F, S or D;
and/or (ii) the
sequence motif GGG, preferably the sequence motif FGGG (SEQ ID NO:6), more
preferably
the sequence motif DVFGGG (SEQ ID NO: 15) (or alternatively, the sequence
motif
TVFGGG (SEQ ID NO: 131), and in particular the sequence motif DVFGGGT (SEQ ID
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NO:19) (or alternatively one of the sequence motifs of SEQ ID NO's: 134 to
139); and most
preferably both these sequence motifs.
The above amino acid sequence is also preferably as further described herein.
In another aspect, the invention relates to an amino acid sequence that:
a) has at least 50%, preferably at least 65 %, more preferably at least 70%,
even more
preferably at least 75%, such as at least 80%, such as at least 90%, but not
100%,
sequence identity (as defined herein) with the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:1);
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: I);
c) comprises an Arg (R) residue, in particular an Arg (R) residue that is
capable of
forming a hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135
of
human serum albumin and/or capable of forming electrostatic interactions with
the
main-chain oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human
serum
albumin;
d) comprises a Trp (W) residue, in particular a Trp (W) residue that is
capable of forming
electrostatic interactions with the Arg (R) 138 residue of human serum
albumin;
and that
e) contains , upstream of said Arg residue, at least one hydrophobic and/or
aromatic
amino acid residue such that said at least one of said hydrophobic and/or
aromatic
amino acid residues can bind (in)to a subpocket in (human) serum albumin that
comprises (at least) one or more of the following amino acid residues of human
serum
albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493 (numbering
as described in Example 8 of WO 09/127691 ).
In particular, such an amino acid sequences of the invention may contain a
stretch of
amino acid residues upstream of said Arg residue that is as described herein
(including the
preferred aspects of such a stretch of amino acid residues, also as described
herein).
This amino acid sequence preferably also comprises (i) the sequence motif
RXWD, in
which X may be any amino acid sequence but is preferably W, Y, F, S or D;
and/or (ii) the
sequence motif GGG, preferably the sequence motif FGGG (SEQ ID NO:6), more
preferably
the sequence motif DVFGGG (SEQ ID NO: 15) (or alternatively, the sequence
motif
TVFGGG (SEQ ID NO:131), and in particular the sequence motif DVFGGGT (SEQ ID
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NO:] 9) (or alternatively one of the sequence motifs of SEQ ID NO's: 134 to
139); and most
preferably both these sequence motifs.
Again, these amino acid sequences are also preferably as further described
herein. For
example, the above amino acid sequences of the invention are again preferably
such that they
bind to human serum albumin (as determined using Biacore) with an affinity
(expressed as
KD) better than 100 nM, preferably better than 5OnM, more preferably better
than 30 nM,
such as equal to or better than 20nM, measured either using the amino acid
sequence of the
invention per se or (preferably) measured using a fusion of the amino acid
sequence of the
invention to another protein or peptide, for example as a fusion with a
NANOBODY (such.
as the Nanobody 2D3 used as an example herein).
Some preferred, but non-limiting sequence motifs that may be present in the
amino
acid sequences of the invention are:
- a stretch of amino acid residues upstream of position 3 that is as described
herein
(including the preferred aspects of such a stretch of amino acid residues,
also as
described herein). This may for example be one of the sequences of SEQ ID NO's
78 to
98 (or a sequence that has 2 or only I "amino acid difference" - as defined
herein -
with at least one of these sequences, provided they (preferably) still meet
the
(preferred) aspects set out herein).
- the amino acid sequence RXWDXDVFGGG (SEQ ID NO, 23), in which the first
(from
the N-terminal end) amino acid residue indicated by X is chosen from Y, S or
D; and
the second amino acid residue indicated by X is chosen from Y or F.
- the amino acid sequence RXWDXDVFGGGT (SEQ ID NO: 24), in which the first
(from the N-terminal end) amino acid residue indicated by X is chosen from Y,
S or D;
and the second amino acid residue indicated by X is chosen from Y or F.
- the amino acid sequence RXWDXDVFGGGTF (SEQ ID NO: 25), in which the first
(from the N-terminal end) amino acid residue indicated by X is chosen from Y,
S or D;
and the second amino acid residue indicated. by X is chosen from Y or F.
- the amino acid sequence RXWDXDVFGGGTPG (SEQ ID NO: 26), in which the first
(from the N-terminal end) amino acid residue indicated by X is chosen from Y,
S or D;
and the second amino acid residue indicated by X is chosen from Y or F.
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24
- the amino acid sequence RX.WDXDVFGGGTPGG (SEQ ID NO: 27), in which the first
(from the N-terminal end) amino acid residue indicated by X is chosen from Y,
S or D;
and the second amino acid residue indicated by X is chosen from Y or F.
an amino acid sequence chosen from RYWDYDVFGGG (SEQ ID NO: 28);
RDWDFDVFGGG (SEQ ID NO: 29); RSWDFDVFGGG (SEQ ID NO: 30) or
RYWDFDVFGGG (SEQ ID NO: 31); and in particular chosen from
RDWDFDVFGGG (SEQ ID NO, 28); RSWDFDVFGGG (SEQ ID NO: 29) or
RYWDFDVFGGG (SEQ ID NO:30).
- an amino acid sequence chosen from RYWDYDVFGGGT (SEQ ID NO: 32);
RDWDFDVFGGGT (SEQ ID NO: 33); RSWDFDVFGGGT (SEQ ID NO: 34) or
RYWDFDVFGGGT (SEQ ID NO: 35); and in particular chosen from
RDWDFDVFGGGT (SEQ ID NO: 33); RSWDFDVFGGGT (SEQ ID NO, 34) or
RYWDFDVFGGGT (SEQ ID NO: 35).
- an amino acid sequence chosen from RYWDYDVFGGGTP (SEQ ID NO: 36);
RDWDFDVFGGGTP (SEQ ID NO: 37); RSWDFDVFGGGTP (SEQ ID NO: 38) or
RYWDFDVFGGGTP (SEQ ID NO: 39); and in particular chosen from
RDWDFDVFGGGTP (SEQ ID NO: 37); RSWDFDVFGGGTP (SEQ ID NO: 38) or
RYWDFDVFGGGTP (SEQ ID NO: 39)
- an amino acid sequence chosen from R.YWDYDVFGGGTPV (SEQ ID NO: 40);
RDWDFDVFGGGTPV (SEQ ID NO: 41); RSWDFDVFGGGTPV (SEQ ID NO: 42)
or RYWDFDVFGGGTPV (SEQ ID NO. 43); and in particular chosen from
RDWDFDVFGGGTPV (SEQ ID NO: 41); RSWDFDVFGGGTPV (SEQ ID NO: 42)
or RYWDFDVFGGGTPV (SEQ ID NO: 43).
Again, all the sequence motifs of SEQ ID NO's: 23 to 43 may contain one or
more
substitutions as described herein, such as (for example and without
limitation) one or more
of the substitutions listed in Table I below.
Thus, some other (non-limiting) sequence motifs that may be present in the
amino
acid sequences of the invention are:
- an amino acid sequence according to any of sequence motifs of SEQ ID NO's:
24 to 27
or 32 to 43, in which the threonine (T) residue at position 14 has been
replaced by
another amino acid residue (preferably but without limitation, A, N or D);
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an amino acid sequence according to any of sequence motifs of SEQ ID NO's: 23
to 43,
in which the aspartate (D) at position 8 has been replaced by a threonine (T);
an amino acid sequence according to any of sequence motifs of SEQ ID NO's: 24
to 27
or 32 to 43, in which (i) the threonine (T) residue at position 14 has been
replaced by
another amino acid residue (preferably but without limitation, A, N or D), and
(ii) the
aspartate (D) at position 8 has been replaced by a threonine (T);
Again, these other sequence motifs may contain one or more other suitable
substitutions, such as (for example and without limitation) one or more of the
substitutions
listed in Table I below.
10 In the context of the present invention, an amino acid sequence of the
invention is
deemed to "bind better" to serum albumin (such as human serum albumin or serum
albumin
from another species of mammal, such as serum albumin of cynomolgus monkey)
than the
amino acid sequence of SEQ ID NO: 1. (and in particular, better than. at least
one and
preferably all of SEQ ID NO: 75, 76 and/or 77):
- when it binds to said serum albumin with a higher specificity than the amino
acid
sequence of SEQ ID NO:I (and in particular, than at least one and preferably
all of SEQ
ID NO: 75, 76 and/or 77); and/or
- when it binds to said serum albumin with a higher affinity (as defined
herein, and
expressed as a KD, KA, k0õ or koff rate, and determined using one of the
methods described
20 herein) than the amino acid sequence of SEQ ID NO:I (and in particular,
than at least
one and preferably all of SEQ ID NO: 75, 76 and/or 77); and/or
- when it binds to said serum albumin with a higher avidity (i.e. when the
amino acid
sequence of the invention is used as a concatamer) than the amino acid
sequence of SEQ
ID NO:1 (i.e. when it is used in the form of a comparable concatamer) (and in
particular,
than at least one and preferably all of SEQ ID NO, 75, 76 and/or 77); and/or
- when a compound of the invention (as defined herein) that comprises one or
more of said
amino acid sequences of the invention binds to said serum albumin with a
higher
specificity, affinity and/or avidity than a corresponding compound of the
invention that
comprises one or more amino acid sequences of SEQ ID NO:I (and in particular,
than at
least one and preferably all of SEQ ID NO: 75, 76 and/or 77), for example as
determined
using the BlAcoreTM measurement used in Example 2. For example, and without
limitation, an. amino acid sequence of the invention is said to bind better to
serum albumin
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26
when a fusion protein in which the relevant amino acid sequence is fused
(optionally via a
suitable linker) to the Nanobody 2D3 (SEQ ID NO: 47) binds with a higher
specificity,
affinity and/or avidity to serum albumin than a corresponding fusion protein
in which the
Nanobody 2D3 is fused (optionally via the same suitable linker) to the amino
acid
sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1) (for example as determined
using the BlAcoreTM measurement used in Example 2). For the purposes of this
comparison, the relevant amino acid sequence may for example (but without
limitation)
be linked to the C-terminus of 2D3 (optionally via the same suitable linker).
A specific
but non-limiting example of all this is given in Example 2.
In particular, "binding" as described herein may be determined using the
solution
binding competition assay described in Example 3 or Example 9 of WO 09/127691;
or, when
the amino acid sequences is expressed as a fusion with the Nanobody 2D3 as
described in
Example 7 or 10 of WO 09/127691 or as described in Example 2 herein, in the
Biacore
assays described in these Examples of WO 09/127691 and/or in Example 2 below.
As mentioned herein, the amino acid sequences of the invention are further
preferably
such that they bind to human serum albumin (as determined using Biacore) with
an affinity
(expressed as KD) better than 100 nM, preferably better than 5OnM, more
preferably better
than 30 nM, such as equal to or better than 20nM (measured either using the
amino acid
sequence of the invention per se or measured using a fusion of the amino acid
sequence of the
invention to another protein or peptide, such as the Nanobody 2D3 used as an
example
herein).
Some non-limiting examples of amino acid sequences of the invention are given
in
Table II and SEQ ID NO's: 54 to 74. Of these, those sequences binding to serum
albumin
(per se and/or as a fusion with 2D3) better than 30 nM (such as one of the
amino acid
sequences of of SEQ ID NO's: 56, 59, 64, 68, 69, 70, 71, 72 or 74) , and in
particular equal to
or better than 20nM, are particularly preferred (such as one of the amino acid
sequences of
SEQ ID NO's: 56, 59, 68, 70, 72 or 74).
The paragraphs below give a description of some preferred, but non-limiting
aspects,
features, amino acid residues, sequence motifs and examples of the part of the
amino acid
sequences of the invention downstream from (and including) position 3 and
further
downstream. Again, these parts of the sequence can be combined with the
parts/sequences
upstream of position 3 as described herein, to provide an amino acid sequence
of the
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27
invention. In general, the preferences mentioned herein for the part of the
amino acid
sequences of the invention downstream from (and including) position 3 and the
preferences
mentioned herein for the part of the amino acid sequences of the invention
upstream of
position 3 both apply. Thus, in particularly preferred amino acid sequences of
the invention,
the part of the amino acid sequences of the invention downstream from (and
including)
position 3 is according to a preferred description/aspect thereof (as set out
herein), and part
of the amino acid sequences of the invention upstream from (and including)
position 3 is
according to a preferred description/aspect thereof (as set out herein). Based
on this, the
skilled person will be able to combine preferred aspects of the sequences
upstream and
downstream of position 3 into a single, preferred, amino acid sequence of the
invention.
As also described in WO 09/127691, generally, the amino acid sequences of the
invention will, in the part downstream from (and including) position 3,
contain (within the
overall limitations set out herein) one or more "amino acid differences" (as
defined herein)
with the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1), such that the
resulting amino acid sequence of the invention binds better (as defined
herein) to human
serum albumin than the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1).
Such substitutions may for example be an R at position 3 and/or a W at
position 5, as
described herein and in WO 09/127691.
Generally, and within the overall limitations set out herein, such an amino
acid
difference may comprise an insertion, deletion or substitution or one or more
amino acid
residues at one or more positions, compared to the sequence of SEQ ID NO:1.
Usually,
compared to the sequence of SEQ ID NO: 1, an amino acid sequence of the
invention contains
at least one amino acid substitution (such as those mentioned herein); and
optionally also one
or more amino acid insertions and/or one or more amino acid deletions.
Suitable substitutions, insertions and/or deletions (and combinations thereof)
will be
clear to the skilled person based on the disclosure herein, and for example
include one or
more of the substitutions, insertions and/or deletions that are present in the
peptides that are
described in WO 09/127691 (see for example SEQ ID NOs: 2 to 115 and in SEQ ID
NO's:
147 to 157 and Figure 1 from WO 09/127691), or any suitable combination of
these
substitutions, insertions and/or deletions.
Some preferred, but non-limiting, examples of possible substitutions that can
be
present from position 3 and further downstream in an amino acid sequence of
the invention
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28
(compared to the amino acid sequence of SEQ ID NO: l.) are listed in Table I
below (it being
understood that an amino acid sequence of the invention can, within the limits
set out herein,
contain one or more further suitable amino acid substitutions, insertions or
deletions).
It should be noted that in the most preferred amino acid sequences of the
invention,
position 3 is most preferably R, position 5 is W (preferably in combination
with a D on
position 6); position 7 is preferably F (but may also be Y or W); position 15
is P and position
16 is V.
By comparison, in the sequence of SEQ ID NO:1, position 3 is S; position 5 is
S;
position 7 is Y; position 15 is D, position 15 is D; and position 16 is F.
The most preferred amino acid sequences of the invention share the following
residues with the sequence of SEQ ID NO: 1: the Y at position 4 (although, in
the sequences
of the invention, this may also be F, W, S or D); the D at position 6; the
DVFGGG motif at
positions 8-13 (although this may also be DAFGGG in the preferred sequences of
the
invention), and the T at position 14; as well as the G at position 17.
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29
Table I: Examples of possible substitutions that can be present in an amino
acid
sequence of the invention (from position 3 and further downstream, with the
amino acid
residues upstream of position 3 being as further described herein).
a.a. in
Position SEQ ID Examples of possible substitutions in an amino acid
sequence of the invention
NO:1
3 S R (preferred), L, F, Y, W, P, T, S, M, A, D, I, K, Q or V;
4 Y Y, F, W, S or D
Y,R,W.F,L,D,P,G,H,K,M,S,T;ofwhichWis
S
much preferred in combination with a D on position 6
7 Y Y. F or W; of which an F preferred
9 V V (preferred) or A
I G G (preferred) or A
14 T A, N, D (optionally in combination with a mutation 8D
to 8T, in particular if the residue at position 1.4 is a D).
D P (preferred), A, D, S, V, E, G, Q, R, W or Y
16 F V,L,E,G,S,R,K,A,P,Q,D,M,F,I,T
18 P G, E, A, V. S, D, T, N, I, Q, R or W
Optionally, based on. the disclosure herein and in WO 09/1.27691, the skilled
person
will also be able to determine other (or additional) suitable substitutions,
insertions and/or
deletions (or combinations thereof) by means of limited trail-and-error, for
example by
testing a candidate amino acid sequence that comprises the intended
substitutions, insertions
10 and/or deletions for binding to human serum albumin, for example using the
assays described
herein and/or in Example 2 and/or Example 3 from WO 09/127691 (in which said
candidate
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amino acid sequence may then optionally be compared to the amino acid sequence
of SEQ ID
NO:1 and/or to one or more of the amino acid sequences from WO 09/12769 1)
and/or
Example 2 herein.
Again, from position 3 and further downstream, the amino acid sequences of the
invention are preferably as further described herein. Thus, for example, such
amino acid
sequences preferably comprise (i) an Arg (R) residue, in particular an Arg (R)
residue that is
capable of forming a hydrogen bond with the amino acid residues Asn (N) 1.33 &
Asn (N)
135 of human serum albumin and/or capable of forming electrostatic
interactions with the
main-chain oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human
serum
1.0 albumin; and/or (ii) a Trp (W) residue, in particular a Trp (W) residue
that is capable of
forming electrostatic interactions with the Arg (R) 13 8 residue of human
serum albumin;
and/or (iii) the sequence motif GGG; and preferably at least any two and more
preferably all
three of (i), (ii) and (iii). In particular, such amino acid sequences of
preferably (at least)
contain (i) the sequence motif RXWD, in which X may be any amino acid sequence
but is
preferably W, Y, F, S or D; and/or (ii) the sequence motif GGG, preferably the
sequence
motif FGGG (SEQ ID NO:6), more preferably the sequence motif DVFGGG (SEQ ID
NO: 15), and in particular the sequence motif DVFGGGT (SEQ ID NO: 19); and
most
preferably both these sequence motifs (i) and (ii).
Generally, when an amino acid sequence of the invention contains one or more
amino
20 acid substitutions compared to the amino acid sequence AASYSDYDVFGGGTDFGP
(SEQ
ID NO: 1), these may be conservative amino acid substitutions (as defined
herein) or non-
conservative amino acid substitutions (it being understood by the skilled
person that suitable
non-conservative amino acid substitutions will generally be more likely to
improve, or further
improve, the binding to human serum albumin.).
The substitutions may also be one or more substitutions that either allow for
and/or
promote, or that prevent and/or decrease, post-translational modification(s)
of the amino acid
sequence or peptide or the invention, when it is expressed (either as such or
as part of a
compound or polypeptide of the invention) in a host organism. As will be clear
to the skilled
person, such post-translational modifications (and the substitutions that can
either
30 allow/promote or prevent/decrease them) will depend on the host organism
(and in some
cases also on the expression system) in which the amino acid sequence,
peptide, compound or
polypeptide is expressed. Some non-limiting examples of such post-
translational modification
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31
are (enzymatic/proteolytic) cleavage, oxidation events, pyroglutamate
formation,
phosphorylation, and/or isomerisation or deamidation of aspartic acids or
asparagins (all of
which are usually undesired, so that in the invention, mutations may be
introduced in the
peptides of the invention that prevent or reduce such post-translational
modification), as well
as glycosylation (which may be either undesired, in which case mutations may
be introduced
that prevent or reduce the same, for example removing glycosylation sites; or
which in some
cases may be desired, in which case mutations may be introduced which allow or
promote the
same, for example by introducing glycosylation sites). Again, it will be clear
to the skilled
person that the post-translational modifications that may occur will depend on
the host
organism and expression system used, and the skilled person will be able to
select suitable
mutations, optionally after limited trial and error.
It will also be clear to the skilled person that, where certain post-
translational
modifications are to be prevented or reduced, that it may be possible to do so
by expressing
the amino acid sequence, peptide, compound or polypeptide of the invention in
a host or host
organism that is not capable of making such. post-translational modifications.
For example,
where glycosylation is to be prevented or reduced, it is possible (optionally
in combination
with one or more of the substitutions referred to herein, although that may
not be necessary)
to express the amino acid sequence, peptide, compound or polypeptide of the
invention in a
bacterial strain such as E. Coli or in a glycosylation-deficient mutant of a
host-organism that
would otherwise glyclosylate certain sites in the amino acid sequence,
peptide, compound or
polypeptide of the invention.
For example and without limitation, in one aspect of the invention, an amino
acid
sequence, peptide, compound or polypeptide of the invention may be expressed
in a suitable
yeast strain such as suitable strains of Saccharomyces cerevisiae, suitable
strains of
Aspergillus and in particular suitable strains of Pichia pastoris (preferred).
This may for
example be any suitable suitable strain of Saccharomyces cerevisiae,
Aspergillus or Pichia
(preferred) that is suitable for expression, manufacture or production of
proteins or
(poly)peptides that are intended for administration to humans, and. examples
of the same will
be clear to the skilled person (including, for example and without limitation,
suitable
protease-deficient strains of the same).
Thus, as one non-limiting example of a post-translational modification that
may be
prevented or reduced in the invention, when a threonine (T) residue is present
at position 14
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and when said threonine residue at position 14 is susceptible to
phosphorylation in the
intended host organism (such as a strain of Pichia pastoris), it may be
possible or desirable to
prevent or reduce such phosphorylation, for example by replacing the threonine
with a amino
acid residue that is less or (essentially) not susceptible to phosphorylation
in said host
organism. For example and without limitation, when the host organism is a
strain of Pichia
pastoris, the threonine (T) at position 14 may be replaced with an alanine
(A), asparagine (N)
or aspartate (D) residue. Some preferred, but non-limiting examples of amino
acid sequences
of the invention in which the threonine at position 14 has been so replaced
are 89D03V 1
(SEQ ID NO: 103, which contains the mutation 14T to 14A), 89D03V2 (SEQ ID NO:
104,
which contains the mutation 14T to 14N) and 89D03V3 (SEQ ID NO: 105, which
contains
the mutation 14T to 14A and in addition the mutation 8D to 8T).
Alternatively, it may be possible to prevent or reduce such phosphorylation by
expressing such an amino acid sequence/peptide (or compound comprising the
same) in
another host organism or in a strain of Pichia in which said. threonine
residue is not
phosphorylated (or phosphorylated to a lesser degree). Some preferred but non-
limiting
examples of amino acid sequences of the invention that are less susceptible to
phosphorylation are given in SEQ ID NO's: 103 to 108.
Other amino acid sequences of the invention may be provided by introducing
suitable
amino acid substitutions, insertions and/or deletions (or combinations
thereof), as further
described herein and/or in WO 09/127691. Again, these may be conservative
amino acid
substitutions (as defined herein) or non-conservative amino acid substitutions
(it being
understood by the skilled person that suitable conservative amino acid
substitutions will
generally be more likely to ensure that the favourable binding to human serum
albumin is
retained, or even improved).
From the disclosure herein, it will be clear that the amino acid sequences of
the
invention preferably either contain, from position 3 onwards and further
downstream, and
compared to the sequence of SEQ ID NO: 1, no amino acid substitutions or
deletions (and
preferably also no insertions) at the positions 4, 6, 7, 8, 9, 10, 12, 13, 14
or 17; or only a
limited number (i.e. 3, 2 or preferably only 1) amino acid substitutions or
deletions compared
to the sequence of SEQ ID NO:1 (which then preferably are conservative
substitutions as
defined herein). The reason for this is that, from the alanine scanning
experiment described in
Example 4 of WO 09/127691, it has become clear that introducing amino acid
substitutions
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or deletions, although not excluded from the scope of the invention, may carry
an increased
risk of reducing the binding to human serum albumin.
In another preferred, but non-limiting aspect, the amino acid sequences of the
invention preferably contain, from position 3 onwards and further downstream,
a least one
proline residue, such as 1, 2, 3 or 4 praline residues. In particular, the
amino acid sequences
of the invention may contain (a) proline residue(s) at one or more (such as
any one, two, three
or four) of the positions 1, 2, 3, 5, 11, 15, 16 or 18 (and in particular 3,
5, 15, 16 and/or 18).
Proline residues may also be inserted next to or near these positions.
According to one preferred, but non-limiting aspect, an amino acid sequence of
the
invention may comprise, from position 3 onwards and further downstream, one or
more
(such as any two, any three, any four or all five) of the following amino acid
substitutions
compared to the amino acid sequence of SEQ ID NO.1:
-- the serine residue (S) at position 3 of SEQ ID NO: I is replaced by an
amino acid residue
chosen from arginine (R), praline (P),an aromatic amino acid residue (F, Y, W
or H; in
particular F, Y or W) or a hydrophobic amino acid residue (L, 1, V or M);
and/or
- the serine residue (S) at position 5 of SEQ ID NO:1 is replaced by an amino
acid residue
chosen from arginine (R), proline (P), or an aromatic amino acid residue (F.
Y, W or H;
in particular F, Y or W);
and/or
- the aspartate residue (D) at position 15 of SEQ ID NO:1 is replaced by an
amino acid
residue chosen from praline (P) or a small amino acid residue (A, G, S or T);
and/or
- the phenylalanine residue (F) at position 16 of SEQ ID NO: I is replaced by
proline (P), a
hydrophobic amino acid residue (L, I, V or M), or a or a small amino acid
residue (A, G,
S or T);
and/or
the proline residue (P) at position 18 of SEQ ID NO:1 is maintained or
replaced by a
(partially) negative amino acid residue (D, E, Q or N) or a small amino acid
residue (A,
G, S or T);
and optionally one or more further suitable amino acid insertions, deletions
and/or
substitutions (as further described herein).
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In a particularly preferred subclass of amino acid sequences of the invention,
compared to the serine residue (S) at position 3 of SEQ ID NO:1, the serine
residue (S) at
position 3 is replaced by arginine (R). These amino acid sequences may
comprise one or
more further amino acid insertions, deletions and/or substitutions as
described herein.
In particular, from position 3 onwards and further downstream, in amino acid
sequences of the invention with an R at position 3:
- the serine residue (S) at position 5 of SEQ ID NO: I is replaced by an amino
acid residue
chosen from proline (P) or an. aromatic amino acid residue (F, Y, W or H; in
particular F,
Y or W);
and/or
- the aspartate residue (D) at position 15 of SEQ ID NO:I is replaced by an
amino acid
residue chosen from proline (P) or a small amino acid residue (A, G, S or T);
and/or
- the phenylalanine residue (F) at position 16 of SEQ ID NO:I is replaced by
proline (P), a
hydrophobic amino acid residue (L, I, V or M), or a or a small amino acid
residue (A, G,
S or T);
and/or
the praline residue (P) at position 18 of SEQ ID NO: I. is maintained or
replaced by a
(partially) negative amino acid residue (D, E, Q or N) or a small amino acid
residue (A,
G, S or T);
and optionally one or more further suitable amino acid insertions, deletions
and/or
substitutions (as further described herein).
Some preferred amino acid sequences within the amino acid sequences of the
invention are the amino acid sequences of SEQ ID NO, 54 to 77, or amino acid
sequences
that have not more than 3, such as 3, 2, or 1 amino acid differences with one
of the amino
acid sequences of SEQ ID NO: 2 to 115 and/or SEQ ID NO's: 54 to 77 (in which
said amino
acid differences are preferably as generally described herein for the amino
acid sequences of
the invention).
Some more preferred amino acid sequences within the amino acid sequences of
the
invention are the amino acid sequences from Table 11 that bind to human serum
albumin
(either per se and/or as a fusion with 2D3) with an affinity better than 30
nM, and preferably
equal to or better than 20nM; or amino acid sequences that have not more than
3, such as 3, 2,
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or I amino acid differences with one of these amino acid sequences (in which
said amino acid
differences are preferably as generally described herein and/or in WO
09/127691), and which
again bind to human serum albumin (either per se and/or as a fusion with 2D3)
with an
affinity better than 30 nM, and preferably equal to or better than 20nM.
Again, all the above amino acid sequences of the invention are preferably such
that
they bind to human serum albumin (as determined using Biacore) with an
affinity (expressed
as KD) better than 100 nM, preferably better than 50nM, more preferably better
than 30 nM,
such as equal to or better than 20nM, measured either using the amino acid
sequence of the
invention per se or (preferably) measured using a fusion of the amino acid
sequence of the
10 invention to another protein or peptide, for example as a fusion with a
NANOBODY (such
as the Nanobody 2D3 used as an example herein).
The amino acid sequences of the invention can also be provided and/or used in
the
form of a peptide in which. the amino acid sequence is linked to a small
flanking sequence
(e.g. of no more than 10, preferably of no more than 5 amino acid residues) at
the C-terminus,
the N-terminus, or both. These may for example be present because the amino
acid sequence
of the invention (or a compound of the invention in which said amino acid
sequence is
present) has been obtained by expression of a corresponding nucleotide
sequence, in which
the nucleotide sequence that encodes the amino acid sequence of the invention
is either
preceded by (i.e. at the 5'-end) and/or followed by (i.e. at the 3'-end) by a
small nucleotide
20 sequence that encodes a restriction site or that forms part of a cloning
site (and that leads to
the presence of the flanking sequence(s) in the expressed peptide). Examples
of such flanking
sequences are the amino acid sequences GSA and AAA.
The amino acid sequences described herein can bind to serum albumin in a "non-
constrained" format (i.e. not comprising any disulphide bridges), and can
advantageously be
used in such a non-constrained format. It is however included in the scope of
the invention
that the amino acid sequences described herein are provided in, and/or are
used in, a
"constrained" format, for example in the form of a peptide in which an amino
acid sequence
of the invention is flanked by two flanking sequences that can form a
disulphide bridge
between them (for a further description hereof, reference is made to
PCT/EP2007/063348).
30 The amino acid sequence of the invention is preferably such that it binds
to serum
albumin (and in particular to human serum albumin) in such a way that the half-
life of the
serum albumin molecule is not (significantly) reduced.
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Preferably, the amino acid sequence of the invention binds to serum albumin or
at
least one part, fragment, epitope or domain thereof; and in particular to
human serum albumin
or at least one part, fragment, epitope or domain thereof. When the amino acid
sequence of
the invention binds to (human) serum albumin, it preferably is capable of
binding to amino
acid residues on serum albumin that are not involved in binding of (human)
serum albumin to
FeRn; and/or of binding to amino acid residues on serum albumin that do not
form part of
domain III of (human) serum albumin. Reference is made to WO 06/0122787.
Generally, the amino acid sequences of the invention are such that they bind
better to
human serum albumin than the amino acid sequence of SEQ ID NO: 1. Preferably,
the amino
acid sequences of the invention are such that they bind equally well or better
to human serum
albumin than at least one and preferably all of the amino acid sequences of
SEQ ID NO:75,
76 and/or 77 (which are reference compounds taken WO 09/127691).. As
mentioned,
"binding" as described herein may in. particular be determined using the
solution binding
competition assay described in Example 3 or Example 9 of WO 09/127691; or,
when the
amino acid sequences is expressed as a fusion with the Nanobody 2D3 as
described in
Example 7 or 10 of WO 09/127691, in the Biacore assays described in these
Examples or in
Example 2 herein.
An amino acid sequence of the invention preferably has a total size of between
12 and
35 amino acid residues, such as between 1.5 and 32 amino acid residues, for
example
between 15 and 27 and in particular 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino
acid residues).
Also, preferably, amino acid sequences of the invention are such that, when
they are
linked or fused to a therapeutic moiety, compound, protein or other
therapeutic entity, the
compound of the invention (as defined herein) thus obtained has a longer half-
life (as defined
herein) than a corresponding compound or construct in which said therapeutic
moiety,
compound, protein or other therapeutic entity is linked or fused to the amino
acid sequence of
SEQ ID NO:l, and preferably compared to a corresponding compound or construct
that
comprises one of the amino acid sequences of SEQ ID NO's: 75, 76 and/or 77
(i.e. instead of
the amino acid sequence of the invention). This may in particular be
determined by fusing the
amino acid sequence of the invention to the Nanobody 2D3 in the manner
described in
Example 6 or Example 10 of WO 09/127691 (see also Example 2 below), and then
by
determining the pharmacokinetic profile as described in Example 7 or Example
13 of WO
09/127691 (see again also Example 2 below).
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In particular, in a preferred aspect, the amino acid sequences of the
invention. are such
that, when they are linked or fused to a therapeutic moiety, compound, protein
or other
therapeutic entity, the compound of the invention (as defined herein) thus
obtained has a
similar or longer half-life (as defined herein) than a corresponding compound
or construct in
which said therapeutic moiety, compound, protein or other therapeutic entity
is linked or
fused to at least one and preferably all of the amino acid sequences of SEQ ID
NO:75, 76
and/or 77 (which are reference compounds taken WO 09/127691).
The amino acid sequences of the invention are preferably also cross-reactive
(as
defined herein) with the serum albumin from at least one species of mammal
other than man;
an in particular cross-reactive with serum albumin from cynomolgus monkey.
Generally, the amino acid sequences of the invention are also preferably such
that
they compete with the peptide of SEQ ID NO:1 and/or with at least one and
preferably all of
the amino acid sequences of SEQ ID NO:75, 76 and/or 77 (which are reference
compounds
taken WO 09/127691), and/or such that they cross-block (as defined herein) the
binding of
the peptide of SEQ ID NO:1 and/or the binding of at least one and preferably
all of the amino
acid sequences of SEQ ID NO:75, 76 and/or 77to human serum albumin.
The amino acid sequences of the invention (and in particular, the part(s) of
their
sequences from position 3 and further downstream) are preferably such that
they can bind to
one or more of the following amino acid residues of human serum albumin
(numbering as
indicated in Example 8): Asn (N) 133; Pro (P) 134; Asn (N) 135; Leu (L) 136;
Leu (L) 139;
Arg (R) 141; Tyr (Y) 162; Glu (E) 165; Ile (I) 166; His (H) 1.70; Phe (F) 173;
Phe (F) 181;
Gly (G) 213; Lys (K) 214; Ser (S) 217; Gln (Q) 483; and/or Lys (K) 543; and/or
such that
they can compete with the amino acid sequence of SEQ ID NO:1 and/or with at
least one and
preferably all of the amino acid sequences of SEQ ID NO: 75, 76 and/or 77 for
binding to one
or more of these amino acid residues; and/or such that they can cross-block
the binding of the
amino acid sequence of SEQ ID NO:1 and/or the binding of at least one and
preferably all of
the amino acid sequences of SEQ ID NO:75, 76 arid/or 77 to one or more of
these amino acid
residues.
More in particular, the amino acid sequences of the invention (and in
particular, the
part(s) of their sequences from position 3 and further downstream) are
preferably such that
they can bind to an epitope on human serum albumin that comprises either (i)
the stretch of
amino acid residues that comprises the residues Asn (N) 133; Pro (P) 134; Asn
(N) 135; Leu
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(L) 136; Leu (L) 139 and Arg (R) 141; and/or (ii) the stretch of amino acid
residues that
comprises the residues Tyr (Y) 162; Glu (E) 165; He (I) 166; His (H) 170; Phe
(F) 173; Phe
(F) 181; and/or (iii) the stretch of amino acid residues that comprises the
residues Gly (G)
213; Lys (K) 214 and Ser (S) 217; and/or such that they can compete with the
amino acid
sequence of SEQ ID NO:1 and/or with at least one and preferably all of the
amino acid
sequences of SEQ ID NO:75, 76 and/or 77 for binding to one of these stretches
of amino acid
residues; and/or such that they can cross-block. the binding of the amino acid
sequence of
SEQ ID NO:1 and/or the binding of at least one and preferably all of the amino
acid
sequences of SEQ ID NO:75, 76 and/or 77 to one or more of these stretches of
amino acid
1.0 residues.
Even more in particular; the amino acid sequences of the invention are
preferably
such that they can bind to a hydrophobic subpocket on human serum albumin that
is
comprises (amongst others) residues the residues Leu (L) 139, Glu (E) 165, Ile
(I) 166, His
(H) 170, Phe (F) 173, Phe (F) 181, Gly (G) 213, Lys (K) 214, Ser (S) 217 and
Gln (Q) 483;
and/or such that they can compete with the amino acid sequence of SEQ ID NO:1
and/or with
at least one and preferably all of the amino acid sequences of SEQ ID NO:75.
76 and/or 77
for binding to this subpocket; and/or such that they can cross-block the
binding of the amino
acid sequence of SEQ ID NO:1 and/or the binding of at least one and preferably
all of the
amino acid sequences of SEQ ID NO:75, 76 and/or 77 to this subpocket.
20 Also, the amino acid sequences of the invention (and in particular, the
part(s) of their
sequences upstream from position 3) are preferably further such that they can
bind to one or
more of the following amino acid residues of human serum albumin (numbering as
indicated
in Example 8): D131, N133, N135, V442, S443,P445, T446, E449, L484, L487,
H488,
K490, T491, V493 and/or 1547, and in particular with (one or more of the amino
acid
sequences that form) the hydrophobic subpocket on human serum albumin that
comprises the
amino acids V442, S443, T446, E449, L484, L487, H488, K490, T491 and/or V493;
and/or
such that they can compete with at least one of the amino acid sequences
mentioned in Table
II (and in particular at least one of the sequences of SEQ ID NO's: 56, 59,
68, 69, 70, 71, 72
and/or 74) for binding to one or more of these amino acid residues; and/or
such that they can
30 cross-block the binding of at least one of the amino acid sequences
mentioned in Table II
(and in particular at least one of the sequences of SEQ ID NO's: 56, 59, 68,
69, 70, 71, 72
and/or 74) to one or more of these amino acid residues on human serum albumin.
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39
Thus, the invention also relates to amino acid sequences that can bind to
human serum
albumin and that are such that they are as described in the preceding
paragraphs in terms of
competing for binding with human serum albumin and/or in terms of cross-
blocking binding
to human serum albumin. Again, such amino acid sequences can be as further
described
herein.
In one specific aspect, the invention does not comprise the amino acid
sequences that
are mentioned in Figure 4 or Figure 8 of PCT/EP2007/063348.
The amino acid sequences of the invention (or a compound of the invention
comprising at least one such amino acid sequence, as further described herein)
are preferably
such that they can bind to a serum albumin, and in particular to human serum
albumin:
- with a dissociation constant (Ko) in the range of 10-s to 1.0-12 moles/liter
or less, and
preferably in the range of 10-7 to 10-12 moles/liter or less and more
preferably in the
range of 1.0-8 to 10-12 moles/liter (i.e. with an association constant (KA) of
in the range
of 105 to 10i2 liter/ moles or more, and preferably in the range of 107 to
1012 liter/moles
or more, and more preferably in the range of 108 to 1012 liter/moles), such
that said
dissociation constant is better (i.e. smaller/lower) than the dissociation
constant with
which the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1) binds to
human serum albumin;
and/or
- with a ko,-rate in the range of between 102 M-1 s-' to about 107 M-1 s"1,
preferably in the
range between 103 M"1s ' and 107 M-'s 1, more preferably in the range between
104 M"
's' 1 and 107 M-'s 1, such as between 10' M's-1 and 107 M 1s-',such that said
k,,,,-rate is
better (i.e. higher) than the kon-rate with which the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: 1) binds to human serum albumin;
and/or
- with a k,,ff rate in the range between I s-1 (t 1 /2=0.69 s) and 10`6 s4
(providing a near
irreversible complex with a t1/2 of multiple days), preferably in the range
between 10-2 s
1. and 10-6 s 1, more preferably in the range between 10' s"1 and 10-' s-1,
such as in the
range between 10"4 s"' and 1() -6 s1, such that said k0 '-rate is better (i.e.
higher) than the
koff-rate with which the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO:1) binds to human serum albumin.
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Preferably, an amino acid sequence of the invention (or a compound of the
invention
comprising one such amino acid sequence, as further described herein) is such
that it will
bind to human serum albumin with an affinity less than 1000 nM, preferably
less than 500
nM, preferably less than 200 nM, preferably better than 50nM, and in
particularly better than
30 nM, such as equal to or better than 20nM, again as measured using the
solution binding
competition assay described in Example 3 or Example 9 of WO 09/127691; or,
when the
amino acid sequences is expressed as a fusion with the Nanobody 2D3 as
described in
Example 7 or 10 of WO 09/127691, in the Biacore assays described in these
Examples or in
Example 2 herein.
1.0 The amino acid sequences of the invention (as well as compounds of the
invention
comprising the same, as defined herein) are preferably such that they bind to
or otherwise
associate with human serum albumin in such a way that, when the amino acid
sequence (or
compound) is bound to or otherwise associated with a human serum albumin in
man, it
exhibits a serum half-life of at least about 50% (such as about 50% to 70%),
preferably at
least 60% (such as about 60% to 80%), or preferably at least 70% (such as
about 70% to
90%), more preferably at least 80% (such as about 80% to 90%), or preferably
at least about
90% of the natural half-life of the human serum albumin in man.
The amino acid sequences of the invention may bind to serum albumin (such as
human serum albumin) in a conditional manner (as described in the
International application
20 PCT/EP2007/060850 of Ablynx N.V.), i.e. such that:
a) they bind to human. serum albumin molecule under a first biological
condition with a
dissociation constant (KD) of 10-' moles/liter or less; and
b) they bind to human serum albumin under a second biological condition with a
dissociation constant (KD) that is at least 10 fold different from (and in
particular
more than) the dissociation constant with which said amino acid sequence binds
to
said desired molecule under said first biological condition.
in which the first and second biological conditions may be as described in the
International
application PCT/EP2007/060850 of Ablynx N.V.. In particular, as described in
the
International application PCT/EP2007/060850, the first biological condition
and the second
30 biological condition may differ in respect of pH, in which said first
biological condition may
comprise a physiological pH of more than 7Ø for example a pH of more than
7.1 or a pH of
more than 7.2, such as a pH in the range of 7.2 to 7.4; and the second
biological condition
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41
may comprise a physiological pH of less than 7.0, for example a pH of less
than 6.7 or a pH
of less than 6.5, such as a pH in the range of 6.5 to 6.0 (or visa versa).
Preferably, however, amino acid sequences of the invention may bind to serum
albumin (such as human serum albumin) in a manner that is "essentially
independent of the
pH" (as described in the International application PCT/EP2007/060849 of Ablynx
N.V., and
as further defined herein).
In one non-limiting aspect, the amino acid sequences of the invention are
preferably
cross-reactive (as defined herein) with serum albumin from at least one other
species of
mammal, for example from mouse, rabbit, rat, or a primate. In particular, the
amino acid
sequences of the invention may be cross-reactive with serum albumin from a
primate chosen
from the group consisting of monkeys from the genus Macaca (such as, and in
particular,
cynomolgus monkeys (Macaca faseicularis) and/or rhesus monkeys (Macaca
mulatta) and
baboon (Papio ursinus), and preferably at least with cyno serum albumin. Also,
when an
amino acid sequence of the invention is cross-reactive with serum albumin from
such a
species of primate, it is preferably such that, when it is bound to or
associated with a serum
albumin molecule in said primate, it exhibits a serum half-life of at least
about 50% (such as
about 50% to 70%), preferably at least about 60% (such as about 60% to 80%),
or preferably
at least about 70% (such as about 70% to 90%), more preferably at least about
80% (such as
about 80% to 90%). or preferably at least about 90% of the natural half-life
of said serum
albumin in said primate.
The invention also relates to a compound or construct which comprises at least
one
amino acid sequence of the invention and at least one therapeutic moiety (also
referred to
herein as "compounds of the invention"). These compounds or constructs may be
as further
described herein, and may for example be polypeptide or protein constructs
that comprise or
essentially consist of at least one amino acid sequence of the invention that
is linked to at
least one therapeutic moiety, optionally via one or more suitable linkers or
spacers. Such
polypepti.de or protein constructs may for example (without limitation) be a
fusion protein, as
further described herein.
Such compounds of the invention may contain one, two, three or more amino acid
sequences of the invention, suitably linked to the at least one therapeutic
moiety (and
optionally to each other), optionally via one or more suitable linkers (as
described herein).
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Also, when a compound of the invention comprises two, three or more amino acid
sequences
of the invention, these may be the same or different.
In one specific aspect, such compounds of the invention may comprise one amino
acid
sequence of the invention, suitably linked to the at least one therapeutic
moiety, optionally
via one or more suitable linkers (as described herein). For example, in such a
case, when the
therapeutic moiety is a protein or polypeptide (such that the resulting
compound of the
invention is a fusion protein), the amino acid sequence of the invention may
either be linked
to the C-terminus of the therapeutic moiety or to the N-terminus of the
therapeutic moiety
(again, optionally via a suitable linker).
In another specific aspect, such compounds of the invention may comprise two
amino
acid sequence of the invention, suitably linked to the at least one
therapeutic moiety (and
optionally to each other), optionally via one or more suitable linkers (as
described herein).
More specifically, such compounds of the invention may comprise two amino acid
sequence of the invention, that are each suitably linked to the at least one
therapeutic moiety
(i.e. on different attachment sites of the therapeutic moiety), again
optionally via suitable
linkers. For example, in such a case, when the therapeutic moiety is a protein
or polypeptide
(such that the resulting compound of the invention is a fusion protein), one
amino acid
sequence of the invention may for example be linked to the C-terminus of the
therapeutic
moiety (again, optionally via a suitable linker) and one amino acid sequence
of the invention
may for example be linked to the N-terminus of the therapeutic moiety (again,
optionally via
a suitable linker).
Alternatively, such compounds of the invention may comprise two (or more)
amino
acid sequences of the invention that are linked to each other (again,
optionally via a suitable
linker) so as to form a "tandem repeat", which tandem repeat may then be
suitably linked to
the at least one therapeutic moiety (again optionally via a suitable linker).
For example, in
such a case, when the therapeutic moiety is a protein or polypeptide (such
that the resulting
compound of the invention is a fusion protein), the tandem repeat of the two
or more amino
acid sequences of the invention may either be linked to the C-terminus of the
therapeutic
moiety or to the N-terminus of the therapeutic moiety (again, optionally via a
suitable linker).
Other suitable combinations of two or more amino acid sequences of the
invention
and one or more therapeutic moieties (again, optionally linked via suitable
linkers) will be
clear to the skilled person based on the disclosure herein.
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In another aspect, the compounds of the invention comprise two or more (such
as two,
three or four) therapeutic moieties (which may be the same or different), and
one or more
(such as two, three, four or more) amino acid sequences of the invention
(which may also be
the same or different), in which the two or more (such as two, three or four)
therapeutic
moieties and/or the one or more (such as two, three, four or more) amino acid
sequences of
the invention may be suitably linked to each other (again optionally via one
or more suitable
linkers) so as to form a compound of the invention. For example, in such
compounds of the
invention, the two or more therapeutic moieties may be suitably linked to each
other (again
optionally via one or more suitable linkers), and one or more of the amino
acid sequences of
the invention (and/or one or more tandem repeats of two or more amino acid
sequences of the
invention., as described herein) may be linked (again, optionally via one or
more suitable
linkers) to any (or all) of the therapeutic moieties.
Also, in a further aspect, one or more of the linker(s) used to link the two
or more
therapeutic moieties to each other may comprise one or more of the amino acid
sequences of
the invention, and such linkers comprising one or more amino acid sequences of
the
invention (optionally comprising one or more further linking amino acid
sequences to link the
acid sequences of the invention to each other and/or to one or more
therapeutic moieties)
form a further aspect of the invention.
For example, when a compound of the invention comprises two therapeutic
moieties
(which may be the same or different), some examples of possible but non-
limiting
configurations of the above compounds of the invention are:
[TM]-[L]-[AA]-[L]-[TM]
[AA]-[L]-[TM]-[L]-[TM]
[TM]-[L]-[TM]-[L]-[AA]
[TM]-[L]-[AA]-[L]-[AA]-[TM]
[AA]-[L]-[TM]-[L]-[TM]-[L]-[AA]
[AA]-[L]-[AA]-[TM]-[L]-[TM]
[TM] -[L] - [TM] -[L] - [AA] - [AA]
[AA]-[L]-[TM]-[L]-[AA]-[L]-[TM]-[L]-[AA]
[AA]-[L]-[TM]-[L]-[AA]-[L]-[AA]-[L]-[TM]-[L]-[AA]
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in which "[TM]" refers to the therapeutic moiety, "[L]" refers to a linker
(which in each case
is optional), and "[AA]" refers to an amino acid sequence of the invention.
Other suitable
configuration will be clear to the skilled person based on the disclosure
herein. Again, in
these constructs, when there are two or more linkers and/or amino acid
sequences of the
invention present, these may be the same or different. Again, when the
therapeutic moieties
and the linkers are proteins or (polypeptides), the above constructs may be
fusion proteins or
fusion constructs (which may for example be suitably obtained by suitable
expression of a
corresponding nucleic acid or nucleotide sequence).
In another aspect, the invention relates to a polypeptide construct that
comprises two
or more (and in particular two or three, and preferably two) amino acid
sequences of the
invention, in which the two or more amino acid sequences of the invention
present in said
polypeptide may be the same or different; and in which the two or more amino
acid
sequences of the invention may be either linked directly to each other, or
linked to each other
via a suitable linker (as further described herein). Such a "tandem repeat"
construct of the
invention may again be linked to one or more therapeutic moieties, in the same
way as a
single amino acid sequence of the invention. In some cases, the use of a
tandem repeat may
provide for an (even further) improved affinity to human serum albumin
(compared to the use
of a single amino acid sequence of the invention) and/or for an (even further)
improved half-
life for the compounds of the invention that contain such a tandem repeat
(compared to a
compound of the invention that comprises a single amino acid sequence of the
invention). A
non-limiting example of the use of such a tandem repeat and of a compound of
the invention
that comprises such a tandem. repeat is given in Example 14. Also, as
described herein, such a
tandem repeat construct may be used as a linker.
Such tandem repeats preferably contain two or more of the preferred amino acid
sequences of the invention (which may be the same or different), and in
particular the
particularly preferred amino acid sequences of the invention, such as (for
example) those
mentioned in Table II and in particular those mentioned in Table II that bind
(either per se
and/or as a fusion with 2D3) with an affinity less than 30 nM and preferably
less than 20nM.
The invention also relates to compounds and constructs that comprise such
tandem repeats
(which may again be fusion proteins); to nucleotide sequences or nucleic acids
encoding such
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tandem repeats of such fusion proteins, and to uses of such tandem repeats
(e.g. to extend
half-life and/or as linkers).
Thus, in another aspect, the invention relates to a polypeptide construct that
comprises
two or more (and in particular two or three, and preferably two) amino acid
sequences of the
invention, in which the two or more amino acid sequences of the invention
present in said
polypeptide may be the same or different; and in which the two or more amino
acid
sequences of the invention may be either linked directly to each other, or
linked. to each other
via a suitable linker (as further described herein); and in which each amino
acid sequence
present therein:
10 a) is one of the amino acid sequences of SEQ ID NO: 54 to 74; or
b) has at least 65 %, more preferably at least 70%, even more preferably at
least 75%,
such as at least 80%, for example at least 85% or at least 90% with at least
one of the
amino acid sequences amino acid sequences of SEQ ID NO: 54 to 74; and/or
c) has no more than. 6, preferably no more than 5, in particular no more than
4, such as 3,
2 or I amino acid difference(s) (as defined herein) with at least one of the
amino acid
sequences of SEQ ID NO: 54 to 74;
and preferably:
d) binds to human serum albumin with an affinity (expressed as KD) better than
100 nM,
preferably better than 5OnM, more preferably better than 30 nM, such as equal
to or
20 better than 20nM, measured either using the amino acid sequence of the
invention per
se or (preferably) measured using a fusion of the amino acid sequence of the
invention
to another protein or peptide, for example as a fusion with a NANOBODY (such
as
the Nanobody 2D3 used as an example herein).
Again, the amino acid sequences present in such a tandem repeat may be as
further
described herein, and the tandem repeat may be linked to one or more
therapeutic moieties, in
the manner described herein.
Thus, in another aspect, the invention relates to a polypeptide construct that
comprises
two or more (and in particular two or three, and preferably two) amino acid
sequences of the
invention, in which the two or more amino acid sequences of the invention
present in said
30 polypeptide may be the same or different; and in which the two or more
amino acid
sequences of the invention may be either linked directly to each other, or
linked to each other
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via a suitable linker (as further described herein); and in which each amino
acid sequence
present therein:
a) is one of the amino acid sequences of SEQ ID NO's: 56, 59, 64, 68, 69, 70,
71, 72 or
74; and preferably one of the amino acid sequences of SEQ ID NO's: 56, 59, 68,
70, 72
or 74;
b) has at least 65 %, more preferably at least 70%, even more preferably at
least 75%,
such as at least 80%, for example at least 85% or at least 90% with at least
one of the
amino acid sequences of SEQ ID NO's: 56, 59, 64, 68, 69, 70, 71, 72 or 74; and
preferably one of the amino acid sequences of SEQ ID NO's: 56, 59, 68, 70, 72
or 74;
and/or
c) has no more than 6, preferably no more than 5, in particular no more than
4, such as 3,
2 or 1 amino acid difference(s) (as defined herein) with at least one of the
amino acid
sequences of SEQ ID NO's: 56, 59, 64, 68, 69, 70, 71, 72 or 74; and preferably
one of
the amino acid sequences of SEQ ID NO's: 56, 59, 68, 70, 72 or 74;
and preferably:
d) binds to human serum albumin with an affinity (expressed as Ko) better than
100 nM,
preferably better than SOnM, more preferably better than 30 nM, such as equal
to or
better than20nM, measured either using the amino acid sequence of the
invention per
se or (preferably) measured using a fusion of the amino acid sequence of the
invention
to another protein or peptide, for example as a fusion with a NANOBODY (such
as
the Nanobody 2D3 used as an example herein).
Again, the amino acid sequences present in such a tandem repeat may be as
further
described herein, and the tandem repeat may be linked to one or more
therapeutic moieties, in
the manner described herein.
The at least one therapeutic moiety present in the compounds of the invention
preferably comprises or essentially consists of an amino acid sequence, and
may in particular
comprise or essentially consist of an immunoglobulin sequence or an antigen-
binding
fragment thereof (for example, an antibody or an antigen-binding fragment
thereof), such as
an immunoglobulin variable domain or an antigen-binding fragment thereof (for
example, a
VH-domain, a VL-domain, a VHH-domain or an antigen-binding fragment thereof);
or a
protein or polypeptide comprising the same (for example, an scFv construct).
For such
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47
constructs, reference is for example made to the review by Holliger and
Hudson, Nat
Biotechnol. 2005 Sep; 23(9):1126-36 and the further prior art cited therein.
According to one specific, but non-limiting aspect, the therapeutic moiety
comprises
or essentially consists of a (single) domain antibody, a "dAb", or a NANOBODY
, and
preferably a Nanobody (which, as stated in WO 08/142164 and other applications
by Ablynx
N.V., may be a VHH, a humanized VIII-t or a camelized VH such as a camelized
human VH).
When the one or more therapeutic moieties are directed against one or more
pharmaceutically relevant targets, they may be directed against any suitable
target known per
se. For example, when the therapeutic moiety comprises or essentially consists
of a (single)
domain antibody, a "dAb", or a NANOBODY , it may for example be a dAb or
NANOBODY , IGN-gamma (see for example WO 04/041863), IgE (see for example WO
04/041867), EGFR (see for example WO 05/044858; WO 07/066106 or WO 07/080392);
vWF (see for example WO 04/062551 or WO 06/1222825); IGF-IR (see for example
WO
07/042289); IL-6 (see for example WO 07/110219); IL-6R (see for example WO
08/020079);
GPCR's (see for example WO 08/074839); chemokin.es (see for example WO
08/077945);
VEGF or its receptors (see for example WO 07/080392; WO 08/101985; WO
08/149147;
WO 08/149146; or WO 08/149150); RANK-L (see for example WO 08/142164); IL-Rl
(see
for example WO 06/059108; WO 07/06' )3 11; WO 07/063308; or WO 08/149149); TNF-
R1
(see for example WO o6/038027; WO 07/049017; WO 08/149148 or WO 08/149144); IL-
4
or IL-13 (see for example WO 07/085815); CD40L (see for example WO 06/030220).
The therapeutic moieties may also be other proteins or peptides with a known
therapeutical and/or pharmacological actions, such as, for example and without
limitation,
GLP-1; insulin; EPO; human growth hormone (e.g., somatropin); interferons,
interleukins
and (other) cytokines and/or protein drugs used in cancer therapy.
In a compound of the invention the one or more amino acid sequences of the
invention may be either directly linked to the at least one therapeutic moiety
or linked to the
at least one therapeutic moiety via one or more suitable linkers or spacers.
Suitable linkers
will be clear to the skilled person, for example based on the further
disclosure herein. Some
preferred, but non-limiting linkers are those mentioned on pages 127 and 128
of the
International application WO 08/020079 of Ablynx N.V., and include the "gly-
ser linkers"
mentioned therein.
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48
When the one or more therapeutic moieties are amino acid sequences, the
linkers or
spacers preferably comprise or essentially consist of amino acid sequences, so
that the
resulting compound or construct essentially consists of a (fusion) protein or
(fusion)
polypeptide (also referred to herein as a "polypeptide of the invention").
In a further aspect, the invention relates to a compound of the invention (as
further
defined herein) that comprises at least one amino acid sequence of the
invention that is as
further described herein, wherein said compound of the invention binds to
human serum.
albumin with an affinity (expressed as Kn) better than 1.00 nM, preferably
better than 5OnM,
more preferably better than 30 nM, such as equal to or better than 20nM,
In a further aspect, the invention relates to a compound of the invention (as
further
defined herein) that comprises at least one amino acid sequence of the
invention that is as
further described herein, wherein said compound of the invention has a longer
half-life (as
defined herein) than a corresponding compound that, instead of said amino acid
sequence(s),
contains one of the amino acid sequences of SEQ ID NO: 75, 76 and/or 77. In a
further
aspect, the invention relates to a compound of the invention that comprises at
least two amino
acid sequences of the invention. In another aspect, the invention relates to a
compound of the
invention that comprises at least one tandem repeat (as defined herein) of at
least two amino
acid sequences of the invention. Preferably, said compound of the invention.
has a longer half-
life (as defined herein) than a corresponding compound that, instead of said
amino acid
sequences, contains the same number of copies of one of the amino acid
sequences of SEQ
ID NO: 75, 76 and/or 77.
Some other aspects of the invention relate to the following peptides. Again,
such
peptides are incorporated into the meaning of the term "amino acid sequences
of the
invention" as used in its broadest sense herein; and these peptides are
preferably as further
described herein for the amino acid sequences of the invention. Also, again,
the peptides
according to the following aspects may contain one or more substitutions as
described herein,
such as (for example and without limitation) one or more of the substitutions
listed in Table I.
Thus, some other (non-limiting) peptides that form aspects of the invention
are
- peptides according to (one or more of) the following aspects that contain
one of the
sequence motifs of SEQ ID NO's: 24 to 27 or 32 to 43, in which the threonine
(T)
residue at position 14 has been replaced by another amino acid residue
(preferably but
without limitation, A, N or D);
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49
- peptides according to (one or more of) the following aspects that contain
one of the
sequence motifs of SEQ ID NO's: 15, 17 to 22 or 23 to 43, in which the
aspartate (D) at
position 8 has been replaced by a threonine (T);
peptides according to (one or more of) the following aspects that contain one
of the
sequence motifs of SEQ ID NO's: 24 to 27 or 32 to 43, in which (i) the
threonine (T)
residue at position 14 has been replaced by another amino acid residue
(preferably but
without limitation, A, N or D), and (ii) the aspartate (D) at position 8 has
been replaced
by a thr eonine (T);
and again such peptides are preferably as further described. herein for the
amino acid
sequences of the invention. In particular, as further described herein, these
peptides may
contain, instead of one of the sequence motifs of SEQ ID NO's: 15, 17 to 22 or
23 to 43,
respectively, one of the corresponding sequence motifs from SEQ ID NO's: 131
to 148,
respectively (for example and without limitation. the sequence motif of SEQ ID
NO: 131
instead of the sequence motif of SEQ ID NO: 15, or one of the sequence motifs
of SEQ ID
NO's: 134 to 139 instead of the sequence motif of SEQ ID NO: 19). Again, these
peptides
may contain one or more other suitable substitution, such as - for example and
without
limitation - one or more of the substitutions listed in Table 1. Also, again,
these peptides are
incorporated into the meaning of the term "amino acid sequences of the
invention" as used in
its broadest sense herein.
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises an Arg (R) residue; and the
sequence
motif DVFGGG (SEQ ID NO: 15), in particular the sequence motif DVFGGGT (SEQ ID
NO: 19); and that contains a stretch of amino acid residues upstream of
position 3 (and/or said
R residue) that is as described herein (including the preferred aspects of
such a stretch of
amino acid residues, also as described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises an Arg (R) residue that is
capable of
forming a hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135
of human
serum albumin and/or capable of forming electrostatic interactions with the
main-chain
oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum
albumin; and the
sequence motif DVFGGG (SEQ ID NO: 15), in particular the sequence motif
DVFGGGT
(SEQ ID NO:19); and that contains a stretch of amino acid residues upstream of
position 3
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(and/or said R residue) that is as described herein (including the preferred
aspects of such a
stretch of amino acid residues, also as described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises a Trp (W) residue; and the
sequence
motif DVFGGG (SEQ ID NO: 15), in particular the sequence motif DVFGGGT (SEQ ID
NO: 19); and that contains a stretch of amino acid residues upstream of
position 3 (and/or said
R residue) that is as described herein (including the preferred aspects of
such a stretch of
amino acid residues, also as described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
10 herein) for human serum albumin and that comprises a Trp (W) residue that
is capable of
forining electrostatic interactions with the Arg (R) 138 residue of human
serum albumin; and
the sequence motif DVFGGG (SEQ ID NO: 15), in particular the sequence motif
DVFGGGT
(SEQ ID NO:19); and that contains a stretch of amino acid residues upstream of
position 3
(and/or said R residue) that is as described herein (including the preferred
aspects of such a
stretch of amino acid residues, also as described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises an Arg (R) residue; a Trp
(W) residue;
and the sequence motif DVFGGG (SEQ ID NO: 15), in particular the sequence
motif
DVFGGGT (SEQ ID NO: 19); and that contains a stretch of amino acid residues
upstream of
20 position 3 (and/or said R residue) that is as described herein (including
the preferred aspects
of such a stretch of amino acid residues, also as described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises an Arg (R) residue; an
aromatic amino
acid residue that is capable of forming electrostatic interactions with the
Arg (R) 138 residue
of human serum albumin; and the sequence motif DVFGGG (SEQ ID NO: 15), in
particular
the sequence motif DVFGGGT (SEQ ID NO: 19); and that contains a stretch of
amino acid
residues upstream of position 3 (and/or said R residue) that is as described
herein (including
the preferred aspects of such a stretch of amino acid residues, also as
described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
30 herein) for human serum albumin and that comprises an Arg (R) residue that
is capable of
forming a hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135
of human
serum albumin and/or capable of forming electrostatic interactions with the
main-chain
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51
oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum
albumin; a Trp
(W) residue that is capable of forming electrostatic interactions with the Arg
(R) 138 residue
of human serum albumin; and the sequence motif DVFGGG (SEQ ID NO:15), in
particular
the sequence motif DVFGGGT (SEQ ID NO: 19); and that contains a stretch of
amino acid
residues upstream of position 3 (and/or said R residue) that is as described
herein (including
the preferred aspects of such a stretch of amino acid residues, also as
described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises the amino acid sequence
RXWDXDVFGGG (SEQ ID NO:23), in which the first (from the N-terminal end) amino
acid residue indicated by X is chosen from Y, S or D; and the second amino
acid residue
indicated by X is chosen from Y or F; and that contains a stretch of amino
acid residues
upstream of position 3 (and/or said R residue) that is as described herein
(including the
preferred aspects of such a stretch of amino acid residues, also as described
herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and. that comprises the amino acid sequence
RXWDXDVFGGGT (SEQ ID NO: 24), in which the first (from the N-terminal end)
amino
acid residue indicated by X is chosen from Y. S or D; and the second amino
acid residue
indicated by X is chosen from Y or F; and that contains a stretch of amino
acid residues
upstream of position 3 (and/or said R residue) that is as described herein
(including the
preferred aspects of such a stretch of amino acid residues, also as described
herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises the amino acid sequence
RXWDXDVFGGGTP (SEQ ID NO: 25), in which the first (from the N-terminal end)
amino
acid residue indicated by X is chosen from Y, S or D; and the second amino
acid residue
indicated by X is chosen from Y or F; and that contains a stretch of amino
acid residues
upstream of position 3 (and/or said R residue) that is as described herein
(including the
preferred aspects of such a stretch of amino acid residues, also as described
herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises the amino acid sequence
RXWDXDVFGGGTPG (SEQ ID NO: 26), in which the first (from the N-terminal end)
amino acid residue indicated by X is chosen from Y, S or D; and the second
amino acid
residue indicated by X is chosen from Y or F; and that contains a stretch of
amino acid
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52
residues upstream of position 3 (and/or said R residue) that is as described
herein (including
the preferred aspects of such a stretch of amino acid residues, also as
described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and. that comprises the amino acid sequence
RXWDXDVFGGGTPGG (SEQ ID NO: 27), in which the first (from the N-terminal end)
amino acid residue indicated by X is chosen from Y, S or D; and the second
amino acid
residue indicated by X is chosen from Y or F; and that contains a stretch of
amino acid
residues upstream of position 3 (and/or said R residue) that is as described
herein (including
the preferred aspects of such a stretch of amino acid residues, also as
described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises an amino acid sequence
chosen from
RYWDYDVFGGG (SEQ ID NO:28); RDWDFDVFGGG (SEQ ID NO:29);
RSWDFDVFGGG (SEQ ID NO: 30) or RYWDFDVFGGG (SEQ ID NO: 31); and in
particular chosen from RDWDFDVFGGG (SEQ ID NO:29); RSWDFDVFGGG (SEQ ID
NO: 30) or RYWDFDVFGGG (SEQ ID NO: 31); and that contains a stretch of amino
acid
residues upstream of position 3 (and/or said R residue) that is as described
herein (including
the preferred aspects of such a stretch of amino acid residues, also as
described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises an amino acid sequence
chosen from
RYWDYDVFGGGT (SEQ ID NO: 32); RDWDFDVFGGGT (SEQ ID NO: 33);
RSWDFDVFGGGT (SEQ ID NO: 34) or RYWDFDVFGGGT (SEQ ID NO: 35); and in
particular chosen from RDWDFDVFGGGT (SEQ ID NO: 33); RSWDFDVFGGGT (SEQ ID
NO: 34) or RYWDFDVFGGGT (SEQ ID NO: 35); and that contains a stretch of amino
acid
residues upstream of position 3 (and/or said R residue) that is as described
herein (including
the preferred aspects of such a stretch of amino acid residues, also as
described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises an amino acid sequence
chosen from
RYWDYDVFGGGTP (SEQ ID NO: 36); RDWDFDVFGGGTP (SEQ ID NO: 37);
RSWDFDVFGGGTP (SEQ ID NO: 38) or RYWDFDVFGGGTP (SEQ ID NO: 39); and in
particular chosen from RDWDFDVFGGGTP (SEQ ID NO: 37); RSWDFDVFGGGTP (SEQ
ID NO: 38) or RYWDFDVFGGGTP (SEQ ID NO: 39); and that contains a stretch of
amino
acid residues upstream of position 3 (and/or said R residue) that is as
described herein
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53
(including the preferred aspects of such a stretch of amino acid residues,
also as described
herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin. and that comprises an amino acid sequence
chosen from
RYWDYDVFGGGTPV (SEQ ID NO: 40); RDWDFDVFGGGTPV (SEQ ID NO: 41);
RSWDFDVFGGGTPV (SEQ ID NO: 42) or RYWDFDVFGGGTPV (SEQ ID NO: 43); and
in particular chosen from RDWDFDVFGGGTPV (SEQ ID NO. 41); RSWDFDVFGGGTPV
(SEQ ID NO: 42) or RYWDFDVFGGGTPV (SEQ ID NO: 43); and that contains a stretch
of
amino acid residues upstream of position 3 (and/or said R residue) that is as
described herein
(including the preferred aspects of such a stretch of amino acid residues,
also as described
herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises the sequence motif RXWD (in
which X
is chosen from W, Y, F, S or D) and the sequence motif FGGG (SEQ ID NO:6); and
that
contains a stretch of amino acid residues upstream of position 3 (and/or said
R residue) that is
as described herein (including the preferred aspects of such a stretch of
amino acid residues,
also as described herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin. and. that comprises the sequence motif RXWD
(in which X
is preferably chosen from W, Y, F, S or D) and the sequence motif DVFGGG (SEQ
ID NO:
15) or DAFGGG (SEQ ID NO: 192); and that contains a stretch of amino acid
residues
upstream of position 3 (and/or said R residue) that is as described herein
(including the
preferred aspects of such a stretch of amino acid residues, also as described
herein).
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin and that comprises the sequence motif RXWD (in
which X
is preferably chosen from W, Y, F, S or D) and the sequence motif DVFGGGT (SEQ
ID
NO:19), DVFGGGS (SEQ ID NO: 45) of DAFGGG T (SEQ ID NO, 46); and that contains
a
stretch of amino acid residues upstream of position 3 (and/or said R residue)
that is as
described herein (including the preferred aspects of such a stretch of amino
acid residues,
also as described herein).
Again, all the above amino acid sequences of the invention are preferably such
that
they bind to human serum albumin (as determined using Biacore) with an
affinity (expressed
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54
as KD) better than 100 nM, preferably better than 5OnM, more preferably better
than 30 nM,
such as equal to or better than 20nM, measured either using the amino acid
sequence of the
invention per se or (preferably) measured using a fusion of the amino acid
sequence of the
invention to another protein or peptide, for example as a fusion with a
NANOBODYQ (such
as the Nanobody 2D3 used as an example herein).
Also, where the above peptides are said to contain the sequence motif RXWD,
either
(i) the Arg (R) residue in this motif is capable of fonning a hydrogen bond
with the amino
acid residues Asn (N) 1' )3 & Asn (N) 1.3 5 of human serum albumin and/or
capable of
forming electrostatic interactions with the main-chain oxygen atoms of the Pro
(P) 134 and
Leu (L) 136 residues of human serum albumin; and/or (ii) the Trp (W) residue
in this motif is
capable of forming electrostatic interactions with the Arg (R) 138 residue of
human serum
albumin; and preferably both (i) and (ii) apply.
As mentioned, all these peptides may be as further described herein for the
amino acid
sequences of the invention.
In another aspect, the invention relates to a peptide that is specific for (as
defined
herein) for human serum albumin that comprises:
a) the sequence motif RXWD (in which X may be any amino acid, but is most
preferably
chosen from W, Y, F, S or D ), in which (i) the Arg (R) residue in this motif
is capable of
forming a hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135
of
human serum albumin and/or capable of forming electrostatic interactions with
the main-
chain oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum
albumin; and/or (ii) the Trp (W) residue in this motif is capable of forming
electrostatic
interactions with the Arg (R) 138 residue of human serum albumin; and
preferably both
(i) and (ii.) apply; and
b) contains upstream of the RXWD motif, a stretch of amino acid residues of
between 2 and
10 amino acid residues, which comprises at least one hydrophobic and/or
aromatic amino
acid residue at least one hydrophobic and/or aromatic amino acid residue such
that at least
one of said hydrophobic and/or aromatic amino acid residues can bind (in)to a
subpocket
in (human) serum albumin that comprises (at least) one or more of the
following amino
acid residues of human serum albumin: V442, S443, T446, L484, L487, H488,
K490,
T491 and/or V493.
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This peptide preferably further contains the sequence motif FGGG (SEQ ID
NO:6),
more preferably the sequence motif DVFGGG (SEQ ID NO:15), and even more
preferably
the sequence motif DVGGGGT (SEQ ID NO:19). Also, again, said stretch of amino
acid
residues upstream of position 3 can be as further described herein, and is
preferably
according to one of the preferred aspects described herein.
Again, all the above amino acid sequences of the invention are preferably such
that
they bind to human serum albumin (as determined using Biacore) with an
affinity (expressed
as KD) better than 100 nM, preferably better than 5OnM, more preferably better
than 30 nM,
such as equal to or better than 20nM, measured either using the amino acid
sequence of the
10 invention per se or (preferably) measured using a fusion of the amino acid
sequence of the
invention to another protein or peptide, for example as a fusion with a
NANOBODY (such
as the Nanobody 2D3 used as an example herein).
In another aspect, the invention relates to a peptide that competes with the
peptide of
SEQ ID NO:I and/or with one or more of the peptides 59F2 (WO 09/127691: SEQ ID
NO:
149/ SEQ ID NO: 76 herein); 59H12 (WO 09/127691: SEQ ID NO: 1551 SEQ ID NO: 77
herein); and/or 59C2 (WO 09/127691: SEQ ID NO: 156/SEQ ID NO: 75 herein) for
binding
to human serum albumin, and/or that cross-blocks (as defined herein) the
binding of the
peptide of SEQ ID NO: 1, and/or the binding of one or more of the peptides
59F2 (WO
09/127691: SEQ ID NO, 149); 59H12 (WO 09/127691: SEQ ID NO: 155); and/or 59C2
to
20 human serum albumin; and that binds to human serum albumin with an affinity
(expressed as
Kai) better than 100 nM, preferably better than 5OnM, more preferably better
than 30 nM,
such as equal to or better than 20nM, measured either using the amino acid
sequence of the
invention per se or (preferably) measured using a fusion of the amino acid
sequence of the
invention to another protein or peptide, for example as a fusion with a
NANOBODY (such
as the Nanobody 2D3 used as an example herein).
Such peptides may be as further described herein. Also, and. in particular.
the above
peptides may compete with at least one of the peptides described in Table 11,
respectively, for
binding to one or more of the following amino acid residues of human serum
albumin
(numbering as indicated in Example 8):
30 - Asn (N) 133; Pro (P) 134; Asn (N) 135; Leu (L) 136; Leu (L) 139; Arg (R)
141; Tyr (Y)
162; Gin (E) 165; Ile (I) 166; His (11) 170; Phe (F) 173; Phe (F) 181; Gly (G)
213; Lys
(K) 214; Ser (S) 217; Gln (Q) 483; and/or Lys (K) 543; more in particular to
an epitope
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56
on human serum albumin that comprises either (i) the stretch of amino acid
residues that
comprises the residues Asn (N) 133; Pro (P) 134; Asn (N) 135; Leu (L) 136; Leu
(L) 139
and Arg (R) 141; and/or (ii) the stretch of amino acid residues that comprises
the residues
Tyr (Y) 162; G1u (E) 165; Ile (I) 166; His (H) 170; Phe (F) 173; Phe (F) 181;
and/or (iii)
the stretch of amino acid residues that comprises the residues Gly (G) 213;
Lys (K) 214
and Ser (S) 217; and even more in particular with a hydrophobic subpocket on
human
serum albumin that is comprises (amongst others) residues the residues Leu (L)
139, Glu
(E) 165, Ile (1) 166, His (H) 170, Phe (F) 173, Phe (F) 181, Gly (G) 213, Lys
(K) 214, Ser
(S) 217 and Gln. (Q) 483;
and/or
- the stretch of amino acids (subpocket) in (human) serum albumin that
comprises (at least)
one or more of the following amino acid residues of human serum albumin V442,
S443,
T446, L484, L487, H488, K490, T491 and/or V493.
The invention also relates to a nucleotide sequence or nucleic acid that
encodes an
amino acid sequence of the invention or a polypeptide of the invention (also
referred to
herein as a "nucleotide sequence of the invention" or a "nucleic acid of the
invention").
The invention also relates to a host or host cell that contains a nucleotide
sequence or
nucleic acid of the invention and/or that expresses (or is capable of
expressing) an amino acid
sequence of the invention or a polypeptide of the invention.
The invention also relates to methods for preparing the amino acid sequences
and
compounds of the invention, which methods are as further described herein.
The invention further relates to a composition that comprises at least one
amino acid
sequence of the invention or compound of the invention; and optionally one or
more further
suitable components or constituents. In particular, the invention relates to a
pharmaceutical
composition that comprises at least one amino acid sequence of the invention,
compound of
the invention, or nucleic acid of the invention; and optionally at least one
pharmaceutically
acceptable carrier, diluent or excipient.
The invention also encompasses some other methods for preparing the constructs
and
compounds of the invention, which generally comprise the step of linking at
least one amino
acid sequence of the invention to at least one therapeutic moiety, optionally
via one or more
suitable linkers or spacers. This may be performed in any suitable manner
known per se, for
example depending on the linker(s) used (if any), and may for example comprise
techniques
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57
for chemical linking known per se in the art, for example by formation of one
or more
covalent bonds. The one or more amino acid sequences of the invention and the
one or more
therapeutic moieties may be as further described herein. Again, the one or
more amino acid
sequences of the invention preferably comprise a disulphide bridge as
described herein.
The invention also relates to compound or construct that is obtained via any
of the
above methods; and also to a pharmaceutical composition that comprises at
least one such
compound or construct and optionally at least one pharmaceutically acceptable
carrier,
diluent or excipient.
The invention also relates to uses of the amino acid sequences of the
invention.
Generally, these uses comprise any use known per se for binding units, binding
domains or
amino acid sequences that can bind to serum proteins in general, and serum
albumin in
particular. Such uses will be clear to the skilled person, and not only
include increasing the
half-life to therapeutic moieties, entities or drugs; but also (or in
addition) directing
therapeutic moieties, entities or drugs to parts of the body or tissues where
serum albumin is
present and/or accumulates in the body, such as inflammation sites or joints.
The invention further relates to therapeutic uses of polypeptide or protein
constructs
or fusion proteins and to pharmaceutical compositions comprising such
polypeptide or
protein constructs or fusion proteins.
Detailed Description of the Invention
In the present description, examples and claims:
a) Unless indicated otherwise herein (for example, in Example 8), amino acid
residues and
positions in the amino acid sequences of the invention will be numbered with.
reference
to the corresponding amino acid residues and positions in the
AASYSDYDVFGGGTDFGP (SEQ ID NO:1).
b) Unless indicated otherwise herein (for example, in Example S), amino acid
substitutions will be mentioned with reference to the amino acid residue
present at the
corresponding position in the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ
ID NO, 1). For example, S3R refers to a substitution, compared to the amino
acid
sequence AASYSDYDVFGGGTDFGP (SEQ ID NO: 1), of the serine residue S at
position 3 into arginine (R).
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c) Unless indicated or defined otherwise, all terms used have their usual
meaning in the
art, which will be clear to the skilled person. Reference is for example made
to the
standard handbooks mentioned in paragraph a) on page 46 of WO 08/020079 of
Ablynx
N.V. entitled "Amino acid sequences directed against II.-6R and polypeptides
comprising the same for the treatment of diseases and disorders associated
with 11-6
mediated signalling".
d) Unless indicated otherwise, the terms "immunoglobulin sequence",
"Sequence",
"nucleotide sequence" and "nucleic acid" are as described in paragraph b) on
page 46
of WO 08/020079.
e) Unless indicated otherwise, all methods, steps, techtmiques and
manipulations that are
not specifically described in detail can be performed and have been performed
in a
manner known per se, as will be clear to the skilled person. Reference is for
example
again made to the standard handbooks and the general background art mentioned
herein
and to the further references cited therein; as well as to for example the
following
reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56; Levin and Weiss,
Mol.
Biosyst. 2006, 2(1): 49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-
2), 31-45;
Schmitz et al., Placenta, 2000, 21 Suppl. A, SI 06-12, Gonzales et al., Tumour
Biol.,
2005, 26(1), 31-43, which describe techniques for protein engineering, such as
affinity
maturation and other techniques for improving the specificity and other
desired
properties of proteins such as immunoglobulins.
f) Amino acid residues will be indicated according to the standard three-
letter or one-
letter amino acid code, as mentioned in Table A;
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Table A: one-letter and three-letter amino acid code
Nonpolar, Alanine Ala A
uncharged Valine Val V
(at pH 6.0 - Leucine Lena. L
7.0)(3) Isoleucine Ile I
Phenylalanine Phe F
Methionine Met M
Tryptophan Trp W
Proline Pro P
Polar, Glycine Gly G
uncharged Serine Ser S
(at pH 6.0-7.0) Threonine Thr T
Cysteine Cys C
Asparagine Asn N
Glutamie Gin Q
Tyrosine Tyr Y
Polar, Lysine Lys K
charged Arginine Arg R
(at pH 6.0-7.0) Histidine His H
Aspartate Asp D
Glutamate Glu E
Notes:
W Sometimes also considered to be a polar uncharged amino acid.
~2)
Sometimes also considered to be a nonpolar uncharged amino acid.
(3) As will be clear to the skilled person, the fact that an amino acid
residue is referred to in
this Table as being either charged or uncharged at pH 6.0 to 7.0 does not
reflect in any
way on the charge said amino acid residue may have at a pH lower than 6.0
and/or at a
pH higher than 7.0; the amino acid residues mentioned in the Table can be
either charged
i j
and/or uncharged at such. a higher or lower pH, as will be clear to the
skilled person.
a~ As is known in the art, the charge of a His residue is greatly dependant
upon even small
shifts in pH, but a His residue can generally be considered essentially
uncharged at a pH
of about 6.5.
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g) For the purposes of comparing two or more nucleotide sequences, the
percentage of
`.sequence identity" between a first nucleotide sequence and a second
nucleotide
sequence may be calculated or determined as described in paragraph c) on page
49 of
WO 08/020079 (incorporated herein by reference), such as by dividing [the
number of
nucleotides in the first nucleotide sequence that are identical to the
nucleotides at the
corresponding positions in the second nucleotide sequence] by [the total
number of
nucleotides in the first nucleotide sequence] and multiplying by [100%], in
which each
deletion, insertion, substitution or addition of a nucleotide in the second
nucleotide
sequence - compared to the first nucleotide sequence - is considered as a
difference at a
10 single nucleotide (position); or using a suitable computer algorithm or
technique, again
as described in paragraph c) on pages 49 of WO 08/020079 (incorporated herein
by
reference),
h) For the purposes of comparing two or more amino acid sequences, the
percentage of
"sequence identity" between a first amino acid sequence and a second amino
acid
sequence (also referred to herein as "amino acid identity") may be calculated
by
dividing [the number of amino acid residues in the first amino acid sequence
that are
identical to the amino acid residues at the corresponding positions in the
second amino
acid sequence] by [the total number of amino acid residues in the first amino
acid
sequence] and multiplying by [100%], in which each deletion, insertion,
substitution or
20 addition of an amino acid residue in the second amino acid sequence -
compared to the
first amino acid sequence - is considered as a difference at a single amino
acid residue
(position), i.e. as an "amino acid difference" as defined herein.
Alternatively, the degree of sequence identity between two amino acid
sequences may
be calculated using a known computer algorithm, such as those mentioned above
for
determining the degree of sequence identity for nucleotide sequences, again
using
standard settings.
Usually, for the purpose of determining the percentage of "sequence identity"
between
two amino acid sequences in accordance with the calculation method outlined
hereinabove, the amino acid sequence with the greatest number of amino acid
residues
30 will be taken as the "first" amino acid sequence, and the other amino acid
sequence
will be taken as the "second" amino acid sequence.
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Also, in determining the degree of sequence identity between two amino acid
sequences, the skilled person may take into account so-called "conservative"
amino
acid substitutions, which can generally be described as amino acid
substitutions in
which. an amino acid residue is replaced with another amino acid residue of
similar
chemical structure and which has little or essentially no influence on the
function,
activity or other biological properties of the polypeptide. Such conservative
amino acid
substitutions are well known in the art, for example from WO 04/037999, GB-A-3
357
768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or
combinations of such substitutions may be selected on the basis of the
pertinent
teachings from WO 04/037999 as well as WO 98/49185 and from the further
references
cited therein.
Such conservative substitutions preferably are substitutions in which one
amino acid
within the following groups (a) - (e) is substituted by another amino acid
residue within
the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala,
Ser, Thr,
Pro and Gly; (b) polar, negatively charged residues and their (uncharged)
amides: Asp,
Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys;
(d) large
aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic
residues: Phe,
Tyr and Trp.
Particularly preferred conservative substitutions are as follows: Ala into Gly
or into
Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln
into Asn;
Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; Ile into Leu
or into
Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into
Leu, into Tyr
or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser;
Trp into Tyr;
Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
Any amino acid substitutions applied to the polypeptides described herein may
also be
based on the analysis of the frequencies of amino acid variations between
homologous
proteins of different species developed by Schulz et at, Principles of Protein
Structure,
Springer-Verlag, 1978, on. the analyses of structure forming potentials
developed by
Chou and Pasnzan, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149,
1978,
and on the analysis of hydrophobicity patterns in proteins developed by
Eisenberg et
al.. Proc. Natl. Acad. Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J Molec.
Biol.
157: 105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353,
1986,
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all incorporated herein in their entirety by reference. Information on the
primary,
secondary and tertiary structure of NANOBODIES is given in the description
herein
and in the general background art cited above. Also, for this purpose, the
crystal
structure of a V1H domain from a llama is for example given by Desmyter et
al., Nature
Structural Biology, Vol. 3, 9, 803 (1996); Spinelli et al., Natural Structural
Biology
(1996); 3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361 (1999).
Further
information about some of the amino acid residues that in conventional VH
domains
form the VH/VL, interface and potential camelizing substitutions on these
positions can
be found in the prior art cited above.
i) Amino acid sequences and nucleic acid sequences are said to be "exactly the
same" if
they have 100% sequence identity (as defined herein) over their entire length;
j) When comparing two amino acid sequences, the term "amino acid difference"
refers to
an insertion, deletion or substitution of a single amino acid residue on a
position of the
first sequence, compared to the second sequence; it being understood that two
amino
acid sequences can contain one, two or more such amino acid differences;
k) When a nucleotide sequence or amino acid sequence is said to "comprise"
another
nucleotide sequence or amino acid sequence, respectively, or to "essentially
consist of
another nucleotide sequence or amino acid sequence, this has the meaning
given. in
paragraph i) on pages 51-52 of WO 08/020079.
1) The term "in essentially isolated form" has the meaning given to it in
paragraph j) on
pages 52 and 53 of WO 08/020079.
m) The terms "domain" and "binding domain" have the meanings given to it in
paragraph
k) on page 53 of WO 08/020079.
n) The terms "antigenic determinant" and "epitope", which may also be used
interchangeably herein. have the meanings given to it in paragraph 1) on page
53 of WO
08/020079,
o) As further described in paragraph m) on page 53 of WO 08/020079, an amino
acid
sequence (such as a NANOBODY(T, an antibody, a polypeptide of the invention,
or
generally an antigen binding protein or polypeptide or a fragment thereof)
that can
(specifically) bind to, that has affinity for and/or that has specificity for
a specific
antigenic determinant, epitope, antigen or protein (or for at least one part,
fragment or
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epitope thereof) is said to be "against" or "directed against" or "specific/or
" said
antigenic determinant, epitope, antigen or protein.
p) The terms "specificity" and "specific for " have the meaning given to it in
paragraph n)
on pages 53-56 of WO 08/020079; and as mentioned therein refers to the number
of
different types of antigens or antigenic determinants to which a particular
antigen-
binding molecule or antigen-binding protein (such as a NANOBODY or a
polypeptide of the invention) molecule can bind. The specificity of an antigen-
binding
protein can be determined based on affinity and/or avidity, as described on
pages 53-56
of WO 08/020079 (incorporated herein by reference), which also describes some
preferred techniques for measuring binding between an antigen-binding molecule
(such
as a NANOBODY or polypeptide of the invention) and the pertinent antigen.
Typically, antigen-binding proteins (such as the amino acid sequences and/or
compounds of the invention) will bind to their antigen with a dissociation
constant (KD)
of 10-5 to 10-12 moles/liter or less, and preferably 10--f to 10-12
moles/liter or less and
more preferably 10 to 10-12 moles/liter (i.e. With an association constant
(KAY) of 105 to
1012 liter/ moles or more, and preferably 1.02 to 1012 liter/moles or more and
more
preferably 108 to 1012 liter/moles). Any KD value greater than 104 mol/liter
(or any KA
value lower than 104 M-1) liters/mol is generally considered to indicate non-
specific
binding. Preferably, an amino acid sequence or compound of the invention will
bind to
the desired serum protein with an affinity less than 1000 nM, preferably less
than 500
nM, preferably less than 200 nM, more preferably less than 10 nM, such as less
than
500 pM. Specific binding of an antigen-binding protein to an antigen or
antigenic
determinant can be determined in any suitable manner known per se, including,
for
example, Scatchard analysis and/or competitive binding assays, such as
radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition
assays, and the different variants thereof known per se in the art; as well as
the other
techniques mentioned herein.
As will be clear to the skilled person, and as described on pages 53-56 of WO
08/020079, the dissociation constant may be the actual or apparent
dissociation
constant.- Methods for determining the dissociation constant will be clear to
the skilled
person, and for example include the techniques mentioned on pages 53-56 of WO
08/020079
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q) The half-life of an amino acid sequence, compound or polypeptide of the
invention can
generally be defined as the time taken for the serum concentration of the
amino acid
sequence, compound or polypeptide to be reduced by 50%, in vivo, for example
due to
degradation of the sequence or compound and/or clearance or sequestration of
the
sequence or compound by natural mechanisms. The in vivo half-life of an amino
acid
sequence, compound or polypeptide of the invention can be determined in any
manner
known per se, such as by phannacokinetic analysis. Suitable techniques will be
clear to
the person skilled in the art, and may for example generally involve the steps
of
suitably administering to a warm.-blooded animal (i.e. to a human or to
another suitable
mammal, such as a mouse, rabbit, rat, pig, dog or a primate, for example
monkeys from
the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca
fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio
ursinus)) a
suitable dose of the amino acid sequence, compound or polypeptide of the
invention;
collecting blood samples or other samples from said animal; determining the
level or
concentration of the amino acid sequence, compound or polypeptide of the
invention in
said blood sample; and calculating, from (a plot of) the data thus obtained,
the time
until the level or concentration of the amino acid sequence, compound or
polypeptide
of the invention has been reduced by 50% compared to the initial level upon
dosing.
Reference is for example made to the Experimental Part below, as well as
Dennis et at.,
J. Biol. Chem. 277:35035-42 (2002) to the standard handbooks, such as Kenneth,
A et
al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and
Peters et
al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also
made to
"Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev.
edition (1982).
As will also be clear to the skilled person (see for example pages 6 and 7 of
WO
04/003019 and in the further references cited therein), the half-life can be
expressed
using parameters such as the tl/2-alpha, tl/2-beta and the area under the
curve (AUC).
In the present specification, an "increase in half life" refers to an increase
in any one of
these parameters, such as any two of these parameters, or essentially all
three these
parameters. As used herein "increase in half-life" or "increased half-life" in
particular
refers to an increase in the tl/2-beta, either with or without an increase in
the tl/2-alpha
and/or the AUC or both.
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r) In the context of the present invention, "modulating" or "to modulate"
generally means
either reducing or inhibiting the activity of, or alternatively increasing the
activity of, a
target or antigen, as measured using a suitable in vitro, cellular or in vivo
assay. In
particular, "modulating" or "to modulate" may mean either reducing or
inhibiting the
activity of, or alternatively increasing a (relevant or intended) biological
activity of, a
target or antigen, as measured using a suitable in vitro, cellular or in vivo
assay (which
will usually depend on the target or antigen involved), by at least M,
preferably at least
5%, such as at least 10% or at least 25%, for example by at least 50%, at
least 60%, at
least 70%, at least 80%, or 90% or more, compared to activity of the target or
antigen in
10 the same assay under the same conditions but without the presence of the
construct of
the invention.
As will be clear to the skilled person, "modulating" may also involve
effecting a change
(which may either be an increase or a decrease) in affinity, avidity,
specificity and/or
selectivity of a target or antigen for one or more of its ligands, binding
partners,
partners for association into a homomultimeric or heteromultimeric form, or
substrates;
and/or effecting a change (which may either be an increase or a decrease) in
the
sensitivity of the target or antigen for one or more conditions in the medium
or
surroundings in which the target or antigen is present (such as pH, ion
strength, the
presence of co-factors, etc.), compared to the same conditions but without the
presence
20 of the construct of the invention. As will be clear to the skilled person,
this may again
be determined in any suitable manner and/or using any suitable assay known per
se,
depending on the target or antigen involved.
"Modulating" may also mean effecting a change (i.e. an activity as an agonist,
as an
antagonist or as a reverse agonist, respectively, depending on the target or
antigen and
the desired biological or physiological effect) with respect to one or more
biological or
physiological mechanisms, effects, responses, functions, pathways or
activities in
which the target or antigen (or in which its substrate(s), ligand(s) or
pathway(s) are
involved, such as its signalling pathway or metabolic pathway and their
associated
biological or physiological effects) is involved. Again, as will be clear to
the skilled
30 person, such an action as an agonist or an antagonist may be determined in
any suitable
manner and/or using any suitable (in vitro and usually cellular or in assay)
assay known
per se, depending on the target or antigen involved. In particular, an action
as an
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agonist or antagonist may be such that an intended biological or physiological
activity
is increased or decreased, respectively, by at least I%, preferably at least
5%, such as at
least 10% or at least 25%. for example by at least 50%, at least 60%, at least
70%, at
least 80%, or 90% or more, compared to the biological or physiological
activity in the
same assay under the same conditions but without the presence of the construct
of the
invention.
Modulating may for example also involve allosteric modulation of the target or
antigen;
and/or reducing or inhibiting the binding of the target or antigen to one of
its substrates
or ligands and/or competing with a natural ligand, substrate for binding to
the target or
antigen. Modulating may also involve activating the target or antigen or the
mechanism
or pathway in which it is involved. Modulating may for example also involve
effecting
a change in respect of the folding or confirmation of the target or antigen,
or in respect
of the ability of the target or antigen to fold, to change its confirmation
(for example,
upon binding of a ligand), to associate with other (sub)units, or to
disassociate.
Modulating may for example also involve effecting a change in the ability of
the target
or antigen to transport other compounds or to serve as a channel for other
compounds
(such as ions).
Modulating may be reversible or irreversible, but for pharmaceutical and
pharmacological purposes will usually be in a reversible manner.
s) In respect of a target or antigen, the term "interaction site" on the
target or antigen
means a site, epitope, antigenic determinant, part, domain or stretch of amino
acid
residues on the target or antigen that is a site for binding to a ligand,
receptor or other
binding partner, a catalytic site, a cleavage site, a site for allosteric
interaction, a site
involved in multimerization (such as homo(di)meri.zation or
hetero(di)merization) of
the target or antigen; or any other site, epitope, antigenic determinant,
part, domain or
stretch of amino acid residues on the target or antigen that is involved in a
biological
action or mechanism of the target or antigen. More generally, an "interaction
site" can
be any site, epitope, antigenic determinant, part, domain or stretch of amino
acid
residues on the target or antigen to which an amino acid sequence or
polypeptide of the
invention can bind such that the target or antigen (and/or any pathway,
interaction,
signalling, biological mechanism or biological effect in which the target or
antigen is
involved) is modulated (as defined herein).
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t) An amino acid sequence or polypeptide is said to be "specific. for" a first
target or
antigen compared to a second target or antigen when is binds to the first
antigen with an
affinity (as described above, and suitably expressed as a Kr value, KA value,
Koff rate
and/or Kx,z rate) that is at least 10 times, such as at least 1.00 times, and
preferably at
beast 1000 times, and up to 10,000 times or more better than the affinity with
which
said amino acid sequence or polypeptide binds to the second target or
polypeptide. For
example, the first antigen may bind to the target or antigen with a KD value
that is at
least 10 times less, such as at least 100 times less, and preferably at least
1000 times
less, such as 10,000 times less or even less than that, than the KD with which
said amino
acid sequence or polypeptide binds to the second target or polypeptide.
Preferably,
when an amino acid sequence or polypeptide is "specific for" a first target or
antigen
compared to a second target or antigen, it is directed against (as defined
herein.) said
first target or antigen, but not directed against said second target or
antigen.
u) An amino acid sequence is said to be "cross-reactive" for two different
antigens or
antigenic determinants (such as serum albumin from two different species of
mammal,
such as human serum albumin and cyno serum albumin) if it is specific for (as
defined
herein) both these different antigens or antigenic determinants.
v) By binding that is "essentially independent of the pH" is generally meant
herein that the
association constant (KA) of the amino acid sequence with respect to the serum
protein
(such as serum albumin) at the pH value(s) that occur in a cell of an animal
or human
body (as further described herein) is at least 5%, such as at least 10%,
preferably at
least 25%, more preferably at least 50%, even more preferably at least 60%,
such as
even more preferably at least 70%, such as at least 80% or 90% or more (or
even more
than 100%, such as more than 110%, more than 120% or even 130% or more, or
even
more than 150%, or even more than 200%) of the association constant (KA) of
the
amino acid sequence with respect to the same serum protein at the pH value(s)
that
occur outside said cell. Alternatively, by binding that is "essentially
independent of the
pH" is generally meant herein that the koff rate (measured by Biacore - see
e.g.
Experiment 2) of the amino acid sequence with respect to the serum protein
(such as
serum albumin) at the pH value(s) that occur in a cell of an animal or human
body (as
e.g. further described herein, e.g. pH around 5.5, e.g. 5.3 to 5.7) is at
least 5%, such as
at least 10%, preferably at least 25%, more preferably at least 50%, even more
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preferably at least 60%. such as even more preferably at least 70%, such as at
least 80%
or 90% or more (or even more than 1.00%, such as more than 110%, more than
120% or
even 130% or more, or even more than 150%, or even more than 200%) of the koff
rate
of the amino acid sequence with respect to the same serum protein at the pH
value(s)
that occur outside said cell, e.g. PH 7.2 to 7.4. By "the pH value(s) that
occur in a cell
of an animal or human body" is meant the pH value(s) that may occur inside a
cell, and
in particular inside a cell that is involved in the recycling of the serum
protein. In
particular, by "the pH value(s) that occur in a cell of an animal or human
body" is
meant the pH value(s) that may occur inside a (sub)cellular compartment or
vesicle that
is involved in recycling of the serum protein (e.g. as a result of
pinocytosis,
endocytosis, transcytosis, exocytosis and phagocytosis or a similar mechanism
of
uptake or internalization into said cell), such as an endosome, lysosome or
pinosome.
w) The terms "cross-block", "cross-blocked" and "cross-blocking" are used
interchangeably herein to mean the ability of an amino acid sequence or other
binding
agents (such as a NANOBODY , polypeptide or compound or construct of the
invention) to interfere with the binding of other amino acid sequences or
binding agents
of the invention to a given target. The extend to which. an amino acid
sequence or other
binding agents of the invention is able to interfere with the binding of
another to the
relevant, and therefore whether it can be said to cross-block according to the
invention,
can be determined using competition binding assays. One particularly suitable
quantitative cross-blocking assay uses a Biacore machine which can measure the
extent
of interactions using surface plasmon resonance technology. Another suitable
quantitative cross-blocking assay uses an ELISA-based approach to measure
competition between amino acid sequences or other binding agents in terms of
their
binding to the target.
The following generally describes a suitable Biacore assay for determining
whether an
amino acid sequence or other binding agent cross-blocks or is capable of cross-
blocking
according to the invention. It will be appreciated that the assay can be used
with any of
the amino acid sequences or other binding agents described herein. The Biacore
machine (for example the Biacore 3000) is operated in line with the
manufacturer's
recommendations. Thus in one cross-blocking assay, the target protein is
coupled to a
CMS Biacore chip using standard amine coupling chemistry to generate a surface
that is
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coated with the target. Typically 200- 800 resonance units of the target would
be
coupled to the chip (an amount that gives easily measurable levels of binding
but that is
readily saturable by the concentrations of test reagent being used). Two test
amino acid
sequences (termed A* and B*) to be assessed for their ability to cross-block
each other
are mixed at a one to one molar ratio of binding sites in a suitable buffer to
create the
test mixture. When calculating the concentrations on a binding site basis the
molecular
weight of an amino acid sequence is assumed to be the total molecular weight
of the
amino acid sequence divided by the number of target binding sites on that
amino acid
sequence. The concentration of each amino acid sequence in the test mix should
be high
enough to readily saturate the binding sites for that amino acid sequence on
the target
molecules captured on the Biacore chip. The amino acid sequences in the
mixture are at
the same molar concentration (on a binding basis) and that concentration would
typically be between 1.00 and 1.5 micromolar (on a binding site basis).
Separate
solutions containing A* alone and B* alone are also prepared. A* and B* in
these
solutions should be in the same buffer and at the same concentration as in the
test mix.
The test mixture is passed over the target-coated Biacore chip and the total
amount of
binding recorded. The chip is then treated in such a way as to remove the
bound amino
acid sequences without damaging the chip-bound target. Typically this is done
by
treating the chip with 30 mM HCl for 60 seconds. The solution of A* alone is
then
passed over the target-coated surface and the amount of binding recorded. The
chip is
again treated to remove all of the bound amino acid sequences without damaging
the
chip-bound target. The solution of B* alone is then passed over the target-
coated
surface and the amount of binding recorded. The maximum theoretical binding of
the
mixture of A* and B* is next calculated, and is the sum of the binding of each
amino
acid sequence when passed over the target surface alone. If the actual
recorded binding
of the mixture is less than this theoretical maximum then the two amino acid
sequences
are cross-blocking each other. Thus, in general, a cross-blocking amino acid
sequence
or other binding agent according to the invention is one which will bind to
the target in
the above Biacore cross-blocking assay such that, during the assay and in the
presence
of a second amino acid sequence or other binding agent of the invention, the
recorded
binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical
binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum
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theoretical binding, and more specifically between 70% and 0.1% (e.g. 70% to
4%) of
maximum theoretical binding (as just defined above) of the two amino acid
sequences
or binding agents in combination. The Biacore assay described. above is a
primary assay
used to determine if amino acid sequences or other binding agents cross-block
each
other according to the invention. On rare occasions particular amino acid
sequences or
other binding agents may not bind to target coupled via amine chemistry to a
CM5
Biacore chip (this usually occurs when the relevant binding site on target is
masked or
destroyed by the coupling to the chip). In such cases cross-blocking can be
determined
using a tagged version of the target, for example a N-terminal His-tagged
version. In
10 this particular format, an anti-His amino acid sequence would be coupled to
the Biacore
chip and then the His-tagged target would be passed over the surface of the
chip and
captured by the anti-His amino acid. sequence. The cross blocking analysis
would be
carried out essentially as described above, except that after each chip
regeneration
cycle, new His-tagged target would be loaded back onto the anti-His amino acid
sequence coated surface. In addition to the example given using N-terminal His-
tagged
target, C-terminal His-tagged target could alternatively be used. Furthermore,
various
other tags and tag binding protein combinations that are known in the art
could be used
for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG
tag
with anti-FLAG antibodies; biotin tag with streptavidin).
20 The following generally describes an ELISA assay for determining whether an
amino
acid sequence or other binding agent directed against a target cross-blocks or
is capable
of cross-blocking as defined herein. It will be appreciated that the assay can
be used
with any of the amino acid sequences (or other binding agents such as
polypeptides of
the invention) described herein. The general principal of the assay is to have
an amino
acid sequence or binding agent that is directed against the target coated onto
the wells
of an ELISA plate. An excess amount of a second, potentially cross-blocking,
anti-
target amino acid sequence is added in solution (i.e. not bound to the ELISA
plate). A
limited amount of the target is then added to the wells. The coated amino acid
sequence
and the amino acid sequence in solution compete for binding of the limited
number of
30 target molecules. The plate is washed to remove excess target that has not
been bound
by the coated amino acid sequence and to also remove the second, solution
phase amino
acid sequence as well as any complexes formed between the second, solution.
phase
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amino acid sequence and target. The amount of bound target is then measured
using a
reagent that is appropriate to detect the target. An amino acid sequence in
solution that
is able to cross-block the coated amino acid sequence will be able to cause a
decrease in
the number of target molecules that the coated amino acid sequence can bind
relative to
the number of target molecules that the coated amino acid sequence can bind in
the
absence of the second, solution phase, amino acid sequence. In the instance
where the
first amino acid sequence, e.g. an Ab-X, is chosen to be the immobilized amino
acid
sequence, it is coated onto the wells of the ELISA plate, after which the
plates are
blocked with a suitable blocking solution to minimize non-specific binding of
reagents
that are subsequently added. An excess amount of the second amino acid
sequence, i.e.
Ab-Y. is then added to the ELISA plate such that the moles of Ab-Y target
binding sites
per well are at least 10 fold higher than the moles of Ab-X target binding
sites that were
used, per well, during the coating of the ELISA plate. Target is then added
such that the
moles of target added per well are at least 25-fold lower than the moles of Ab-
X target
binding sites that were used for coating each well. Following a suitable
incubation
period the ELISA plate is washed and a reagent for detecting the target is
added to
measure the amount of target specifically bound by the coated anti[target
amino acid
sequence (in this case Ab-X). The background signal for the assay is defined
as the
signal obtained in wells with the coated amino acid sequence (in this case Ab-
X),
second solution phase amino acid sequence (in this case Ab-Y), target buffer
only (i.e.
without target) and target detection reagents. The positive control signal for
the assay is
defined as the signal obtained in wells with the coated amino acid sequence
(in this case
Ab-X), second solution phase amino acid sequence buffer only (i.e. without
second
solution phase amino acid sequence), target and target detection reagents. The
ELISA
assay may be run in such a manner so as to have the positive control signal be
at least 6
times the background signal. To avoid any artefacts (e.g. significantly
different
affinities between Ab-X and Ab-Y for the target) resulting from the choice of
which
amino acid sequence to use as the coating amino acid. sequence and which to
use as the
second (competitor) amino acid sequence, the cross-blocking assay may to be
run in
two formats: 1) format 1 is where Ab-X is the amino acid sequence that is
coated onto
the ELISA plate and Ab-Y is the competitor amino acid sequence that is in
solution and
2) format 2 is where Ab-Y is the amino acid sequence that is coated onto the
ELISA
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plate and Ab-X is the competitor amino acid sequence that is in solution. Ab-X
and Ab-
Y are defined. as cross-blocking if, either in format 1 or in format 2, the
solution phase
anti-target amino acid sequence is able to cause a reduction of between 60%
and 100%,
specifically between 70% and 100%, and more specifically between 80% and 100%,
of
the target detection signal {i.e. the amount of target bound by the coated
amino acid
sequence) as compared to the target detection signal obtained in the absence
of the
solution phase anti- target amino acid sequence (i.e. the positive control
wells).
x) Any Figures, Sequence Listing and the Experimental Part/Examples are only
given to
further illustrate the invention and should not be interpreted or construed as
limiting the
scope of the invention and/or of the appended claims in any way, unless
explicitly
indicated otherwise herein.
For a general description of heavy chain antibodies and the variable domains
thereof,
reference is inter alia made to the prior art cited herein, to the review
article by Muyldermans
in Reviews in Molecular Biotechnology 74(2001), 277-302; as well as to the
following patent
applications, which are mentioned as general background art: WO 94/04678, WO
95/04079
and WO 96/341.03 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681,
WO
00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and
WO
02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and
WO 03/055527 of the Vlaarns Instituut voor Biotechnologie (VIB); WO 03/050531
of
Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council
of
Canada; WO 03/025020 (= EP 1 433 793) by the Institute of Antibodies; as well
as WO
04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO
05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO
06/122825, by Ablynx N.V. and the further published patent applications by
Ablynx N.V.
Reference is also made to the :further prior art mentioned in these
applications, and in
particular to the list of references mentioned on pages 41-43 of the
International application
WO 06/040153, which list and references are incorporated herein by reference.
The amino acid sequences of the invention may be prepared in a manner known
per
se. For example, a desired amino acid sequence may be prepared by peptide
synthesis or by
suitably expressing a nucleic acid encoding said amino acid sequence. A
desired nucleotide
sequence may be prepared by techniques of nucleic acid synthesis known per se.
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One method for preparing the amino acid sequences or polypeptides of the
invention
generally comprises at least the step of:
a) expressing a nucleotide sequence or nucleic acid of the invention;
and optionally further comprises:
b) isolating the amino acid sequence of the invention or the polypeptide of
the invention,
respectively, so expressed.
Another method for preparing the amino acid sequences or polypeptides of the
invention generally comprises at least the step of:
a) cultivating or maintaining a host or host cell as described herein under
conditions such
that said host or host cell produces an amino acid sequence or polypeptide of
the
invention,
and optionally further comprising:
b) isolating the amino acid sequence of the invention or polypeptide of the
invention
respectively, thus obtained.
Where an amino acid sequence of the invention is to be used in a constrained
format
(i.e. comprising a disulphide bridge between the flanking sequences that flank
the amino acid
sequence of the invention). the above methods may also comprise a further step
of forming
such a disulphide bridge, as further described. in PCT/EP2007/063348.
The invention also relates to the amino acid sequences, compounds, construct
or
polypeptides obtained via the above methods.
The amino acid sequences disclosed herein can be used with advantage as a
fusion
partner in order to increase the half-life of therapeutic moieties such as
proteins, compounds
(including, without limitation, small molecules) or other therapeutic
entities.
Thus, in another aspect, the invention provides amino acid sequences that can
be used
as small peptides or peptide moieties for linking or fusing to a therapeutic
compound in order
to increase the half-life thereof, and constructs and fusion proteins
comprising such peptides
or peptide moieties, that can bind to a serum protein in such a way that, when
the amino acid
sequence, construct, or fusion protein of the invention is bound to a serum
protein molecule,
the half-life of the serum protein molecule is not (significantly) reduced
(i.e. compared to the
half-life of the serum protein molecule when the amino acid sequence,
construct, or fusion
protein is not bound thereto). In this aspect of the invention, by "not
significantly reduced" is
meant that the half-life of the serum protein molecule (as measured using a
suitable technique
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known per se) is not reduced by more than. 50%, preferably not reduced by more
than 30%,
even more preferably not reduced by more than 1.0%, such as not reduced by
more than 5%,
or essentially not reduced at all.
In another preferred, but non-limiting aspect, the amino acid sequences of the
invention are preferably such that they bind to or otherwise associate with
human serum
albumin. in such a way that, when the amino acid sequences are bound to or
otherwise
associated with a human serum albumin, the amino acid sequences exhibit a
serum half-life
in human of at least about 9 days (such as about 9 to 14 days), preferably at
least about 10
days (such as about 1.0 to 15 days), or at least about 11 days (such as about
11 to 16 days),
more preferably at least about 12 days (such as about 12 to 18 days or more),
or more than 14
days (such as about 14 to 19 days).
In another aspect, the invention provides polypeptide or protein constructs
that
comprise or essentially consist of an amino acid sequence as disclosed herein.
The invention also relates to a compound or construct which comprises at least
one
amino acid sequence of the invention and at least one therapeutic moiety (also
referred to
herein as "compounds of the invention").
For example, and without limitation, a compound of the invention may comprise
the
at least one therapeutic moiety, that is linked to one, two, three, four or
more amino acid
sequences of the invention. For example, when the therapeutic moiety is a
protein or
polypeptide, the one or more amino acid sequences of the invention may be
linked to the C-
terminus of the protein or polypeptide (either directly or via a suitable
spacer or linker); to the
N-terminus of the protein or polypeptide (again either directly or via a
suitable spacer or
linker); or both to the C-terminus and the N-terminus. When a compound of the
invention
comprises two or more amino acid sequences of the invention, these may be the
same or
different.
The therapeutic moiety may also be linked (either at its C-terminus, its N-
terminus, or
both, and again either directly or via a suitable spacer or linker) to a
multirer or concatazner
that comprises at least two (such as two, three or four) amino acid sequences
of the invention
(which may be the same or different), that may either be linked directly to
each other, or via a
suitable linker or spacer. Such (bivalent, trivalent or multivalent) multimers
or concatamers
(and nucleotide sequences encoding the same, as well as compounds of the
invention
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comprising the same) form a further aspect of the invention, and may bind to
serum albumin
with a higher avidity than a monomeric amino acid sequence of the invention.
Also, when a compound of the invention comprises two or more therapeutic
moieties,
each of these therapeutic moieties (or both) may be linked to one or more
amino acid
sequences of the invention, as further described herein. Also, the two or more
therapeutic
moieties may be linked to each other via a linker that comprises or
essentially consists of one
or more amino acid sequences of the invention (and optionally further linking
amino acid
sequences), and such a linker (as well as compounds of the invention
comprising the same)
form a further aspect of the invention.
10 In one aspect, the therapeutic moiety is directed. against a desired
antigen or target, is
capable of binding to a desired antigen (and in particular capable of
specifically binding to a
desired antigen), and/or is capable of interacting with a desired target. In
another
embodiment, the at least one therapeutic moiety comprises or essentially
consists of a
therapeutic protein or polypeptide. In a further embodiment, the at least one
therapeutic
moiety comprises or essentially consists of an immunoglobulin or
immunoglobulin sequence
(including but not limited to a fragment of an immunoglobulin), such as an
antibody or an
antibody fragment (including but not limited to an ScFv fragment or Fab
fragment). In yet
another embodiment, the at least one therapeutic moiety comprises or
essentially consists of
an antibody variable domain, such as a heavy chain variable domain or a light
chain variable
20 domain.
In one preferred, but non-limiting aspect, the one or more therapeutic
moieties or
entities may be one or more binding units (as defined in PCT/EP2007/063348) or
binding
domains (as defined herein), i.e, binding units or domain that are capable of
binding to a
desired target, antigen or antigenic deterininant (such as a therapeutically
relevant target). As
such, the compound of the invention may be a monovalent, bivalent, bispecific,
multivalent
or multispecific construct (as defined in PCT/EP2007/063348). The binding unit
may
generally comprise a scaffold-based binding unit or domain, such as binding
scaffolds based
on or derived from immunoglobulins (i.e. other than the immunoglobulin
sequences already
described herein), protein scaffolds derived from protein A domains (such as
AffibodiesTM),
30 tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed
ankyrin repeats,
avimers and PDZ domains (Linz et al., Nat. Biotech 2005, Vol 23:1257). and
binding
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moieties based on DNA or RNA including but not limited to DNA or RNA aptamers
(Ulrich
et al., Comb Chem High Throughput Screen 2006 9(8):619-32).
The amino acid sequences of the invention may also be linked to one of the
"polypeptide drugs" referred to in the International application WO 05/118642
(Domantis
Ltd.) or the International application 06/059106 (Domantis Ltd.); such as to
one of the
polypeptide drugs that are mentioned on pages 45 to 50 of WO 05/118642;
antagonists of the
interleukin I receptor (see pages 11-12 of WO 05/118642) including functional
variants of
IL-Ira; saporins (see pages 12-14 of WO 05/118642); the anticancer peptides
listed in Table
8 of WO 05/118642; and insulinotropic agents or analogues thereof such as GLP-
1 or GLP-1
analogues (see 06/059] 06).
In a preferred aspect, the at least one therapeutic moiety comprises or
essentially
consists of at least one domain antibody or single domain antibody, "dAb" or
NANOBODY . Thus, for example, in a compound of the invention, one or more
amino acid
sequences of the invention may be fused or linked to one or more domain
antibodies, single
domain antibodies, "dAb's" or NANOBODIES , such that the resulting compound of
the
invention is a monovalent, bivalent, multivalent, bispecific or multispecifie
construct (in
which the terms "monovalent", "bivalent", "multivalent", "bispecific" and
"multispecific" are
as described in PCT/EP2007/063348 or in the patent applications of Ablynx N.V.
cited
above).
Thus, one embodiment of the invention relates to a protein or polypeptide
construct or
fusion protein that comprises or essentially consists of at least one amino
acid sequence of the
invention and at least one immunoglobulin sequence, such as a domain antibody,
a single
domain antibody, a "dAb" or a NANOBODY .
Generally, a compound of the invention preferably has a half-life that is more
than I
hour, preferably more than 2 hours, more preferably of more than 6 hours, such
as of more
than 12 hours, and for example of about one day, two days, one week, two weeks
or three
weeks, and preferably no more than 2 months, although the latter may be less
critical.
Preferably, the compounds or polypeptides of the invention that comprise at
least one
amino acid sequence of the invention and at least one therapeutic moiety
preferably have a
half-life that is at least 1.5 times, preferably at least 2 times, such as at
least 5 times, for
example at least 10 times or more than 20 times, greater than the half-life of
the therapeutic
moiety per se. For example, the compounds or polypeptides of the invention may
have a half-
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life that is increased with more than I hours, preferably more than 2 hours,
more preferably
more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72
hours,
compared to the therapeutic moiety per se.
In a preferred, but non-limiting aspect of the invention, such compounds or
polypeptides of the invention have a serum half-life that is increased with
more than I hours,
preferably more than 2 hours, more preferably more than 6 hours, such as more
than 12
hours, or even more than 24, 48 or 72 hours, compared to the therapeutic
moiety per se.
The invention also relates to nucleotide sequences or nucleic acids that
encode amino
acid sequences, compounds, proteins, polypeptides, fusion proteins, or
multivalent or
multi specific constructs described herein. The invention further includes
genetic constructs
that include the foregoing nucleotide sequences or nucleic acids and one or
more elements for
genetic constructs known per se. The genetic construct may be in the form of a
plasmid or
vector. Such and other genetic constructs are known by those skilled in the
art.
The invention also relates to hosts or host cells that contain such nucleotide
sequences
or nucleic acids, and/or that express (or are capable of expressing) amino
acid sequences,
compounds, proteins, polypeptides, fusion proteins, or multivalent or
multispecific constructs
described herein. Again, such hosts or host cells are known by those skilled
in the art.
The invention also generally relates to a method for preparing amino acid
sequences,
compounds, proteins, polypeptides, fusion proteins, or multivalent or
multispecific constructs
as described herein, which method comprises cultivating or maintaining a host
cell as
described herein under conditions such that said host cell produces or
expresses an amino
acid sequence, compound, protein, polypeptide, fusion protein, or multivalent
or
multispecific construct as described herein, and optionally further comprises
isolating the
amino acid sequence, compound, protein, polypeptide, fusion protein, or
multivalent or
multispecific construct so produced. Again, such methods can be performed as
generally
described in the co-pending patent applications by Ablynx N.V. described
herein, such as
WO 04/041862 or WO 06/122825.
In one specific, but non-limiting embodiment, the amino acid sequence,
compound,
protein, polypeptide, fusion protein, or multivalent or multispecific
construct may be
expressed in a suitable strain of Pichia pastoris (such as, for example and
without limitation,
a protease-deficient strain or another suitable strain). As mentioned, when
Pichia pastoris is
used, it may be advantageous to use an amino acid sequence of the invention
that does not
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78
contain a threonine residue at (or close to) position 14, because this residue
may be
susceptible to phosphorylation (depending on the specific Pichia strain used).
Thus, in one aspect, a method of the invention for expressing an amino acid
sequence,
compound, protein, polypeptide, fusion protein, or multivalent or
inultispecific construct (as
described herein) may comprise the following step a): expressing a nucleotide
sequence or
nucleic acid of the invention, in which said nucleotide sequence or nucleic
acid is expressed
in a suitable yeast strain (and in particular, a suitable Pichia strain, such
as a suitable strain of
Pichia pastoris), and in which said nucleotide sequence or nucleic acid of the
invention
encodes an amino acid sequence or peptide of the invention (or a compound,
protein,
polypeptide, fusion protein, or multivalent or multispecific construct
comprising the same)
that does not contain a threonine residue that is susceptible to
phosphorylation when
expressed in said yeast strain. For example, such an amino acid sequence or
peptide of the
invention may comprise an alanine (A), asparagine (N) or aspartate (D) residue
on position
14 (or any other suitable amino acid residue, for example glutamine (Q),
glutamate (E),
glycine (G), isoleucine (I), leucine (L), phenylalanine (F), proline (P),
tryptophan (W) or
valine V), e.g. instead of a threonine residue as is the case in for example
the sequence motifs
of SEQ ID NO's: 7, 16, 19, 21, 22, 24 to 27, and 32 to 43. For example, as
further described
herein, they may suitably contain one of the sequence motifs of SEQ ID NO's:
135 to 140 or
143 to 148.
In one even more specific aspect, a method of the invention for expressing a
amino
acid sequence, compound, protein, polypeptide, fusion protein, or multivalent
or
multispecific construct (as described herein) may comprise the following step
a): expressing a
nucleotide sequence or nucleic acid of the invention, in which said nucleotide
sequence or
nucleic acid is expressed in a suitable yeast strain (and in particular, a
suitable Pichia strain,
such as a suitable strain of Pichia pastoris), and in which said nucleotide
sequence or nucleic
acid of the invention encodes an amino acid sequence of the invention (or a
compound,
protein, polypeptide, fusion protein, or multivalent or multispecific
construct comprising the
same) that comprises at least one of the sequence motifs of SEQ ID NO's: 126
to 148, and in
particular one of the sequence motifs of SEQ ID NO's: 132 to 148 (in which
said amino acid
sequence may again be as further described herein).
Some preferred, but non-limiting examples of amino acid sequences that do not
contain a threonine at (or around) position 14 are given in SEQ ID NO's: 104
to 108; and
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79
SEQ ID NO's: l l l to 125 give some examples of constructs comprising the same
based on
the 5F7 Nanobody (used as an example of a Nanobody).
In another aspect, a method of the invention. for expressing a amino acid
sequence,
compound, protein, polypeptide, fusion protein, or multivalent or
multispecific construct (as
described herein) may comprise the following step a): expressing a nucleotide
sequence or
nucleic acid of the invention, in which said nucleotide sequence or nucleic
acid is expressed
in a suitable yeast strain (and in particular, a suitable Pichia strain, such
as a suitable strain of
Pichia pastoris) that either shows reduced phosphorylation (i.e when used to
express an
amino acid sequence of the invention or a compound comprising the same), and
in particular
reduced (i.e. essentially no) phosphorylation of threonine (T) residues; or in
which said
nucleotide sequence or nucleic acid is expressed in a yeast strain (and in
particular, a suitable
Pichia strain, such as a suitable strain of Pichia pastoris) that has been
genetically modified
to show reduced (i.e. essentially no) phosphorylation (i.e when used to
express an amino acid
sequence of the invention or a compound comprising the same), and in
particular reduced
phosphorylation of threonine (T) residues. This aspect of the invention may
generally be used
to express any amino acid sequence of the invention (or compound comprising
the same),
including without limitation amino acid sequences of the invention that
comprise a threonine
(T) residue that is susceptible to phosphorylation (including without
limitation amino acid
sequences of the invention with threonine (T) residue on position 14).
In a method of the invention that comprises the step of a): cultivating or
maintaining a
host or host cell as described herein under conditions such that said host or
host cell produces
an amino acid sequence or polypeptide of the invention, similar considerations
apply. Thus,
said step a) may comprise: cultivating or maintaining a host or host cell as
described herein
under conditions such that said host or host cell produces an amino acid
sequence or
polypeptide of the invention, in which said host or host is a suitable yeast
strain (and in
particular, a suitable Pichia strain, such as a suitable strain of
Pichiapastoris), and in which
said amino acid sequence or polypeptide of the invention that does not contain
a threonine
(T) residue that is susceptible to phosphorylation when expressed in said
yeast strain.
Alternatively, said step a) may comprise the step of cultivating or
maintaining a host or host
cell as described herein under conditions such that said host or host cell
produces an amino
acid sequence or polypeptide of the invention, in which said host or host is a
suitable yeast
strain (and in particular, a suitable Pichia strain, such as a suitable strain
of Pichia pastoris)
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that either shows reduced (i.e. essentially no) phosphorylation or that has
been genetically
modified to show reduced (i.e. essentially no) phosphorylation.
In the above context, "essentially no" phosphorylation means that less than
5%,
preferably less than 3%, such as less than 2%, less than 1%, or less than 0.5%
by weight of
the product obtained after expression is phosphorylated on an amino acid
residue comprised
within an amino acid sequence of the invention.
Methods for isolating and purifying an amino acid sequence, compound, protein,
polypeptide, fusion protein, or multivalent or multispecific construct of the
invention may be
performed in any suitable manner known per se, as will be clear to the skilled
person.
10 Reference is again also made to WO 09/127691 and WO 08/068280.
In one specifically preferred, but non-limiting embodiment, the step of
isolating/purifying the amino acid sequence of the invention or polypeptide of
the invention
comprises at least one step of affinity purification/chromatography using an
affinity matrix is
specific for the amino acid sequence of the invention that is present in said
compound,
protein, polypeptide, fusion protein, or multivalent or multispecific
construct of the invention.
Such an affinity matrix may for example comprise a suitable resin to which is
linked (in a
manner known per se, and optionally using a suitable linker) at least one
ligand, binding
domain or binding unit that is directed against/specific for the amino acid
sequence of the
invention. Such a ligand may be any suitable ligand known per se (although the
use of
20 albumin or a fragment of albumin may in some instances be less preferred
because it may not
provide the desired specificity for the amino acid sequence of the invention),
and may for
example in a preferred aspect be a VHa1 or nanobody that has been raised
against an amino
acid sequence of the invention (such as the amino acid sequence present in in
said compound,
protein, polypeptide, fusion protein, or multivalent or multispecific
construct of the
invention) or a desired (antigenic) fragment, epitope or determinant thereof,
for example by
immunizing a camelid with said amino acid sequence of the invention, obtaining
an immune
library of VHH's from. said camelid, screening said immune library for VHH's
specific for said
amino acid sequence of the invention (for example using phage display or
another suitable
screening technique) and obtaining/expressing/isolating one or more VHH's
specific for said
30 amino acid sequence of the invention, which may then be linked to a
suitable resin to provide
an affinity resin suitable for use in this aspect of the invention. The
aforementioned steps may
all be performed in a manner known per se and/or using techniques known per
se. For
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example, suitable techniques for generating VHH's against an amino acid
sequence of the
invention are described in the prior art mentioned on page 59 of WO 08/020079
and to the
list of references mentioned on pages 41-43 of the International application
WO 06/0401.53,
which prior art and references are incorporated herein by reference.
An affinity resin that is directed against an amino acid sequence of the
invention and
its use in isolating or purifying an amino acid sequence, compound, protein,
polypeptide,
fusion protein, or multivalent or multispecific construct of the invention
form further aspects
of the invention.
In one specific aspect of the invention, the affinity matrix comprises a
ligand, binding
domain or binding unit (such as a VHH) that is directed against/specific for a
part, epitope or
antigenic determinant of the amino acid sequence of the invention that is
situated at or
towards the C-terminus of an amino acid sequence of the invention (for
example, at one or
more positions downstream of the GGG motif).
Thus, some further aspects of the invention are:
- an amino acid sequence of the invention that comprises, downstream of the (C-
terminal)
GGG motif at positions 11 to 13, a part, epitope or antigenic determinant that
is
recognized by (at least one ligand, binding domain or binding unit on) an
affinity matrix;
- a compound, protein, polypeptide, fusion protein, or multivalent or
multispecific
construct of the invention that comprises such an amino acid sequence of the
invention;
- a ligand, binding domain or binding unit that is directed towards,
recognizes and/or can
specifically hind a part, epitope or antigenic determinant that is present in
an amino acid
sequence of the invention (i.e. most preferably downstream of the (C-terminal)
GGG
motif at positions 11 to 13). As mentioned, the ligand may for example and
without
limitation be a VHH that has been raised against said amino acid sequence of
the invention
(or against another amino acid sequence of the invention that comprises
essentially the
same part, epitope or antigenic determinant);
- an affinity matrix that (contains at least one ligand, binding domain or
binding unit that)
is directed towards, recognizes and/or can specifically bind an amino acid
sequence of the
invention; and in particular an affinity matrix that (contains at least one
ligand, binding
domain or binding unit that) is directed towards, recognizes and/or can
specifically bind a
part, epitope or antigenic determinant in an amino acid sequence of the
invention that is
present downstream of the GGG motif at positions 11 to 13.
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- a method for isolating and/or purifying a compound, protein, polypeptide,
fusion protein,
or multivalent or multispecific construct of the invention (i.e. that
comprises at least one
amino acid sequence of the invention), which comprises at least one step of
contacting (a
composition or mixture comprising) such a compound, protein, polypeptide,
fusion
protein, or multivalent or multispecific construct of the invention with an
affinity matrix
that (contains at least one ligand, binding domain or binding unit that) is
directed towards,
recognizes and/or can specifically bind the amino acid sequence of the
invention that is
comprised within such a compound, protein, polypeptide, fusion protein, or
multivalent or
multispecific construct of the invention; and in particular with an affinity
matrix that
(contains at least one ligand, binding domain or binding unit that) is
directed towards,
recognizes and/or can specifically bind a part, epitope or antigenic
determinant in said
amino acid sequence of the invention that is present downstream of the GGG
motif at
positions II to 13.
- the use of an affinity matrix that (contains at least one ligand, binding
domain or binding
unit that) is directed towards, recognizes and/or can specifically bind an
amino acid
sequence of the invention (and in particular, a part, epitope or antigenic
determinant that
is present in said amino acid sequence downstream of the (C-terminal) GGG
motif at
positions I I to 13) in isolating and/or purifying a compound, protein,
polypeptide, fusion
protein, or multivalent or multispecific construct of the invention comprising
such an
amino acid sequence.
Such part, epitope or antigenic determinant may for example (and without
limitation)
comprise the amino acid residues VG, which are located downstream of the GGG
motif at
positions 11.13 (for example and without limitation at positions 16 and 17 or
further
downstream). Thus, for example and without limitation, such amino acid
sequences of the
invention may comprise one of the sequence motifs GGGTPVG (SEQ ID NO: 150),
GGGNPVG (SEQ ID NO: 1.51), GGGNPVG (SEQ ID NO: 152) or GGGDPVG (SEQ ID
NO: 153). Some non-limiting examples of amino acid sequences of the invention
that
comprise such an antigenic determinant that comprises the amino acid residues
VG are given
in SEQ ID NO's: 106 to 108, and some non-limiting examples of compounds of the
invention
comprising the same are given in SEQ ID NO's: 114 to 116. Also, SEQ ID NO's:
117 to 125
give some non-limiting examples of compounds of the invention that comprise a
dimeric
amino acid sequence of the invention that comprises a VG-based antigenic
determinant
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towards the C-terminus (i.e. only in the peptide of the invention that is C-
terminal in the
dimer, and not in the upstream peptide). Each of the dimeric amino acid
sequences of the
invention comprised in the compounds of SEQ ID NO's: 117 to 125 form further
examples of
amino acid sequences of the invention and thus form further aspects of the
invention.
The invention also encompasses medical uses and methods of treatment
encompassing
the amino acid sequence, compound, or multivalent and multispecific compound
of the
invention, wherein said medical use or method is characterized in that said
medicament is
suitable for administration at intervals of at least about 50% of the natural
half-life of human
serum albumin.
The invention also relates to methods for extending or increasing the serum
half-life
of a therapeutic (i.e. a therapeutic moiety, compound, protein or other
therapeutic entity). The
methods include contacting the therapeutic with any of the foregoing amino
acid sequences,
such that the therapeutic is bound to or otherwise associated with the amino
acid sequences,
compounds, fusion proteins or constructs of the invention. In some
embodiments, the
therapeutic is a biological therapeutic, preferably a peptide or a
polypeptide, in which case
the step of contacting the therapeutic can include preparing a fusion protein
by linking the
peptide or polypeptide with the amino acid sequence, compound, fusion proteins
or
constructs of the invention.
These methods can further include administering the therapeutic to a subject
after the
therapeutic is bound to or associated with the amino acid sequence, compound,
fusion protein
or construct of the invention. In such methods, the serum half-life of the
therapeutic is at least
1.5 times the half-life of therapeutic per se, or is increased by at least 1
hour (such as by at
least 6 hours, preferably at least 12 hours, more preferably at least I day,
such as more than 2
days, or even more than 5 days or more) compared to the half-life of
therapeutic per se. In
some preferred embodiments, the serum half-life of the therapeutic is at least
2 times, at least
5 times, at least 10 times, or more than 20 tunes greater than the half-life
of the
corresponding therapeutic moiety per se. In other preferred embodiments, the
serum half-life
of the therapeutic is increased by more than 2 hours, more than 6 hours or
more than 12 hours
compared to the half-life of the corresponding therapeutic moiety per se.
In the above methods, the serum half-life of the therapeutic is preferably
increased or
extended such that said serum half-life (i.e. of the compound of the invention
thus obtained)
is longer than the serum half-life of a corresponding compound or construct
that comprises
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the therapeutic and the amino acid sequence of SEQ ID NO: 1, and preferably
compared to a
corresponding compound or construct that comprises one of the amino acid
sequences of
SEQ ID NO's: 75, 76 and/or 77 (i.e. instead of the amino acid sequence of the
invention).
Preferably, the serum half-life of the compound of the invention is at least
5% longer,
preferably at least 10% longer, more preferably at least 25% longer, or even
more preferably
at least than 50% longer, such as more than 100% longer or even more improved,
compared
to the serum half-life of a corresponding compound or construct that comprises
the
therapeutic and the amino acid sequence of SEQ ID NO: 1, and preferably
compared to a
corresponding compound or construct that comprises one of the amino acid
sequences of
SEQ ID NO's: 75, 76 and/or 77 (i.e. instead of the amino acid sequence of the
invention).
For example, in such methods, the serum half-life of the compound of the
invention
may be at least 1.1, such as at least 1.2 times, more preferably at least 1.5
times the half-life
of the corresponding compound or construct that comprises the therapeutic and
the amino
acid sequence of SEQ ID NO: 1, and preferably compared to a corresponding
compound or
construct that comprises one of the amino acid sequences of SEQ ID NO's: 75,
76 and/or 77
(i.e. instead of the amino acid sequence of the invention), and/or may be
increased by at least
1 hour (such as by at least 6 hours, preferably at least 12 hours, more
preferably at least 1
day, such as more than 2 days, or even more than 5 days or more) compared to
the half-life of
a corresponding compound or construct that comprises the therapeutic and the
amino acid
sequence of SEQ ID NO: 1, and preferably compared to a corresponding compound.
or
construct that comprises one of the amino acid sequences of SEQ ID NO's: 75,
76 and/or 77
(i.e. instead of the amino acid sequence of the invention). In some preferred
embodiments,
the serum half-life of the compound of the invention is at least 2 times, at
least 3 times or at
least 5 times greater than the half-life of the corresponding compound or
construct that
comprises the therapeutic and the amino acid sequence of SEQ ID NO:1, and
preferably
compared to a corresponding compound or construct that comprises one of the
amino acid
sequences of SEQ ID NO's: 75, 76 and/or 77 (i.e. instead of the amino acid
sequence of the
invention).
In another aspect, the invention. relates to a method for modifying a
therapeutic such
that the desired therapeutic level of said therapeutic is, upon suitable
administration of said
therapeutic so as to achieve said desired therapeutic level, maintained for a
prolonged period
of time.
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The methods include contacting the therapeutic with any of the foregoing amino
acid
sequences, such that the therapeutic is bound to or otherwise associated with
the amino acid
sequences, compounds, fusion proteins or constructs of the invention. In some
embodiments,
the therapeutic is a biological therapeutic, preferably a peptide or
polypeptide, in which case
the step of contacting the therapeutic can include preparing a fusion protein
by linking the
peptide or polypeptide with the amino acid sequence, compound, fusion protein,
or constructs
of the invention.
These methods can further include administering the therapeutic to a subject
after the
therapeutic is bound to or otherwise associated with the amino acid sequence,
compound,
10 fusion protein, or construct of the invention, such that the desired
therapeutic level is achieve
upon such administration. In such methods, the time that the desired
therapeutic level of said
therapeutic is maintained upon such administration is at least 1.5 times the
half-life of
therapeutic per se, or is increased by at least 1 hour compared to the half-
life of therapeutic
per se. In some preferred embodiments, the time that the desired therapeutic
level of said
therapeutic is maintained upon such administration is at least 2 times, at
least 5 times, at least
10 times or more than 20 times greater than the half-life of the corresponding
therapeutic
moiety per se. In other preferred embodiments, the time that the desired
therapeutic level of
said therapeutic is maintained upon such administration is increased by more
than 2 hours,
more than 6 hours or more than 12 hours compared to the half-life of the
corresponding
20 therapeutic moiety per se.
Preferably, the time that the desired therapeutic level of said therapeutic is
maintained
upon such administration is increased such that the therapeutic can be
administered at a
frequency that is as defined herein for the compounds of the invention.
In the above methods, the time that the desired therapeutic level of said
therapeutic is
maintained is preferably increased or extended such that said serum half-life
(i.e. of the
compound of the invention thus obtained) is longer than the time that the
desired therapeutic
level of said therapeutic is maintained by a corresponding compound or
construct that
comprises the therapeutic and the amino acid sequence of SEQ ID NO: 1, and
preferably
compared to a corresponding compound or construct that comprises one of the
amino acid
30 sequences of SEQ ID NO's: 75, 76 and/or 77 (i.e. instead of the amino acid
sequence of the
invention). Preferably, the time that the desired therapeutic level of said
therapeutic is
maintained is at least 5% longer, preferably at least 10% longer, more
preferably at least 25%
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longer, or even more preferably at least than 50% longer, such as more than.
100% longer or
even more improved, compared to the time that the desired therapeutic level of
said
therapeutic is maintained by a corresponding compound or construct that
comprises the
therapeutic and the amino acid sequence of SEQ ID NO: 1, and preferably
compared to a
corresponding compound or construct that comprises one of the amino acid
sequences of
SEQ ID NO's: 75, 76 and/or 77 (i.e. instead of the amino acid sequence of the
invention).
For example, in such methods, the time that the desired therapeutic level of
said
therapeutic is maintained may be at least 1. 1, such as at least 1.2 times,
more preferably at
least 1.5 times the time that the desired therapeutic level of said
therapeutic is maintained by
a corresponding compound or construct that comprises the therapeutic and the
amino acid
sequence of SEQ ID NO: 1, and preferably compared to a corresponding compound
or
construct that comprises one of the amino acid sequences of SEQ ID NO" s: 75,
76 and/or 77
(i.e. instead of the amino acid sequence of the invention), and/or may be
increased by at least
1 hour (such as by at least 6 hours, preferably at least 12 hours, more
preferably at least 1
day, such as more than 2 days, or even more than 5 days or more) compared to
the time that
the desired therapeutic level of said therapeutic is maintained by a
corresponding compound
or construct that comprises the therapeutic and the amino acid sequence of SEQ
ID NO: 1,
and preferably compared to a corresponding compound or construct that
comprises one of the
amino acid sequences of SEQ ID NO's: 75, 76 and/or 77 (i.e. instead of the
amino acid
sequence of the invention). In some preferred embodiments, the time that the
desired
therapeutic level of said therapeutic is maintained is at least 2 times, at
least 3 times or at
least 5 times greater than the time that the desired therapeutic level of said
therapeutic is
maintained by a corresponding compound or construct that comprises the
therapeutic and the
amino acid sequence of SEQ ID NO: 1, and preferably compared to a
corresponding
compound or construct that comprises one of the amino acid sequences of SEQ ID
NO's: 75,
76 and/or 77 (i.e. instead of the amino acid sequence of the invention).
In another aspect, the invention relates to the use of a compound of the
invention (as
defined herein) for the production of a medicament that increases and/or
extends the level of
the therapeutic agent in said compound or construct in the serum of a patient
such that said
therapeutic agent in said compound or construct is capable of being
administered at a lower
dose as compared to the therapeutic agent alone (i.e. at essentially the same
frequency of
administration).
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The invention also relates to a pharmaceutical composition that comprises at
least one
amino acid sequence, compound, protein, polypeptide, fusion protein, or
multivalent or
multispecific construct as described herein, and optionally at least one
pharmaceutically
acceptable carrier, diluent or excipient. Such preparations, carriers,
excipients and diluents
may generally be as described in the co-pending patent applications by Ablynx
N.V.
described herein, such as WO 04/041862 or WO 06/122825.
However, since the amino acid sequences, compounds, proteins, polypeptides,
fusion
proteins, or multivalent or multispecific constructs described herein have an
increased half-
life, they are preferably administered to the circulation. As such, they can
be administered in
any suitable manner that allows the amino acid sequences, compounds, proteins,
polypeptides, fusion proteins, or multivalent or multispecific constructs to
enter the
circulation, such as intravenously, via injection or infusion, or in any other
suitable manner
(including oral administration, administration through the skin, intranasal
administration,
administration via the lungs, etc). Suitable methods and routes of
administration will be clear
to the skilled person, again for example also from the teaching of WO
04/041862 or WO
06/122825.
Thus, in another aspect, the invention relates to a method for the prevention
and/or
treatment of at least one disease or disorder that can be prevented or treated
by the use of
amino acid sequences, compounds, proteins, polypeptides, fusion proteins, or
multivalent or
multispecific constructs described herein, which method comprises
administering, to a
subject in need thereof, a pharmaceutically active amount of a amino acid
sequences,
compounds, proteins, polypeptides, fusion proteins, or multivalent or
multispecific constructs
of the invention, and/or of a pharmaceutical composition comprising the same.
As will be
clear to the skilled person, the diseases and disorders that can be prevented
or treated by the
use of amino acid sequences, compounds, proteins, polypeptides, fusion
proteins, or
multivalent or multispecific constructs described herein will. generally be
the same as the
diseases and disorders that can be prevented or treated by the use of the
therapeutic moiety
that is present in the amino acid sequences, compounds, proteins,
polypeptides, fusion
proteins, or multivalent or multispecific constructs of the invention.
In the context of the present invention, the term "prevention and/or
treatment" not
only comprises preventing and/or treating a disease, but also generally
comprises preventing
the onset of a disease, slowing or reversing the progress of a disease,
preventing or slowing
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the onset of one or more symptoms associated with a disease, reducing and/or
alleviating one
or more symptoms associated with a disease, reducing the severity and/or the
duration of a
disease and/or of any symptoms associated therewith and/or preventing a
further increase in
the severity of a disease and/or of any symptoms associated therewith,
preventing, reducing
or reversing any physiological damage caused by a disease, and generally any
pharmacological action that is beneficial to the patient being treated.
The subject to be treated may be any warm-blooded animal, but is in particular
a
mammal, and more in particular a human being. As will be clear to the skilled
person, the
subject to be treated will in particular be a person suffering from, or at
risk from, the diseases
and disorders mentioned herein.
More specifically, the present invention relates to a method of treatment
wherein the
frequency of administering the amino acid sequence, compound, fusion protein
or construct
of the invention is at least 50% of the natural half-life of serum albumin in
said mammal (i.e.
in the case of man, of human serum albumin), preferably at least 60%,
preferably at least
70%, more preferably at least 80%, and most preferably at least 90%.
Specific frequencies of administration. to a mammal, which are within the
scope of the
present invention are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or at
least 100% of the natural half-life of serum albumin in said mammal as defined
above.
In other words, specific frequencies of administration, which are within the
scope of
the present invention are every 4, 5, 6, 7, 8, 9, 1. O, 11, 12, 13, 14, 15,
16, 17, 18, or 19 days.
Without limitation, the frequencies of administration referred to above are in
particular suited for maintaining a desired level of the amino acid sequence,
compound,
fusion protein or construct in the serum of the subject treated with the amino
acid sequence,
compound, fusion protein, or construct, optionally after administration of one
or more
(initial) doses that are intended to establish said desired serum level. As
will be clear to the
skilled person, the desired serum level may inter alia be dependent on the
amino acid
sequence, compound, fusion protein, or construct used and/or the disease to be
treated. The
clinician or physician will be able to select the desired serum level and to
select the dose(s)
and/or amount(s) to be administered to the subject to be treated in order to
achieve and/or
maintain the desired serum level in said subject, when the amino acid
sequence, compound,
fusion protein, or construct of the invention is administered at the
frequencies mentioned
herein.
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In one non-limiting aspect of the invention, the compounds, proteins,
polypeptides,
fusion proteins, or multivalent or multispecific constructs of the invention
are
administered/delivered via the lungs (i.e. via inhalation, intratracheal
administration or other
suitable methods and/or equipment for pulmonary delivery). For this purpose,
the
compounds, proteins, polypeptides, fusion proteins, or multivalent or
multispecific constructs
of the invention may for example be suitably formulated (using one or more
suitable carriers,
diluents, excipients or additives known per se) into a formulation suitable
for administration
to/via the lungs, for example in the form of an aerosol (or a form that is
suitable and/or
intended for delivery as an aerosol), in a form that is suitable and/or
intended for
administration by inhalation, in the form of a (dry) powder that is suitable
and/or intended for
administration to the lungs, or in a form that is suitable and/or intended for
administration
(i.e. to/via the lungs) using a nebulizer, or in a form that is suitable
and/or intended for
intratracheal administration. For this purpose, the formulation may optionally
be included in
suitable holder (for example, a holder that also comprises a pump, valve or
other device
capable of delivering a unit dose of the formulation), or the formulation may
be in the form of
a kit-of-parts with equipment for pulmonary delivery, such as an inhaler or
nebulizer.
When the compounds, proteins, polypeptides, fusion proteins, or multivalent or
multispecific constructs of the invention were administered to or via the
lungs, it has been
found in animal models for pulmonary administration [in which the pulmonary-to-
systemic
delivery of active principles that contain a peptide according to WO
09/127691. (to which the
amino acid sequences/peptides of the invention are an improvement, as
described herein) for
providing half-life extension is compared to the pulmonary-to-systemic
administration of
similar compounds in which the extended half-life is provided by the presence
of a Nanobody
against serum albumin (such as Alb-1 or a humanized variant thereof such as
Alb-8, see for
example WO 2006/122787)], that the pulmonary administration of an active
principle in
which an amino acid sequence/peptide of the invention is present (i.e. for
providing increased
half-life) can result in serum concentrations of the administered active
principle that are
higher than when a similar active principle containing a serum albumin binding
Nanobody
for half-life extension is similarly administered via the pulmonary route.
Reference is for
example made to the data shown in Example 23 of the US patent application
13/018,047 by
Ablynx N.V., filed on January 31, 2011.
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Thus, in another aspect, the invention relates to a pharmaceutical (including
diagnostic) composition, preparation or formulation comprising a compound,
protein,
polypeptide, fusion protein, or multivalent or multispecific construct of the
invention that is
suitable and/or intended for pulmonary-to-systemic administration of the
compound, protein,
polypeptide, fusion protein, or multivalent or multispecific construct of the
invention (i.e.
delivering the same into the circulation via administration to the lungs).
The invention also relates to a method of preventing or treating of a disease
in a
human subject (i.e. a subject in need of such treatment), in which a compound,
protein,
polypeptide, fusion protein, or multivalent or multispecific construct of the
invention suitable
10 for preventing or treating said disease (or a formulation of the same
suitable for pulmonary
administration) is administered to and/or via the lungs of the subject.
In another aspect, the invention relates to a compound, protein, polypeptide,
fusion
protein, or multivalent or multispecific construct of the invention for
administration to or via
the lungs, and in particular for pulmonary-to-systemic delivery.
The invention also relates to a compound, protein, polypeptide, fusion
protein, or
multivalent or multispecific construct of the invention that has been
formulated for for
administration to or via the lungs, and in particular for pulmonary-to-
systemic delivery.
The invention also relates to a pharmaceutical (including diagnostic)
composition that
comprises at least one compound, protein, polypeptide, fusion protein, or
multivalent or
20 multispecific construct of the invention for administration to or via the
lungs, and in
particular for pulmonary-to-systemic delivery.
Thus, the use of a peptide of the invention in compounds, proteins,
polypeptides,
fusion proteins, or multivalent or multispecific constructs that are intended
for administration
via the lungs may also provide an improved alternative to the methods for
pulmonary
delivery (and in particular pulmonary-to-systemic delivery) of active
principles that comprise
Nanobodies, such as the methods described in WO 2003/055527 and WO 2010/081856
of
Ablynx N.V.(for example, by performing the methods described in WO 2003/055527
and
WO 2010/081856 using a compound, protein, polypeptide, fusion protein or
multivalent or
multispecific construct of the invention, or by otherwise administering a
compound, protein,
30 polypeptide, fusion protein or multivalent or multispecific construct of
the invention to and/or
via the lungs using any suitable methods, techniques, formulations and
equipment for
pulmonary delivery known per se).
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Furthermore, it has also been described in the art that the use of binding
domains or
binding units that bind to serum albumin in active principles may provide for
increased
penetration into joints, tissues or tumors. As compounds, proteins,
polypeptides, fusion
proteins, or multivalent or multispecific constructs of the invention have a
smaller size
compared to similar compounds, proteins, polypeptides, fusion proteins, or
multivalent or
multispecific constructs which have been provided with an extended half-life
due to the
presence of a Nanobody against serum albumin, it is also expected that the
compounds,
proteins, polypeptides, fusion proteins, or multivalent or multispecific
constructs of the
invention may have an improved penetration into joints, tissues or tumors
compared to the
corresponding Nanobody-based active principles.
In another embodiment, the invention relates to a method for immunotherapy,
and in
particular for passive immunotherapy, which method comprises administering, to
a subject
suffering from or at risk of the diseases and disorders mentioned herein, a
pharmaceutically
active amount of a fusion protein or construct of the invention, and/or of a
pharmaceutical
composition comprising the same.
The amino acid sequences, compounds, proteins, polypeptides, fusion proteins,
or
multivalent or multispecific constructs and/or the compositions comprising the
same are
administered according to a regime of treatment that is suitable for
preventing and/or treating
the disease or disorder to be prevented or treated. The clinician will
generally be able to
determine a suitable treatment regimen, depending on factors such as the
disease or disorder
to be prevented or treated, the severity of the disease to be treated and/or
the severity of the
symptoms thereof, the specific amino acid sequence, compound, protein,
polypeptide, fusion
protein, or multivalent or multispecific construct of the invention to be
used, the specific
route of administration and pharmaceutical formulation or composition to be
used, the age,
gender, weight, diet, general condition of the patient, and similar factors
well known to the
clinician.
Generally, the treatment regimen will comprise the administration of one or
more
amino acid sequences, compounds, proteins, polypeptides, fusion proteins, or
multivalent or
multispecific constructs of the invention, or of one or more compositions
comprising the
same, in one or more pharmaceutically effective amounts or doses. The specific
amount(s) or
doses to administered can be determined by the clinician, again based on the
factors cited
above.
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Generally, for the prevention and/or treatment of intended diseases and
disorders (i.e.
those diseases and disorders which are usually treated or prevented through
the use of the
therapeutic entity per se) and depending on the specific disease or disorder
to be treated, the
potency and/or the half-life of the specific amino acid sequences, compounds,
proteins,
polypeptides, fusion proteins, or multivalent or multispecific constructs to
be used, the
specific route of administration and the specific pharmaceutical formulation
or composition
used, the amino amino acid sequences, compounds, proteins, polypeptides,
fusion proteins, or
multivalent or multispecific constructs of the invention will generally be
administered in an
amount between I gram and 0.01 microgram per kg body weight per day,
preferably between
0.1 gram and 0.1 microgram per kg body weight per day, such as about 1, 10,
100, 1000, or
2000 microgram per kg body weight per day, either continuously (e.g. by
infusion), as a
single daily dose or as multiple divided doses during the day. The clinician
will generally be
able to determine a suitable daily dose, depending on the factors mentioned
herein. It will
also be clear that in specific cases, the clinician may choose to deviate from
these amounts,
for example on the basis of the factors cited above and his expert judgment.
Generally, some
guidance on the amounts to be administered can be obtained from the amounts
usually
administered for comparable conventional antibodies or antibody fragments
against the same
target administered via essentially the same route, taking into account
however differences in
affinity/avidity, efficacy, biodistribution, half-life and similar factors
well known to the
skilled person.
Usually, in the above method, a single amino acid sequence, compound, protein,
polypeptide, fusion protein, or multivalent or multispecific construct of the
invention will be
used. It is however within the scope of the invention to use two or more amino
acid
sequences, compounds, proteins, polypeptides, fusion proteins, or multivalent
or
multispecific constructs of the invention in combination (e.g. as separate
preparations or
combined in a single preparation).
The amino acid sequences, compounds, proteins, polypeptides, fusion proteins,
or
multivalent or multispecific constructs of the invention may also be used in
combination with
one or more further pharmaceutically active compounds or principles, i.e. as a
combined
treatment regimen, which may or may not lead to a synergistic effect. Again,
the clinician
will be able to select such further compounds or principles, as well as a
suitable combined
treatment regimen, based on the factors cited above and his expert judgement.
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In particular, the amino acid sequences, compounds, proteins, polypeptides,
fusion
proteins, or multivalent or multispecific constructs of the invention may be
used in
combination with other pharmaceutically active compounds or principles that
are or can be
used for the prevention and/or treatment of the diseases and disorders that
can be prevented
or treated with the amino acid sequences, compounds, proteins, polypeptides,
fusion proteins,
or multivalent or multispecific constructs of the invention, and as a result
of which a
synergistic effect may or may not be obtained.
The effectiveness of the treatment regimen used according to the invention may
be
determined and/or followed in any manner known per se for the disease or
disorder involved,
as will be clear to the clinician. The clinician will also be able, where
appropriate and or a
case-by-case basis, to change or modify a particular treatment regimen, so as
to achieve the
desired therapeutic effect, to avoid, limit or reduce unwanted side-effects,
and/or to achieve
an appropriate balance between achieving the desired therapeutic effect on the
one hand and
avoiding, limiting or reducing undesired side effects on the other hand.
Generally, the treatment regimen will be followed until the desired
therapeutic effect
is achieved and/or for as long as the desired therapeutic effect is to be
maintained. Again, this
can be determined by the clinician.
The subject to be treated may be any warm-blooded animal, but is in particular
a
mammal, and more in particular a human being. As will be clear to the skilled
person, the
subject to be treated will in particular be a person suffering from, or at
risk from, the diseases
and disorders mentioned herein.
The invention will now be further illustrated by means of the following non-
limiting
Experimental Part and the non limiting Figures, of which Figure I shows the
sequences
referred to in the present specification, Figure 11 shows the BlAcore results
obtained in
Example 2B, and Figure III shows an augment of some reference sequences and
some amino
acid sequences of the invention.
Experimental part
Example 1: Examples of amino acid sequences of the invention.
Some non-limiting examples of amino acid sequences of the invention are given
as
SEQ ID NO's: 54 to 74 in Table 11 below. Binding to human serum albumin was
determined
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as a fusion of the sequence with the Nanobody 2D3 (see Example 2 and the
further examples
from WO 09/127691 mentioned herein).
Table II: some preferred but non-limiting amino acid sequences of the
invention
Clone ID SEQ ID: Sequence Kn
(nM)
on
PISA
Biacore
EXPGMP 54 LWYMLRDWDFDVFGGGTP n.d.
80C05
EXPGMP 55 LWYLYRDWDFDVFGGGTP n..d.
j 80D11
EXPGMP 56 YWWERRDWDFDVFGGGTP 16
80B10
EXPGMP 57 AWYDYRDWDFDVFGGGTP 59-222
80E03
EXPGMP 58 WWNWRDWDFDVFGGGTP n.d.
80F06
EXPGMP 59 EWWWRRDWDFDVFGGGTP 15
85E01
E EXPGMP 60 VDWFYRDWDFDVFGGGTP 31
85F10
EXPGMP 61 RDWFLRDWDFDVFGGGTP 39
85E07
EXPGMP 62 DWWNRRDWDFDVFGGGTP 46
85D09
EXPGMP 63 YGDWFRDWDFDVFGGGTP 154
85D06
EXPGMP 64 WWTWGRDWDFDVFGGGTP 28
85A10
EXPGMP 65 PIDFWRDWDFDVFGGGTP 94
85F09
EXPGMP 66 WWTSDRDWDFDVFGGGTP 48
85H07
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Table II: continued
EXPGMP 67 QKLYWRDWDFDVFGGGTP 96
85H06
EXPGMP 68 KWWEIRDWDFDVFGGGTPAKSDE 20
86A10
EXPGMP 69 WWSTPRDWDFDVFGGGTPRGEMH 22
86C01.
EXPGMP 70 LFWWERDWDFDVFGGGTP 20
88501
EXPGMP 71 WWLQERDWDFDVFGGGTP 22
88E10
EXPGMP 72 WWEQDRDWDFDVFGGGTP 15
89D03
EXPGMP 73 NQLIVRDWDFDVFGGGTP n.d.
89D11
EXPGMP 74 W WELDRD WDFDV FGGGTP 13
89F02 i
EXPGMP 75 AAARDWDFDVFGGGTPVGG 198-
59C2 648
(reference)
89D0-V1 103 WWEQDRDWDFDVFGGGAP n.d.
89D03V2 104 WWEQDRDWDFDVFGGGNP n.d.
89D03V3 105 WWEQDRDWDFTVFGGGDP n.d.
89D03V 1- n.d.
VG 1.06 WWEQDRDWDFDVFGGGAPVG
89D03V2- n.d.
VG 107 WWEQDRDWDFDVFGGGNPVG
89D03V3- n.d.
VG 108 i WWEQDRDWDFTVFGGGDPVG
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Example 2: Construction of a Nanobody-Expedite fusion proteins and analysis_of
binding to
human serum albumin:
Example 2A: Construction of 2D3-Expedite fusions:
An amino acid sequence of the invention (such as one of the peptides of Table
II) is
genetically fused at the C-tenninus of the Nanobody 2D3:
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSIN
WSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRD
AGTTWFEKSGSAGQGTQVTVSS [SEQ ID NO:47]
via a suitable linker sequence and with the following C-terminal tag:
AAAEQKLI SEEDLNGAAH HHHHH [SEQ ID NO:491.
Non-limiting examples of suitable linker sequences are GGGGSGGGS [SEQ ID NO:
53] (which comprises a Gly4Ser-Gly3Ser linker, also referred to as 9GS
herein);
GGGGSGGGSA [SEQ ID NO: 48] (which comprises a Gly4Ser-Gly3Ser linker with a
flanking amino acid residue A at the C-terminus) and GGGGSGGGGSGGGGSGGGGS
[SEQ ID NO:109] (also referred to as 20GS herein).
The binding of the resulting fusion proteins to human serum albumin was
determined
using surface plasmon resonance analysis. For this purpose, the fusion
proteins were
expressed in E. coli TGl cells. The fusion proteins were purified by IMAC/SEC
and binding
to HSA was assessed in BlAcoreTM 3000, by injecting I pM and 5 pM of the
fusion proteins
on a CM5 chip coated. with -7000 RU human serum albumin (Sigma, 99% pure) and
2460
RU of an irrelevant protein antigen (reference). Coating of the chip (CM5) was
performed by
amine coupling using NHS/EDC for activation and ethanolamine for deactivation
(Biacore
amine coupling kit). HBS-EP was used as flow buffer at a rate of 10q.1 min-1.
20 1 of sample
was injected for 120s. The 2D3 Nanobody was injected as control. The results
are mentioned
in Table II above.
By way of example, a Nanobody construct was prepared as a fusion of the
peptide
80B 10 (SEQ ID NO: 56) and the Nanobody 2D3 (SEQ ID NO: 47), via a Gly4Ser-
Gly3Ser
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("9GS") linker sequence (SEQ ID NO: 53). The sequence of the resulting
Nanobody
construct (referred to as 2D3-9GS-EXP80B10 or EXP 424) was:
EV QLVESGGSLVQPGGSLRLSCAASGFTFDDYAMS WVRQVPGKGLEWV SSIN
WSGTHTDYADS VKGRFTISIJNNANNTLYLQMNSLKSEDTAV YYCAKNWRD
AGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSYWWERRDWDFDVFGGGTP
[SEQ ID NO: 991.
Fusions of other amino acid sequences of the invention (such as those
mentioned in
Table 11) and a Nanobody (such as 2D3) or Nanobody construct can be prepared
in a similar
manner, again optionally using a suitable linker. Some further non-limiting
examples of this
are given in Example 2B and 2C.
Also, instead of a single peptide of the invention, an amino acid sequence of
the
invention that comprises two or more {such as two or three) peptides of the
invention (such as
two peptides as mentioned in Table II, which may be the same or different, and
which may be
linked either directly to each other or via one or more suitable linkers, such
as the linkers of
SEQ ID NO: 48 or 53) may be linked to the Nanobody. For example, a Nanobody
may be
linked to two such peptides which are identical and which are linked to each
other via a linker
so as to form a tandem repeat. Some non-limiting examples of this are also
given in the
following Examples 2B and 2C.
Example 2B: Construction of SF7-Expedite fusions;:
An amino acid sequence of the invention (such as one of the peptides of Table
II) is
genetically fused at the C-terminus of the Nanobody SF7:
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISS
IGDTYYADS VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGT
DYWGQGTQVTVSS [SEQ ID NO, 100]
via a suitable linker sequence (such as the 9GS linker sequence of SEQ ID NO:
53), either a
monovalent peptide or as a tandem repeat sequence (in which two peptides of
the invention.
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By way of example, 5F7 was so linked to 89D03 (SEQ ID NO: 72) in monovalent
format. The resulting Nanobody construct (referred to as 5F7-9GS-EXP89DO3 or
EXP413)
was:
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISS
IGDTYYADSVKGRFTI SRDNAKNTV YLQMNSLKPEDTAV YYCKRFRTAAQGT
DYWGQGTQVTVSSGGGGSGGGSWWEQDRDWDFDVFGGGTP [SEQ ID
NO:101]
5177 was also linked to 89D03 (SEQ ID NO: 72) in a tandem repeat format (in
which
two 89D03 peptides were linked to each other via a 9GS linker). The sequence
of the
resulting Nanobody construct (referred to as 5F7-9GS-EXP89D03-9GS-EXP89D03 or
EXP486) was:
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISS
IGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGT
D Y W GQGTQ V T V S S GGGGS GGGS W WEQDRD WDFD V FGGGTPGGGG S GGGS
WWEQDRDWDFDVFGGGTP [SEQ ID NO:102]
The binding of the monovalent and tandem repeat constructs of SEQ ID NO: 101
and.
102 was compared in BlAcoreTM 3000, by injecting 200nM and I j tM of the
fusion proteins
on a CM5 chip coated with -920 RU human serum albumin (Sigma, 99% pure) or
cynomolgus serum albumin (900 RU). The results are shown in Figure 2. A
slightly higher
response and clearly an avidity effect for the tandem Expedite construct
EXP486 compared to
the single Expedite construct EXP413 was observed. The data showed that the
binding
profiles to human serum albumin and cynomolgus serum albumin are very similar.
Some non-limiting examples of other constructs of the invention (using 5F7 as
an
example of a representative Nanobody) are given in SEQ ID NO's: 110 to 125. In
these
constructs, 5F7 is linked via a 20GS linker (SEQ ID NO: 109) either to a
single peptide of the
invention (in which said peptide is one of the peptides of SEQ ID NO: 72, 103,
104, 105,
106, 107 or 108, respectively), or to a tandem of two peptides of the
invention that are linked
to each other via a 9GS linker (SEQ ID NO: 53).
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In the constructs of SEQ ID NO 's: 110 to 116, a peptide of the invention is
used that
contains a VG sequence at the C-terminal end, which may serve as (part of) a
(C-terminal)
tag for affinity purification (for example, using an affinity matrix that is
based on Vj-1j-j's that
have been raised against peptide of the invention that contains such a tag, as
further described
herein). Similarly, in the constructs of SEQ ID NO's 117 to 125, an amino acid
sequence of
the invention. is used that comprises two peptides of the invention (which are
the same or
different, and which are linked via a 9G S, linker), in which the peptide of
the invention that is
at the C-terminal end of the tandem contains such a VG sequence at the C-
terminal end.
Example 3: in silico modelling of the interactions of the amino acid sequences
of the
invention with human serum albumin.
For the interaction of the amino acid residue at position 3 in the amino acid
sequences
of the invention, and of the further amino acid residues downstream of
position 3, reference is
made to Example 8 of WO 09/12769 1, incorporated herein by reference.
Modelling of the potential interactions of some preferred amino acid residues
that can
be (and preferably are) present upstream of position 3 (i.e. in addition to
those downstream of
position 3 as mentioned in Example 8 of WO 09/127691) was performed ICM-Pro
(Molsoft)
and Discovery Studio (Accelrys) with a force-field that is based on the
parameters as
described in Momany et al. (Momany et al. J. Phys. Chem. 1975, 79, 2361-2381).
In respect of human serum albumin, for the purposes of the disclosure herein,
reference is made to the sequence given under Genbank accession number
AAA98797
(Minghetti et al., J. Biol. Chem. 261 (15), 6747-6757 (1986); SEQ ID NO: 144.)
- see also
Example 8 of WO 09/127691.
The results of the modelling study are summarized in Table Ill, which in the
second
column exemplifies some of the preferred amino acid residues that could be
present at the
positions listed in the first column for interaction wit h the amino acid
residues on human
serum albumin mentioned in the third column. The numbering of the positions is
again as
mentioned above. Underlined residues form a small subpocket on human serum
albumin
wherein a hydrophobic or aromatic residue can reside. As can be seen, and
although the
invention is not limited to any specific explanation or hypothesis, there is a
correlation
between the modelling data shown in Table III and the results of the binding
study shown in
Table 11 above.
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Table III: modelling of some preferred amino acid residues (interaction with
human
serum albumin) that can be present towards the N-terminal end of the amino
acid
sequences of the invention.
Position in Possible amino-acids at said position Potential HSA
peptide partners
6 Hydrophobic, e.g. L, I, V N133, P445, T446,
2
Aromatic, e.g. W Y, F L487, H488, K.490,
Polar, e.g. R, E, Q, N, D 1491,1547
a Hydrophobic, e.g. L, 1, V M N133, N135, P445,
1 m Aromatic, e.g. W, Y, F T446. L487, T491
Polar, e.g,R,E,Q,N,D
N133, V442*, S443,
T446, E449, L484,
0 e Preferably hydrophobic or aromatic L487, H488, K490,
T491, V493
D13
-1 0 Preferably polar or aromatic T491
* Hydrophobic, e.g. L, 1, V, M N135, K490, T491
-2 Aromatic, e.g. W Y, F
Polar, e.g. R, E, D
Again, without being limited to any specific explanation or hypothesis, the
following
observations can be made based on the modelling data,
- When the part of the amino acid sequences of the invention downstream of
(and
including) position 3 undergoes the interactions shown in Example 8 of WO
09/127691,
N-terminal part of the peptide is in close proximity to hydrophobic and
aromatic residues
on human serum albumin: e.g. L487, L485 + residues with significant aliphatic
contribution (e.g. K490). These appear to form part of a hydrophobic subpocket
that,
amongst others, comprise one or more of the underlined amino acid residues in
Table 111,
Thus, it is assumed that preferably, the amino acid. sequences of the
invention contain,
towards the N-terminal end, one or more amino acid residues that can undergo
an
interaction with these residues on human serum albumin (such as, for example,
the
residues mentioned in the second column of Table 1II).
- This subpocket on human serum albumin. also appears to comprise some
partially positive
charged residues (for example, K490 and H488). Thus, it is assumed that
preferably, the
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amino acid sequences of the invention may also contain (or in addition
contain), towards
the N-terminal end, one or more (partially) negatively charged and/or aromatic
amino
acid residues that can undergo an interaction with these residues on human
serum
albumin,
- The N-terminal part of the amino acid sequences of the invention is likely
also in close
proximity to some partially negatively charged residues on human serum
albumin, such
as D 131 and NI 33. Thus, it is assumed that the amino acid sequences of the
invention
may also contain (or in addition contain), towards the N-terminal end, one or
more
(partially) positively charged amino acid residues that can undergo an
interaction with
these residues on human serum albumin,
Exam le 4: Pharmacokinetic profile in male c nomol us monkeys
The pharmacokinetic profile of the following Nanobody constructs (each
hereafter
also referred to as a "construct" or "test item"): 2D3-9GS-EXP80B10 (EXP424),
5F7-9GS-
EXP89DO3 (EXP413) and 5F7-9GS-EXP89DO3-9GS-EXP89DO3 (EXP486) were analysed
in male cynomolgus monkeys of approximately 3 to 4 years old and is compared
to the 2D3
control ("control" or "negative control" hereafter), according to the
following protocol. The
results are listed in Table IV below.
The construct and the control are each injected in three monkeys. Both the
construct
and the control are administered at a dose of 2 mg/kg via intravenous
infusion. Blood
samples are taken at predose, 5 min, 20 min, Ih, 2h, 4h, 8h, and 16h after
administration and at test days 2, 3, 5, 7, 9, 12, 15, 18, 21, 24, 27, 30, 33,
36, 39, 42,
45, 48, 51, 54, and 57 after the start of the infusion. In order to obtain at
least 0.25
mL serum per animal per sampling time, a sufficient volume of whole blood is
withdrawn per sampling time and the serum is isolated after lh of incubation
at 37 C.
The serum samples are stored at -80 C.
Serum samples are tested for serum levels of construct and the control,
respectively,
using the following ELISA assay (see also Examples 7, 13 and 14 of WO
09/127691,
where essentially the same methodology is used).
96-well microliter plates (Maxisorp, Nunc, Wiesbaden, Germany) are coated for
1.
hour at 37 C with Recombinant Human ErbB2/Fc Chimera, CF (R&D Systems,
Minneapolis) in PBS at 3 pg/mL for the negative control and 4.5 ug/mL for the
test
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item. Wells are aspirated and blocked for 30 minutes at room temperature (RT)
with
SuperBloekOO T20 PBS (Pierce, Rockford, IL). After this blocking step, wells
are
washed with PBS-0.05% Tween20.
Preparations for the standards, QC samples and dilutions of the test samples
are
performed in a non-coated (polypropylene) plate.
Standard curve and QC-samples: Solutions at the required concentrations are
prepared
in PBS 0.1% casein and spiked into 100% monkey serum. To prepare standards and
QC samples, a 1/10 dilution of the pure monkey serum dilutions is made in PBS-
0.1%
casein.
Test samples: Dilution factors for the test samples are estimated, and varied
from 1/10
to 1/500. Samples are diluted 1/10 in PBS 0.1% casein in a first step, and if
needed,
further dilution was done in PBS 0.1% casein containing 10% monkey serum.
These
sample dilutions are further serially dilated 115 in PBS 0.1 % casein with 10%
monkey
serum over 2 wells.
Standards, QC samples and the 1 /5 dilutions of the test samples are
transferred onto
the coated plate and incubated for 1 hour at RT. Afterwards the plates are
washed and
rabbit polyclonal anti-VHH K1, purified against protein A and Her2/Fc
depleted, is
added at 1 ig/mL in PBS 0,1% casein, and incubated for 1 hour at RT. After
washing
a 1/2000 dilution in PBS 0.1% casein of horse radish peroxidase labelled goat
anti-
rabbit (Dakocytomation, Denmark) is added to the plate and incubated for a0
minutes
at RT. This enzyme catalyzes a chemical reaction with the substrate sTMB
(3,3',5,5'-
tetramethylbenzidine, SDT reagents, Brussels, Belgium), which results in a
colorimetric change. After stopping this reaction after 15 minutes using HCI
(iN),
the intensity of the colour is measured by a spectrophotometer, which
determines the
optical density of the reaction product, using a 450 nm wavelength of light.
Mean serum concentrations were calculated per dose group and per sampling time
point using Microsoft Excel 2007. In case one out of two values was <LLOQ, the
BQL value was set at zero and the mean calculated; if the mean was <LLOQ, BQL
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was reported, otherwise the mean was reported. If both values were below the
LLOQ,
BQL was reported. For pharmacokinetic analysis nominal times were used as all
actual blood sampling times were within 5% of the protocol specified nominal
times.
Individual serum concentration-time profiles were subjected to non-
compartmental
analysis (NCA) (Model 201; i.v. bolus injection) using WinNonlin Pro 5.1
(Pharsight
Corporation, USA; 2006). The area under the curve (AUC) was estimated using
the
lin up/log down rule. LLOQ values were treated as missing, except when
comprised
between two values above the LLOQ, then they were set to zero. The
concentration at
time zero (CO) was estimated through back-calculation based on the two first
data
points. The terminal elimination half-life (t1/2) was calculated automatically
(best-fit)
using a log-linear regression of the non-zero concentration-time data of the
log-linear
portion of the terminal phase. A minimum of three points were considered for
the
determination of 2,z.
The following main pharmacokinetic parameters were estimated: the predicted
serum
concentration at time zero (Co); the area under the serum concentration-time
curve
extrapolated to infinity (AUC,,,f), total body clearance (CL), the apparent
volume of
distribution at steady-state (VdSS), and the terminal half-life (t112,
terminal).
In Table IV, mean (+/- s.d.; n=3) serum concentration-time profiles of EXP413,
EXP424 and EXP486 in male Cynomolgus monkeys following a single intravenous
(bolus)
dose at 2 mg/kg bodyweight, together corresponding key pharmacokinetic
parameters, are
compiled.
The mean (n=3) predicted dose-normalized maximum serum level (dose norm. Co)
of
EXP424 was 54,1 dug/ml . Similar values were calculated for EXP413 (59,2
g/ml) and
EXP486 (53,9 kg/ml).
The mean (n=3) estimated Clearance and Vdss values of EXP424 were
28.3 mL/day*.kg and 69 mL/kg, respectively. Corresponding values for EXP413
containing
Nanobody 5F7 linked to 89D03 (SEQ ID NO: 72) in monovalent format, were 19,4
mL/day*kg and 48,7 mL/kg, those of EXP486 containing 89D03 (SEQ ID NO: 72) in
a
tandem repeat format (in which two 89D03 peptides were linked to each other
via a 9GS
linker) amounted to 10,3 mL/day*kg and 59,3 mL/kg.
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The estimated mean half-life (tl/2 terminal) for EXP424 was 37,6 hours or 1,6
days. The
mean half-life for EXP413 was 31,2 h or 1,3 days and for EXP486 84,5 hours or
3,5 days.
Table IV: Main pharmacokinetic parameters (mean +/- SD, n=3) of EXP424, EXP413
and EXP486 following i.v. bolus administration of 2 mg/kg EXP424, EXP413 and
EXP486, respectively in the male cynomolgus monkey.
EXP 424 EXP413 EXP 486
Parameter Units Mean SD Mean SD mean SD
CO ,g/rnl 54.1 14,9 59,2 21,5 53,9 9,6
AUCinf Jg*day/ml 74,0 20,8 107,0 26.5 194,0 14,4
CL ml/day*kg 28,3 6,9 19,4 43 10,3 0,76
Vdss ml/kg à 69,2 8,6 48,7 14,6 59,3 6,36
13,9
TV2 h 37,6 18 31,2 3,3 84,5
(terminal)
The terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention in the use of
such terms and
expressions of excluding any equivalents of the features shown and described
or portions
thereof, it being recognized that various modifications are possible within
the scope of the
invention.
All references disclosed herein are incorporated by reference, in particular
for the
teaching that is referenced hereinabove.
