WO2011095545A1 - Peptides capable of binding to serum albumin and compounds, constructs and polypeptides comprising the same - Google Patents

Peptides capable of binding to serum albumin and compounds, constructs and polypeptides comprising the same Download PDF

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Publication number
WO2011095545A1
WO2011095545A1 PCT/EP2011/051559 EP2011051559W WO2011095545A1 WO 2011095545 A1 WO2011095545 A1 WO 2011095545A1 EP 2011051559 W EP2011051559 W EP 2011051559W WO 2011095545 A1 WO2011095545 A1 WO 2011095545A1
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amino acid
seq
acid sequence
sequence
residue
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PCT/EP2011/051559
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French (fr)
Inventor
Hilde Adi Pierrette Revets
Carlo Boutton
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Ablynx Nv
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Priority to CA2788993A priority Critical patent/CA2788993A1/en
Priority to JP2012551620A priority patent/JP2013518853A/en
Priority to AU2011212442A priority patent/AU2011212442A1/en
Priority to CN2011800083085A priority patent/CN102781959A/en
Priority to EP11702631A priority patent/EP2531523A1/en
Publication of WO2011095545A1 publication Critical patent/WO2011095545A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • 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.
  • an Arg (R) residue in particular an Arg (R) residue that is capable of forming a
  • exemplary peptides described in WO 09/127691 may for example contain one or more of the following features:
  • 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);
  • 09/127691 it is an object of the invention to provide amino acid sequences that: - 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:l, 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 09/127691 : SEQ ID NO: 155/ SEQ ID NO: 77 herein); and/or 59C2 (WO 09/127691 : SEQ ID NO: 156/SEQ ID NO: 75 herein);
  • 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
  • serum albumin from a mouse, rat, rabbit, dog or a species of primate such as baboon or rhesus monkey
  • primate such as baboon or rhesus monkey
  • amino acid sequences described, herein can bind to (and in particular, specifically bind to, as defined herein) serum albumin (and in particular to human serum 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.
  • serum albumin and in particular to human serum albumin
  • amino acid sequences are as further defined herein.
  • 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-tenninal end of the peptides may be as further described herein.
  • 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.
  • these 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.
  • 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).
  • 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.
  • 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 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.
  • 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.
  • 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
  • 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.
  • the amino acid sequences of the invention may be as described in WO 09/127691 (with the same preferred aspects and features applying).
  • 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 serom albumin: V442, S443, T446, L484, L487, H488, 490, T491 and/or V493 (numbering as described in Example 8 of WO 09/127691 ).
  • the amino acid sequences of the invention may be as described in WO 09/127691 (with the same preferred aspects and features applying).
  • 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.
  • 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.
  • the amino acid sequences of the invention may be as described in WO 09/127691 (with the same preferred aspects and features applying).
  • 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, 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.
  • 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.
  • 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 K D ) 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 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).
  • 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).
  • the amino acid sequences of the invention may be as described in WO 09/127691 (with the same preferred aspects and features applying).
  • 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, H488, 490, T491 and/or V493.
  • 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).
  • the amino acid sequences of the invention may be as described in WO 09/127691 (with the same preferred aspects and features applying).
  • 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.
  • 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.
  • 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.
  • 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 D) 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).
  • a NANOBODY® such as the Nanobody 2D3 used as an example herein.
  • 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).
  • the amino acid sequences of the invention may be as described in WO 09/127691 (with the same preferred, aspects and features applying).
  • 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, T4 1 and/or V493.
  • 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.
  • 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.
  • 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.
  • 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.
  • further amino acid residues preferably 3, 4, 5, 6 or 7 amino acid residues.
  • 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).
  • 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
  • Another aspect of the invention relates to an amino acid, sequence 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).
  • d) contains a stretch of amino acid residues upstream of posi tion 3 that is as descri bed herein (including the preferred aspects of such a stretch of amino acid residues, also as described herein);
  • amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID NO: l ).
  • 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: l 1) and/or FGGGT (SEQ ID NO: 12);
  • 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);
  • amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID NO: l).
  • amino acid sequence is as further described herein.
  • 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.
  • amino acid sequences of the invention may be replaced 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).
  • T threonine
  • such an amino acid sequence may be as described above, but may instead of the sequence motif D YD VF (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.
  • 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).
  • 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;
  • 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);
  • amino acid sequences of the invention may be replaced 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).
  • T threonine
  • such an amino acid sequence may be as 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 DYTVFG (SEQ ID NO: 129), YTVFGG (SEQ ID NO: 130) or TVFGGG (SEQ ID NO:13i), respectively.
  • 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).
  • a) contains one or more of the following sequence motifs: D YDVFGG (SEQ ID NO: 17),
  • 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;
  • 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);
  • amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP (SEQ ID NOT).
  • amino acid sequence is as further described herein.
  • 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.
  • 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;
  • 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);
  • Trp (W) residue in particular a Trp (W) residue that is capable of forming
  • 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).
  • amino acid sequences of the invention preferably (at least) contain:
  • 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).
  • 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 K D ) 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 invention to another protein or peptide, for example as a fusion with a NANOBODY® (such as the Nanobody 2D3 used as an example herein).
  • a NANOBODY® such as the Nanobody 2D3 used as an example herein.
  • 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
  • c) comprises an Arg (R) residue, in particular an Arg (R) residue that is capable of
  • 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 albumin: V442, S443, T446, L484, L487, H488, K490, T491 and/or V493 (numbering as described in Example 8 of WO 09/127691 ).
  • 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).
  • the above amino acid sequence is also preferably as further described herein.
  • 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
  • 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 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.
  • 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;
  • 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, 490, T491 and/or V493 (numbering as described, in Example 8 of WO 09/127691 ).
  • 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 NO: 19) (or alternatively one of the sequence motifs of SEQ ID NO's: 134 to 139); and most preferably both these sequence motifs.
  • RXWD the sequence motif RXWD
  • X may be any amino acid sequence but is preferably W, Y, F, S or D
  • 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:
  • 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 1 "amino acid difference" - as defined herein - with at least one of these sequences, provided they (preferably) still meet 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.
  • 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.
  • 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.
  • 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).
  • RYWDFDVFGGGT (SEQ ID NO: 35); and in particular chosen from
  • RDWDFDVFGGGTP (SEQ ID NO: 37); RSWDFDVFGGGTP (SEQ ID NO: 38) or RY WDFD VF GGGTP (SEQ ID NO: 39); and in particular chosen from
  • RDWDFDVFGGGTP SEQ ID NO: 37
  • RSWDFDVFGGGTP SEQ ID NO: 38
  • RYWDFDVFGGGTP SEQ ID NO: 39
  • 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.
  • 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.
  • sequence of SEQ ID NO:l (and in particular, than at least one and preferably all of SEQ ID NO: 75, 76 and/or 77); and/or
  • amino acid sequence of the invention is said to bind better to serum albumin 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: l) (for example as determined using the BIAcoreTM measurement used in Example 2).
  • 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.
  • 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.
  • 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 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 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).
  • KD affinity
  • amino acid sequences of the invention are given in Table ⁇ and SEQ ID NO's: 54 to 74.
  • those sequences binding to serum albumin (per se and/or as a fusion with 2D3) better than 30 nM are particularly preferred (such as one of the amino acid sequences of SEQ ID NO's: 56, 59, 68, 70, 72 or 74).
  • 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).
  • 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.
  • 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: l .
  • an amino acid sequence of the inventio 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.
  • substitutions, insertions and/or deletions 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 1 15 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.
  • 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.
  • 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).
  • polypeptides 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).
  • a threonine (T) residue when present at position 14 and when said threonine residue at position 14 is susceptible to phosphorylation in the intended host organism (such as a strain oiPichia 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.
  • the threonine (T) at position 14 may be replaced with an alanine (A), asparagine (N) or aspartate (D) residue.
  • A alanine
  • N asparagine
  • D aspartate
  • amino acid sequences of the invention in which the threonine at position 14 has been so replaced are 89D03V1 (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).
  • 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:l, 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:l (which then preferably are conservative substitutions as defined herein).
  • the aspartate residue (D) at position 15 of SEQ ID NO:l is replaced by an amino acid residue chosen from proline (P) or a small amino acid residue (A, G, S or T);
  • - 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);
  • amino acid sequences of the invention compared to the serine residue (S) at position 3 of SEQ ID NO:l. the serine residue (S) at position 3 is replaced by arginine ( ).
  • amino acid sequences may comprise one or more further amino acid insertions, deletions and/or substitutions as described herein.
  • S serine residue at position 5 of SEQ ID NO:l 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);
  • 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 1 15 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).
  • amino acid sequences of the invention are the amino acid sequences from Table II that bind to human serum albumin
  • 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.
  • 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.
  • 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).
  • amino acid sequences of the invention 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) 1 33; Pro (P) 134: Asn (N) 135; Leu (L) 136; Leu (L) 139; Arg (R) 141 ; Tyr (Y) 162; Glu (E) 165; lie (I) 1 66; His (H) 170; Phe (F) 173 ; Phe (F) 181 ; Gly (G) 213; Lys (K) 214; Ser (S) 217; Gin (Q) 483; and/or Lys (K) 543; and/or such that they can compete with the amino acid sequence of SEQ ID NO: l and/or with at least one and preferably all of the amino acid sequences of SEQ ID NO: 75, 76 and/or 77
  • 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, He (I) 166, His (H) 170, Phe (F) 173, Phe (F) 181 , Gly (G) 213, Lys (K) 214, Ser (S) 217 and Gin (Q) 483; and/or such that they can compete with the amino acid sequence of SEQ ID NO: l and/or with at least one and preferably all of the amino acid sequences of SEQ ID NO: 75.
  • amino acid sequences of the invention 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
  • 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.
  • the invention does not comprise the amino acid sequences that are mentioned in Figure 4 or Figure 8 of PCT/EP2007/063348.
  • amino acid sequences of the invention are preferably such that they can bind to a serum albumin, and in particular to human serum albumin:
  • K D dissociation constant in the range of 10 "5 to 10 '12 moles/liter or less, and preferably in the range of l O "7 to 10 "12 moles/liter or less and more preferably in the range of 10 " to 10 " moles/liter (i.e. with an association constant (K A ) of in the range of 10 5 to lO 12 liter/ moles or more, and preferably in the range of 10 7 to 10 12 liter/moles or more, and more preferably in the range of 10 to 10 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:l) binds to human serum albumin;
  • AASYSDYDVFGGGTDFGP (SEQ ID NO: 1 ) binds to human serum albumin
  • 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 PCT/EP2007/060850 of Ablynx N. V.), i.e. such that:
  • K D dissociation constant
  • 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).
  • serum albumin such as human serum albumin
  • 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.
  • 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 fascicularis) and/or rhesus monkeys (Macaca mulatto) and baboon (Papio rsinus), and preferably at least with cyno serum albumin.
  • an amino acid sequence of the invention when 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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
  • 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.
  • 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);
  • 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,
  • 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 and/or uncharged at such a higher or lower pH, as will be clear to the skilled person.
  • 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.
  • 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 G]y; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, 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 Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn;
  • 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;
  • 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.
  • polypeptide of the invention 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.
  • 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
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • sandwich competition assays sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • the dissociation constant may be the actual or apparent dissociation constants
  • 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
  • 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 pharmacokinetic 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 monlieys from the genus Macac (such as, and in particular, cynomoigus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ur sinus)) 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.
  • the half-life can be expressed using parameters such as the 11/2 -alpha, 11/2 -beta and the area under the curve (AUG).
  • 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.
  • 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.
  • 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.
  • 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 1%, 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 the same assay under the same conditions but without the presence of the construct of the invention.
  • moduleating 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 H, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention.
  • 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.
  • 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
  • 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
  • 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
  • an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, 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 constr uct 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 Hgand, 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
  • 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)merization 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.
  • 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).
  • 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 KD value, KA value, K 0 f rate and/or Ko n rate) that is at least 10 times, such as at least 100 times, and preferably at least 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.
  • an affinity as described above, and suitably expressed as a KD value, KA value, K 0 f rate and/or Ko n rate
  • 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.
  • 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.
  • 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.
  • 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 ser um albumin and cyno serum albumin) if it is specific for (as defined herein) both these different antigens or antigenic determinants.
  • antigens or antigenic determinants such as serum albumin from two different species of mammal, such as human ser um albumin and cyno serum albumin
  • binding that is "essentially independent of the plf ' is generally meant herein that the association constant (K A ) 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 1 10%, more than 120% or even 130% or more, or even more than 150%), or even more than 200%) of the association constant (K A ) of the amino acid sequence with respect to the same serum protein at the pH value(s) that occur outside said cell.
  • the k 0ff rate (measured by Biacore - see 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 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 350%, or even more than 200%) of the karate 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.
  • 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.
  • 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.
  • an amino acid sequence or other binding agents such as a NANOBODY®, polypeptide or compound or construct of the invention
  • the extend to which, an amino acid sequence or other binding agents of the inventio 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 plasroon 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 Biacore machine for example the Biacore 3000
  • the target protein is coupled to a CMS Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target.
  • 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).
  • 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.
  • 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.
  • 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 micromoiar (on a binding site basis).
  • 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 niM HC1 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.
  • 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.
  • 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 CMS 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.
  • 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.
  • C -terminal His-tagged target could alternatively be used.
  • 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).
  • 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 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 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.
  • 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 low r er than the moles of Ab-X target binding sites that were used for coating each well.
  • 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.
  • 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 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)
  • the target detection signal i.e. the amount of target bound by the coated amino acid sequence
  • the target detection signal i.e. the amount of target bound by the coated amino acid sequence
  • amino acid sequences of the invention may be prepared in a manner known per se.
  • 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.
  • One method for preparing the amino acid sequences or polypeptides of the invention generally comprises at least the step of:
  • Another method for preparing the amino acid sequences or polypeptides of the invention generally comprises at least the step of:
  • 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.
  • amino acid sequences disclosed herein can be used with advantage as a fusion partner in order to i ncrease the half-life of therapeutic moieties such as proteins, compounds (including, without limitation, small molecules) or other therapeutic entities.
  • 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).
  • not significantly reduced is meant that the half-life of the serum protein molecule (as measured using a suitable technique 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 10%, such as not reduced by more than 5%, or essentially not reduced at all.
  • 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).
  • 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).
  • 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").
  • 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.
  • 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.
  • 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 multimer or concatamer that comprises at least two (such as two, three or fom-) 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.
  • a multimer or concatamer that comprises at least two (such as two, three or fom-) 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 comprising the same) form a further aspect of the invention, and may bind to serum albumin with a higher avidity than a monomelic amino acid sequence of the invention.
  • each of these therapeutic moieties may be linked to one or more amino acid sequences of the invention, as further described herein.
  • 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.
  • 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.
  • 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.
  • the at least one therapeutic moiety comprises or essentially consists of a therapeutic protein or polypeptide.
  • 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).
  • 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 domain.
  • 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 detenninant (such as a therapeutically relevant target).
  • 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.
  • protein scaffolds derived from protein A domains such as AffibodiesTM
  • tendamistat fibronectin
  • lipocalin CTLA-4
  • T-cell receptors designed ankyrin repeats
  • avimers and PDZ domains Bosset et al., Nat. Biotech 2005, Vol 23: 1257
  • binding 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).
  • amino acid sequences of the invention may also be linked to one of the amino acids
  • 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 05/1 18642; antagonists of the interleukin 1 receptor (see pages 1 1-12 of WO 05/1 18642) including functional variants of IL-lra; saporins (see pages 12-14 of WO 05/118642); the anticancer peptides listed in Table 8 of WO 05/1 18642; and insulinotropic agents or analogues thereof such as GLP-1 or GLP-1 analogues (see 06/059106).
  • the at least one therapeutic moiety comprises or essentially consists of at least one domain antibody or single domain antibody, "dAb" or
  • 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 multispecific 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).
  • 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®.
  • a compound of the invention preferably has a half-life that is more than 1 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.
  • 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.
  • the compounds or polypeptides of the invention may have a half- life that is increased with more than 1 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.
  • such compounds or polypeptides of the invention have a serum half-life that is increased with more than 1 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 multispecific 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.
  • 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.
  • 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).
  • a suitable strain of Pichia pastoris such as, for example and without limitation, a protease-deficient strain or another suitable strain.
  • a method of the invention for expressing an amino acid sequence, compound, protein, polypeptide, fusion protein, or multivalent or multispecific construct 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.
  • 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.
  • they may suitably contain one of the sequence motifs of SEQ ID NO's: 135 to 140 or 143 to 148.
  • a method of the invention for expressing a amino acid sequence, compound, protein, polypeptide, fusion protein, or multivalent 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 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 oiPichia 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 constmct comprising the same) that comprises at least one of the sequence motifs of SEQ ID NO's: 126 to 348, 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).
  • SEQ ID NO's: 104 to 108 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 SEQ ID NO's : 1 1 1 to 125 give some examples of constructs comprising the same based on the 5F7 Nanobody (used as an example of a Nanobody).
  • a method of the invention for expressing a amino acid sequence, compound, protein, polypeptide, fusion protein, or multivalent or multi specific 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 particulai * . 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.
  • a yeast strain and in particular, a suitable Pichia strain, such as a suitable strain of Pichia pastoris
  • a suitable Pichia strain such as a suitable strain of Pichia pastoris
  • 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).
  • 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 ceil produces an amino acid sequence or polypeptide of the invention
  • 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 particulai-, a suitable Pichia strain, such as a suitable strain of Pichia pastoris), 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.
  • T threonine
  • 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) that either shows reduced (i.e. essentially no) phosphorylation or that has been genetically modified to show reduced (i.e. essentially no) phosphorylation.
  • a suitable yeast strain and in particular, a suitable Pichia strain, such as a suitable strain of Pichia pastoris
  • "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.
  • 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.
  • 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 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 V H H 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 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
  • 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.
  • the affinity matrix comprises a ligand, binding domain or binding unit (such as a V HH ) 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).
  • a ligand, binding domain or binding unit such as a V HH
  • an amino acid sequence of the invention that comprises, downstream of the (C-terminal) GGG motif at positions 1 1 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 bind 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).
  • 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);
  • a method for isolating and/or purifying a compound, protein, polypeptide, fusion protein, or multivalent or multispecific construct of the invention i.e.
  • 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 11 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.
  • 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 1 1-13 (for example and without limitation at positions 16 and 17 or further downstream).
  • amino acid sequences of the invention may comprise one of the sequence motifs GGGTPVG (SEQ ID NO: 150),
  • GGGAPVG (SEQ ID NO: 1.51), GGGNPVG (SEQ ID NO: 152) or GGGDPVG (SEQ ID NO: 153).
  • 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: 1 14 to 1 16.
  • SEQ ID NO's: 1 17 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 towards the C -terminus (i.e.
  • 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 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 the therapeutic and 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).
  • 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: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.
  • 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).
  • 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.
  • the invention relates to a pharmaceutical (including diagnostic) composition, preparation or formulation comprising a compound, protein, polypeptide, fusion protein, or multivalent or muitispecific 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 muitispecific construct of the invention (i.e. delivering the same into the circulation via administration to the lungs).
  • amino acid sequences of the invention are given as SEQ ID NO's: 54 to 74 in Table II below. Binding to human serum albumin was determined as a fusion of the sequence with the Nanobody 2D3 (see Example 2 and the further examples from WO 09/127691 mentioned herein).
  • GGGGSGGGSA [SEQ ID NO: 48) (which comprises a GIy4Ser-G3y3Ser linker with a flanking amino acid residue A at the C-terminus) and GGGGSGGGGSGGGGSGGGGS [SEQ ID NO: 109] (also referred to as 20GS herein).
  • 5F7 was so linked to 89D03 (SEQ ID NO: 72) in monovalent format.
  • the resulting Nanobody construct (referred to as 5F7-9GS-EXP89D03 or EXP413) was:
  • 5F7 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:
  • 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 V H H' S that have been raised against peptide of the invention that contains such a tag, as further described herein).
  • Example 3 in silico modelling of the interactions of the amino acid sequences of the invention with human serum albumin.
  • 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 III.
  • 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 III).
  • 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 D131 and N133.
  • 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,
  • Nanobody constructs each hereafter also referred to as a "construct” or “test item": 2D3-9GS-EXP80B10 (EXP424), 5F7-9GS- EXP89D03 (EXP413) and 5F7-9GS-EXP89D03-9GS-EXP89D03 (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
  • 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).
  • 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 diluted 1/5 in PBS 0.1 % casein with 10% monkey serum over 2 wells.
  • 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.
  • 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 was reported, otherwise the mean was reported.
  • 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.
  • the estimated mean half-life (tj /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.

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Abstract

The present invention relates to amino acid sequences that are capable of binding to serum proteins; to compounds, proteins, polypeptides, fusion proteins or constructs comprising or essentially consisting of such amino acid sequences; to nucleic acids that encode such amino acid sequences, compounds, proteins, polypeptides, fusion proteins or constructs; to compositions, and in particular pharmaceutical compositions, that comprise such amino acid sequences, compounds, proteins, polypeptides, fusion proteins or constructs; and to uses of such amino acid sequences, compounds, proteins, polypeptides, fusion proteins or constructs.

Description

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 (ii) a Tip (W) residue, in particular a Tip (W) residue that is capable of forming
electrostatic interactions with the Arg ( ) 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 ciystal 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, whic peptides can be linked or fused to a therapeutic moiety, compound, protein or other therapeutic entity in order to increase the half-life thereof. SEQ ID NO:l is an example of a peptide that has been generated using the methodology described in WO 08/06S280 (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
09/127691. In particular, it is an object of the invention to provide amino acid sequences that: - 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:l, 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 09/127691 : SEQ ID NO: 155/ 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 cynomolgus 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 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-tenninal 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 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)
EWEODRDWPFDVFGGGTP
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 ID NO:96) are said, to be "towards the N-terminal end" or "upstream" of the RXWD motif (it should also be noted that, 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 1 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, I, K, Q or V at position 3, and the same applies to the amino acid sequences of the inventi on).
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
residue Q D R D w D p D V
Position [etc.] A -3 -2 -1 0 1 2 3 4 5 6 8 9 [etc.].
"Upstream" of position 3 « 3 » "Downstream" of position 3 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:l (reference), 59C2 (SEQ ID NO: 75, reference), 80B10 (SEQ ID NO: 56, invention); 89D03 (SEQ ID NO:72, invention), 89D03V1 (SEQ ID NO: 103, invention), 89D03V1VG (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 parti cular, 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 serom albumin: V442, S443, T446, L484, L487, H488, 490, 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 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).
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 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, 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, 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 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 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 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, H488, 490, 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, 490, T491 and/or V493. 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 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, S443, 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 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 inventi on 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, 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, 490, 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 D) 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, T4 1 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 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 inventi on 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, 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 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, YWWER, AWYDY, WWNWR, EWWWR YDWFY, RDWFL, DWWNR, YGDWF, WWTWG, P1DFW, WWTSD, QKLYW, KWWEI. WWSTP. LFWWE, WWLOE,
WWEQD, NQLIV, 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 1 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: l);
and that:
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: l);
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: l); 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 ID 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 posi tion 3 that is as descri bed 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: l ).
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: l 1) 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 NOT):
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: l). 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 D YD VF (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 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
AA S YSD YD VFGGGTDFGP (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 AAS YSD YD VFGGGTDFGP (SEQ ID NO:l). 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 DYDVFG (SEQ ID NO: 13), YDVFGG (SEQ ID NO: 14) or DVFGGG (SEQ ID NO: 15) contain the sequence motif DYTVFG (SEQ ID NO: 129), YTVFGG (SEQ ID NO: 130) or TVFGGG (SEQ ID NO:13i), 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: D YDVFGG (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: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 NOT). 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 D YTVFGG (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, 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 O: 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: l). 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: D YDVFGGGT (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: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: l). 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 DYDVFGGG (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 YDVFGGG A (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 (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, 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 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, S443, 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 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 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 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
AASYSDYDVFGGGTDFGP (SEQ ID NO:l);
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: l);
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 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
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: l);
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO:l);
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 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
A AS YSD YD VF GGGTDFGP (SEQ ID NO:l);
b) binds better to human serum albumin than the amino acid sequence
AASYSDYDVFGGGTDFGP (SEQ ID NO: l);
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, 490, 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 NO: 19) (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 M, 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 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 1 "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 XWDXDVFGGGT (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 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.
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. 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.
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 RY WDFD VF GGGTP (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 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).
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); 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);
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.
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:l. (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:l (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? kon or k0ff rate, and determined using one of the methods described herein) than the amino acid sequence of SEQ ID NO:l (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:l (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:l (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 BIAcore™ 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 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: l) (for example as determined using the BIAcore™ 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 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 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 Π 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 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:l), 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:l). Such substitutions may for example be an R at position 3 and/or a W at position 5, as described herein and in WO 09/327691.
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: l . Usually, compared to the sequence of SEQ ID NO: 1, an amino acid sequence of the inventio 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 1 15 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 (compared to the amino acid sequence of SEQ ID NO: 1) 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:l , 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.
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).
Figure imgf000030_0001
Optionally, based on the disclosure herein and in WO 09/127691 , 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 mtended substitutions, insertions 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 amino acid sequence may then optionally be compared to the amino acid sequence of SEQ ID NO: l and/or to one or more of the amino acid sequences from WO 09/127691) 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 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 (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 acid substitutions compared to the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO: l), 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-transiational 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-transiational modifications (and the substitutions that can either
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-transiational modification 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 inventi on in a bacterial strain such as E. Coli or in a glycosylation-deficient mutant of a host-organism that would otherwise glycosylate 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 pastor is (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 and when said threonine residue at position 14 is susceptible to phosphorylation in the intended host organism (such as a strain oiPichia 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 oiPichia 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 89D03V1 (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 oiPichia 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:l, 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:l (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 or deletions, although not excluded from the scope of the invention, may cany 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 proline 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, 1 1, 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.l :
- the serine residue (S) at position 3 of SEQ ID NO: 1 is replaced by an amino acid residue chosen from arginine (R), proline (P),an aromatic amino acid residue (F, Y, W or H; in particular F, Y or W) or a hydrophobic amino acid residue (L, I, V or M);
and/or
- the serine residue (S) at position 5 of SEQ ID NO:l 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:l 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 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). 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:l. the serine residue (S) at position 3 is replaced by arginine ( ). 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:l 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: l 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:l 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).
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 1 15 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 II 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, or 1 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 20n3VL
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 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-termmus, 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 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).
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. 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 FcRn; 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). 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: l 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: l 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) 1 33; Pro (P) 134: Asn (N) 135; Leu (L) 136; Leu (L) 139; Arg (R) 141 ; Tyr (Y) 162; Glu (E) 165; lie (I) 1 66; His (H) 170; Phe (F) 173 ; Phe (F) 181 ; Gly (G) 213; Lys (K) 214; Ser (S) 217; Gin (Q) 483; and/or Lys (K) 543; and/or such that they can compete with the amino acid sequence of SEQ ID NO: l 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: l 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 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 (L) 136; Leu (L) 139 and Arg (R) 141; and/or (ii) the stretch of amino acid residues thai comprises the residues Tyr (Y) 162; Glu (E) 165; He (I) 166; His (H) 170: Phe (F) 173; Phe (F) 1.81; and/or (iii) the stretch of amino acid residues that comprises the residues Gly (G) 213; Lys ( ) 214 and Ser (S) 217; and/or such that they can compete with the amino acid sequence of SEQ ID NO:l 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:l 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 ammo acid 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, He (I) 166, His (H) 170, Phe (F) 173, Phe (F) 181 , Gly (G) 213, Lys (K) 214, Ser (S) 217 and Gin (Q) 483; and/or such that they can compete with the amino acid sequence of SEQ ID NO: l 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 NQ: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.
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 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. 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 (KD) in the range of 10"5 to 10'12 moles/liter or less, and preferably in the range of l O"7 to 10"12 moles/liter or less and more preferably in the range of 10" to 10" moles/liter (i.e. with an association constant (KA) of in the range of 105 to lO12 liter/ moles or more, and preferably in the range of 107 to 1012 liter/moles or more, and more preferably in the range of 10 to 10 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:l) binds to human serum albumin;
and/or
- with a kon-rate in the range of between 102 M"!s"' to about 107 IVfV1, preferably in the ranee between 103 M and 107 MV, more preferably in the range between 104 M" V1 and 107 Nf's"1, such as between 10^ M"!s"' and 107 M'V1, such that said kon-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 ko f rate in the range between Is"1 (t /2~0.69 s) and 10"6 s"! (providing a near irreversible complex with a t\a of multiple days), preferably in the range between 10"2 s' 1 and 10"& s"!, more preferably in the range between 10"J s"1 and 10"4 s"1, such as in the range between 10"4 s'1 and 1()"6 s"1, such that said k0ff-rate is better (i.e. higher) than the koff-rate with which the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID
NO: l) binds to human serum albumin. 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.
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 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"3 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 biological condition may differ in respect of H, in which said first biological condition may comprise a physiological pH of more than 7.0, for example a pH of more than 7.1 or a pH of more than 7.2, such as a pIT in the range of 7.2 to 7.4; and the second biological condition 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 fascicularis) and/or rhesus monkeys (Macaca mulatto) and baboon (Papio rsinus), 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 polypeptide 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). 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. 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 (suc 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]-fL]-[AA]-[L]-[TM]-[L]-[AA]
[AA]-[L]-[TM]-[L] AA]-[L]-[AA]-[L]-[TM]-[L]-[AA] in which "[TM]" refers to the therapeutic moiety, "[L]" refers to a linker (which in each case is optional), and "[AAJ" 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 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:
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 1 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 D) 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 invention to another protein or peptide, for example as a fusion with a NANOBODY® (such as the Nanobody 2D 3 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 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:
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 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 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-domam, a VL-domain, a VnH-domain or an antigen-binding fragment thereof); or a protein or polypeptide comprising the same (for example, an scFv construct). For such constructs, reference is for example made to the review by Holliger and Hudson, Nat
Biotechnol. 2005 Sep; 23(9): 1 126-36 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 VHH 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 lmown 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 07/1 10219); IL-6R (see for example WO 08/020079); GPCR's (see for example WO 08/074839); chemokines (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); 1L-R1 (see for example WO 06/059108; WO 07/063311 ; 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. 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 KD) better than 100 nM, preferably better than 50nM, more preferably better than 30 nM, such as equal to or better than 20nM5
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); 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 threonine (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 SE Q 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 I. 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 (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 herein) for human serum albumin and that comprises a Trp (W) residue that is capable of fomiing 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 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 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; 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 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 (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 DAFGGGT (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 as B) 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 invention to another protein or peptide, for example as a fusion with a NANOBODY® (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 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.
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 W7, 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. 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 50nM5 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).
In another aspect, the invention relates to a peptide that competes with the peptide of SEQ ID NO: l 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: 155/ 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: l, 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 human serum albumin; and that binds to human serum albumin 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 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 II, respectively, for binding 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; GIu (E) 165; He (I) 166; His (FI) 170; Phe (F) 173; Phe (F) 181 ; Gly (G) 213; Lys (K) 214; Ser (S) 217; Gin (Q) 483; and/or Lys (K) 543; more in particular 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 (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 (Hi) 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, He (I) 166, His (H) 170, Phe (F) 173, Phe (F) 181, Gly (G) 213, Lys (K) 214, Ser (S) 217 and Gin. (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 inventi on 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 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
A AS YSD YD VF GGGTD FGP (SEQ ID NOT).
b) Unless indicated otherwise herein (for example, in Example 8), 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: l). For example, S3R refers to a substitution, compared to the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NOT), of the serine residue S at position 3 into arginine (R). 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 IL-6R and polypeptides comprising the same for the treatment of diseases and disorders associated with Π-6 mediated signalling".
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.
Unless indicated otherwise, all methods, steps, techniques 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.
Amino acid residues will be indicated according to the standard three-letter or one- letter amino acid code, as mentioned in Table A;
Table A: one-letter and three-letter amino acid code
Figure imgf000060_0001
Notes:
Sometimes also considered to be a polar uncharged amino acid.
Sometimes also considered to be a nonpolar uncharged amino acid.
As will be clear to the skilled person, the fact thai 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 and/or uncharged at such a higher or lower pH, as will be clear to the skilled person. 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. 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 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).
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 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 will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence. 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 G]y; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, 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 Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn;
Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; 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 He 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 al., Principles of Protein Structure, Springer- Verlag, 1978, on the analyses of structure forming potentials developed by Chou and Fasman, 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, 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 VHH 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 cameiizing 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®, 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 epitope thereof) is said to be "against" or "directed against" or "specific for" said antigenic determinant, epitope, antigen or protein.
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 NAN O BODY® 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)
■ 12 1 12
of 10 to 10" moles/liter or less, and preferably 10" to 10" moles/liter or less and more preferably 10"8 to 10"12 moles/liter (i.e. with an association constant (KA) of 105 to
1 7 1
10 liter/ moles or more, and preferably 10 to 10 liter/moles or more and more preferably 10s to 1012 liter/moles). Any D value greater than 104 mol/liter (or any KA value lower than 104 M" ') 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 constants 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 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 pharmacokinetic 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 monlieys from the genus Macac (such as, and in particular, cynomoigus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ur sinus)) 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 al., 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 (1.982).
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 11/2 -alpha, 11/2 -beta and the area under the curve (AUG). 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. 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 1%, 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 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 H, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence 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 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 agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, 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 constr uct 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 Hgand, 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.
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)merization 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). 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 KD value, KA value, K0 f rate and/or Kon rate) that is at least 10 times, such as at least 100 times, and preferably at least 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.
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 ser um albumin and cyno serum albumin) if it is specific for (as defined herein) both these different antigens or antigenic determinants.
By binding that is "essentially independent of the plf' 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 1 10%, 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 pfF is generally meant herein that the k0ff 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 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 350%, or even more than 200%) of the karate 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. The terms "cross-block", "cross-blocked^ and "cross-blocking" are used
interchangeabl 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 inventio 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 plasroon 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 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 micromoiar (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 niM HC1 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 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 CMS 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 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).
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 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 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 lowrer 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 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 96/34103 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 Vlaams 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. 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 i ncrease 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 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 10%, 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 multimer or concatamer that comprises at least two (such as two, three or fom-) 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 comprising the same) form a further aspect of the invention, and may bind to serum albumin with a higher avidity than a monomelic 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.
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 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 detenninant (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 Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23: 1257). and binding 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 05/1 18642; antagonists of the interleukin 1 receptor (see pages 1 1-12 of WO 05/1 18642) including functional variants of IL-lra; saporins (see pages 12-14 of WO 05/118642); the anticancer peptides listed in Table 8 of WO 05/1 18642; and insulinotropic agents or analogues thereof such as GLP-1 or GLP-1 analogues (see 06/059106).
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 multispecific 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 1 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- life that is increased with more than 1 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 1 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 multispecific 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 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 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 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 oiPichia 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 constmct comprising the same) that comprises at least one of the sequence motifs of SEQ ID NO's: 126 to 348, 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 SEQ ID NO's : 1 1 1 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 multi specific 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 particulai*. 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 ceil 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 particulai-, a suitable Pichia strain, such as a suitable strain of Pichia pastoris), 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) 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.
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 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 VHH 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 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 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 1 1 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 bind 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 1 1 to 13. 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 11 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 11 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 1 1-13 (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),
GGGAPVG (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: 1 14 to 1 16. Also, SEQ ID NO's: 1 17 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 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 1 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 times 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 the therapeutic and 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). 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 NQ: 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: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), 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 compri ses 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: 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).
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. 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, 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 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: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). 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% 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: 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).
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: 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), 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: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). 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: 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).
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). 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 di sorders 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 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), preferabl y 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, 1.0, 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. 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 A3b-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 , 201 1. 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 muitispecific 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 muitispecific 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 muitispecific construct of the invention suitable 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 m uitispecific 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 muitispecific 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 mui tispecific 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 muitispecific 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 perfomiing the methods described in WO 2003/055527 and WO 2010/G81856 using a compound, protein, polypeptide, fusion protein or multivalent or muitispecific construct of the invention, or by otherwise administering a compound, protein, polypeptide, fusion protein or multivalent or muitispecific construct of the invention to and/or via the lungs using any suitable methods, techniques, formulations and equipment for pulmonary delivery known per se). 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 multi specific 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 anobody-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. 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 1 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. In particular, the amino acid sequences, compounds, proteins, polypeptides, fusion proteins, or multivalent or multi specific 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 II shows the BIAcore results obtained in Example 2B, and Figure III shows an aligment 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 II below. Binding to human serum albumin was determined 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-iimiting amino acid sequences of the invention
Clone ID SEQ ID: Sequence KD
(nM) on HSA
Biacore
EXPGMP 54 LWYMLRDWDFDVFGGGTP n.d.
80C05
EXPGMP 55 LWYLYRDWDFDVFGGGTP n.d.
80D11
EXPGMP 56 YWWERRDWDFDVFGGGTP 16 80B10
EXPGMP 57 AWYDYRDWDFDVFGGGTP 59-222 80E03
EXPGMP 58 W WN WPvD WDFD VF GGGTP n.d.
80F06
EXPGMP 59 EWWWRRDWDFDVFGGGTP 15 85E01
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 Table II: continued
EXPGMP 67 QKLYWPvDWDFDVFGGGTP 96 85H06
EXPGMP 68 KWWEIRDWDFDVFGGGTPAKSDE 20 86A10
EXPGMP 69 WWSTPRDWDFDVFGGGTPRGEMH 22 86C01
EXPGMP 70 LFWWERDWDFDVFGGGTP 20 88B01
EXPGMP 71 WWLQERDWDFDVFGGGTP 22 88E10
EXPGMP 72 W WEQDRD WDFD VF GGGTP 15 89D03
EXPGMP 73 NQLIVRDWDFDVFGGGTP n.d.
89D11
EXPGMP 74 WWELDRDWDFDVFGGGTP 13 89F02
EXPGMP 75 AAARDWDFDVFGGGTPVGG 198-
59C2 648 (reference)
89D03V1 103 WWEQDRDWDFDVFGGGAP n.d.
89D03V2 104 W EQDRDWDFDVFGGGNP n.d.
89D03V3 105 WWEQDRDWDFTVFGGGDP n.d.
89D03V1- n.d.
VG 106 WWEQDRDWDFDVFGGGAPVG
S9D03V2- n.d.
VG 107 WWEQDRDWDFDVFGGGNPVG
89D03V3- n.d.
VG 108 WWEQDRDWDFTVFGGGDPVG Example 2: Construction of a Nanobody-Expedite fusion proteins and analysis of binding to human serum albumin:
Example 2A: Construction of 2D 3 -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 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:49].
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 GIy4Ser-G3y3Ser 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 TGI cells. The fusion proteins were purified by IMAC/SEC and binding to HSA was assessed in BIAcore™ 3000. by injecting 1 μΜ and 5 μΜ of the fusion proteins on a CMS 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 ΙΟμΙ min-1. 20μ1 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 80B10 (SEQ ID NO: 56) and the Nanobody 2D3 (SEQ ID NO: 47), via a Gly4Ser-Gly3Ser ("9GS") linker sequence (SEQ ID NO: 53). The sequence of the resulting Nanobody construct (referred to as 2D3-9GS-EXP80B10 or EXP 424) was:
EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPG GLEWVSSIN WSGTHTD Y AD S VKGRFTIS RNN ANNTL YLQMNSL SEDT A V Y YC AKN WRD AGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSYWWERRDWDFDVFGGGTP
[SEQ ID NO: 99].
Fusions of other amino acid sequences of the invention (such as those mentioned in Table II) 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 IL 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 5F7-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 5F7:
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISS IG DT Y Y AD S VKGRFT1 SRDN A NT V YLQMN SLKPEDT A V Y Y C KRF RTAAQ GT 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. 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-EXP89D03 or EXP413) was:
EVQLVESGGGLVQAGGSL LSCAASGITFSINTMGWY QAPGKQRELVALISS IGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGT DYWGQGTQVTVSSGGGGSGGGSWWEQDRDWDFDVFGGGTP [SEQ ID NO:101] 5F7 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:
EVQLVESGGGLV Q AGG SLRL SC A A S GITF SINTMG WYRQ APGKQREL V ALI S S IGDT Y Y AD S VKGRFTI S RDN AKNT V YLQMN S LKPEDT A V Y YCKRFRT A AQ GT DYWGQGTQVTVSSGGGGSGGGSWWEQDRDWDFDVFGGGTPGGGGSGGGS WWEQDRD WDFD VF GGGTP [SEQ ID NO: 102] The binding of the monovalent and tandem repeat constructs of SEQ ID NO: 101 and.
102 was compared in BlAcore™ 3000, by injecting 200nM and Ι μΜ 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 Iinlced 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). In the constructs of SEQ ID NO's: 1 10 to 1 16, 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 VHH' 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 9GS 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 09/127691, 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 (Moisoft) and Discovery Studio (Accelrys) with a force-field that is based on the parameters as described, in Momany et al. (Momany et al. J. P ys. 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 III, 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 II above. 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.
Figure imgf000101_0001
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 III. 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 III).
This subpocket on human serum albumin also appears to comprise some partially positive charged residues (for example, 490 and H488). Thus, it is assumed that preferably, the 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 D131 and N133. 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,
Example 4: Pharmacokinetic profile in male cynomolgus 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- EXP89D03 (EXP413) and 5F7-9GS-EXP89D03-9GS-EXP89D03 (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, 1 5, 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 Ih 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-weIl microtiter 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 μg mL for the negative control and 4.5 pg/mL for the test item. Wells are aspirated and blocked for 30 minutes at room temperature (RT) with SuperBlock®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 diluted 1/5 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 Kl, purified against protein A and Her2/Fc depleted, is added at 1 g/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 30 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 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 (t½) 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 λζ.
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 (AUQnf), total body clearance (CL), the apparent volume of distribution at steady-state (Vdss), and the tenninal half-life (t , 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 g/ml . Similar values were calculated for EXP413 (59,2
Figure imgf000104_0001
and EXP486 (53,9 μ^πύ).
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. The estimated mean half-life (tj/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.
Figure imgf000105_0001
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.

Claims

C L A I M S
1. Amino acid sequence that contains:
a) 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
b) 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 c) the sequence motif GGG;
and preferably at least any two and more preferably all three of (i), (ii) and (iii);
and that 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, T491 and/or V493
2. Amino acid sequence according to claim 1 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 preferably the sequence motif DVFGGG (SEQ ID NO; 15) or TVFGGG (SEQ ID NO: 131).
3. Amino acid sequence according to any of the preceding claims, wherein said amino acid sequence is such that, when it is linked or fused to a therapeutic moiety, compound, protein or other therapeutic entity, the compound thus obtained has a longer half- life 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 has a half life that is the same or longer than a corresponding compound or construct in which said therapeutic moiety, compound, protein or other therapeutic entity is linked or fused to one of the amino acid sequences of SEQ ID NO:75, 76 and/or 77.
4. Amino acid sequence according to any of the preceding claims, that is cross- reactive with serum albumin from cynomolgus monkeys (Macaca fascicularis).
5. Compound or construct which comprises at least one amino acid sequence of according to any of the preceding claims and at least one therapeutic moiety.
6. Compound or construct which comprises at least two amino acid sequence of according to any of the preceding claims and at least one therapeutic moiety.
7. Compound or construct which comprises at least one tandem repeat- comprising at least two amino acid sequence of according to any of the preceding claims and at least one therapeutic moiety.
8. Compound or construct which comprises at least one amino acid sequence of according to any of the preceding claims and at least one therapeutic moiety, 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 the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO: l); and preferably has an equal or 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's: 75, 76 or 77.
9. Compound or construct according to claim 7 or 8, which is a fusion protein or polypeptide.
10. Compound or construct according to any of claims 7 to 9, in which the at least one therapeutic moiety preferably comprises or essentially consists of an immunoglobulin sequence or an antigen-binding fragment thereof, such as an immunoglobulin variable domain or an antigen-binding fragment thereof.
1 1 . Compound or construct according to claim 13, in which the at least one therapeutic moiety preferably comprises or essentially consists of a (single) domain antibody, a "dAb", or a ANOBODY®.
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