Improved serum albumin binders

Abstract

To provide amino acid sequences that can bind to serum albumin.SOLUTION: The present invention relates to immunoglobulin single variable domains, in particular heavy-chain immunoglobulin single variable domains, that can bind to serum albumin. The immunoglobulin single variable domains provided by the invention are preferably such that they are capable of binding to human serum albumin. More preferably, these immunoglobulin single variable domains are preferably such that they are further cross-reactive between human serum albumin and serum albumin from at least one other mammalian species. The invention also relates to proteins, polypeptides and other constructs, compounds, molecules or chemical entities that comprise at least one of the immunoglobulin single variable domains binding to serum albumin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to amino acid sequences capable of binding to serum albumin.

[0002] In particular, the invention relates to immunoglobulin single variable domains, in particular heavy chain immunoglobulin single variable domains, capable of binding to serum albumin.

[0003] As described herein, the immunoglobulin single variable domains provided by the invention are preferably such that they are capable of (at least) binding (in particular specifically binding) to human serum albumin. More preferably, as further described herein, these immunoglobulin single variable domains are preferably such that they are further cross-reactive (as described herein) between human serum albumin and serum albumin from at least one other mammalian species.

[0004] The present invention also relates to proteins, polypeptides and other constructs, compounds, molecules or chemical entities that comprise at least one of the immunoglobulin single variable domains that bind serum albumin as described herein.

[0005] Immunoglobulin single variable domains are also commonly referred to herein using the abbreviations "ISV" or "ISVD" (used interchangeably herein).

[0006] The immunoglobulin single variable domains that bind serum albumin described herein are also referred to herein as "amino acid sequences of the invention" or "serum albumin binders of the invention". As described further herein, albumin binders of the invention may in particular be nanobodies (as described further herein).

[0007] Proteins, polypeptides and other constructs, compounds, molecules or chemical entities that comprise at least one of the serum albumin binders of the present invention are also referred to herein as "compounds of the present invention" or "polypeptides of the present invention".Preferably, the compounds of the present invention are proteins or polypeptides, and may in particular be fusion proteins.

[0008] Other aspects, embodiments, features, uses and advantages of the present invention will be apparent to those skilled in the art based on the disclosure herein.

[0009] In this application, amino acid residues / positions in immunoglobulin heavy chain variable domains are designated by numbering according to Kabat. For convenience, Figure 1 provides a table listing some amino acid positions specifically mentioned herein and their numbering according to some alternative numbering systems (such as Aho and IMGT. Note: For the purposes of this description and claims, unless expressly indicated otherwise, the Kabat numbering is governing; other numbering systems are provided for reference only).

[0010] Concerning CDRs, as is well known in the art, there are several rules for defining and describing the CDRs of VH or VHH fragments, such as the Kabat definition (based on sequence variability and most commonly used) and the Chothia definition (based on the location of structural loop regions). See, for example, the website (http: / / www.bioinf.org.uk / abs / ). For the purposes of this specification and claims, CDRs according to Kabat may be taken, but it is most preferred that CDRs are defined based on the Abm definition (based on Oxford Molecular's AbM antibody modeling software), since it is considered to be the best compromise between the Kabat definition and the Chothia definition. Again, see the website (http: / / www.bioinf.org.uk / abs / ).

[0011] Thus, in this specification and claims, unless expressly stated otherwise herein, all CDRs are defined according to the rules of Abm.

[0012] ISVDs (particularly nanobodies) capable of binding to serum albumin and their uses are known in the art, for example from WO 2004 / 041865, WO 2006 / 122787, WO 2012 / 175400, WO 2015 / 173325 and PCT / EP 2016 / 077973, which describe serum albumin-binding ISVDs and their uses (as defined in these applications) for extending the serum half-life of therapeutic compounds, moieties and entities. For example, WO 2006 / 122787 discloses a humanized serum albumin-binding nanobody called Alb-8 (see SEQ ID NO:1 herein) as SEQ ID NO:62. WO 2012 / 175400 discloses as SEQ ID NO:6 a humanized serum albumin binding nanobody called Alb-23D (see SEQ ID NO:2 herein). The amino acid sequences of Alb-8 and Alb-23D and their CDRs (which are the same for Alb-8 and Alb-23D) are given in Table A below as SEQ ID NOs:1, 2 and 3-8, respectively.

[0013] Several other references disclosing ISVDs for serum albumin include WO 2003 / 035694, WO 2004 / 003019, EP 2 139 918, WO 2011 / 006915 and WO 2014 / 111550.

[0014] 3A and 3B show the alignment of Alb-8 (reference), Alb-23D (reference), SEQ ID NO:18 (invention) and SEQ ID NO:19 (invention).

[0015] The present invention aims to provide an improved serum albumin binder, in particular one with improved properties compared to serum albumin binders known in the art.In particular, the present invention aims to provide a serum albumin binder that can bind to canine serum albumin and / or has improved cross-reactivity between human serum albumin and canine serum albumin (i.e., compared to prior art serum albumin binders such as Alb-8 and / or Alb-23D).

[0016] [Table 1]

[0017] [Table 2]

[0018] In general, the serum albumin binding ISVDs provided by the present invention are variants of the sequences of SEQ ID NOs: 18 and 19 in that: - they have the same CDRs (or essentially the same CDRs) as the sequences of SEQ ID NO: 18 and / or 19 (provided that, if the CDRs are defined according to Kabat, CDR2 can be AISQNSIHTYYANSVKG (SEQ ID NO: 10) or AISQNSIHTYYADSVKG (SEQ ID NO: 11)); and - they have a certain degree of sequence identity with the sequences of SEQ ID NO: 18 and / or 19 (the degree of sequence identity is as further described herein).

[0019] In particular, the serum albumin binding ISVDs provided by the present invention generally have a (limited) number of "amino acid differences" (as described herein) compared to the sequences of SEQ ID NO: 18 and / or 19. These amino acid differences may be present in the CDRs (as long as the resulting amino acid sequence is such that it retains the additional properties of the amino acid sequences of the present invention as set out herein) and / or in the framework regions, in particular in the framework regions (as defined according to Kabat and / or according to Abm). By way of example and without limitation, these amino acid differences may be, for example, humanization substitutions, substitutions that improve expression in the desired host cell or host organism, substitutions that improve stability and / or resistance to degradation and / or proteases, mutations that reduce binding by existing antibodies, and / or other mutations intended to optimize the sequence of the amino acid sequences of the present invention; or any suitable combination of such amino acid differences. See further disclosure herein.

[0020] In a first aspect, the present invention relates to an ISVD capable of binding, in particular specifically binding, to human serum albumin and comprising: - a CDR1 (according to Kabat) which is the amino acid sequence TYVMG (SEQ ID NO:9) or an amino acid sequence which has two or one amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO:9; and - a CDR2 (according to Kabat) which is the amino acid sequence AISQNSIHTYYANSVKG (SEQ ID NO:10) or AISQNSIHTYYADSVKG (SEQ ID NO:11) or an amino acid sequence which has 3, 2 or 1 amino acid difference (as defined herein) with the amino acid sequence of SEQ ID NO:10 and / or SEQ ID NO:11; and - a CDR3 (according to Kabat), which is the amino acid sequence SRFTSWYTADYEYDY (SEQ ID NO:14) or an amino acid sequence which has 3, 2 or 1 amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO:14.

[0021] In particular, the serum albumin binder according to this aspect of the invention may (and preferably is) as further described herein.

[0022] In a more specific embodiment, the present invention relates to an ISVD capable of binding (in particular specifically binding) to human serum albumin and having: - CDR1 (according to Kabat), which has the amino acid sequence TYVMG (SEQ ID NO:9); and - a CDR2 (according to Kabat) with the amino acid sequence AISQNSIHTYYANSVKG (SEQ ID NO: 10) or AISQNSIHTYYADSVKG (SEQ ID NO: 11); and - CDR3 (according to Kabat), which has the amino acid sequence SRFTSWYTADYEYDY (SEQ ID NO:14).

[0023] Again, the serum albumin binder according to this aspect of the invention may (and preferably is) as further described herein.

[0024] In another embodiment, the present invention relates to an ISVD capable of binding (in particular specifically binding) to human serum albumin and having: - a CDR1 (according to Abm) that is the amino acid sequence GGTFSTYVMG (SEQ ID NO:12) or an amino acid sequence that has two or one amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO:12; and - a CDR2 (according to Abm) that is the amino acid sequence AISQNSIHTY (SEQ ID NO:13) or an amino acid sequence that has 3, 2 or 1 amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO:13; and - a CDR3 (according to Abm) which is the amino acid sequence SRFTSWYTADYEYDY (SEQ ID NO:14) or an amino acid sequence which has 3, 2 or 1 amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO:14.

[0025] In particular, the serum albumin binder according to this aspect of the invention may (and preferably is) as further described herein.

[0026] In a more specific embodiment, the present invention relates to an ISVD capable of binding (in particular specifically binding) to human serum albumin and having: - CDR1 (according to Abm), which has the amino acid sequence GGTFSTYVMG (SEQ ID NO: 12); and - CDR2 (according to Abm) having the amino acid sequence AISQNSIHTY (SEQ ID NO: 13); and - CDR3 (according to Abm), which has the amino acid sequence SRFTSWYTADYEYDY (SEQ ID NO:14).

[0027] Again, the serum albumin binder according to this aspect of the invention may (and preferably is) as further described herein.

[0028] In general, the serum albumin binders according to the different aspects of the invention preferably comprise: - having a degree of sequence identity with the sequences of SEQ ID NO: 18 and / or 19 of at least 85%, preferably at least 90%, more preferably at least 95% (the possible CDRs and any C-terminal extensions are not taken into account when determining the degree of sequence identity); And / or: - a serum albumin binder having no more than seven, preferably no more than five, e.g. only three, two or one "amino acid difference(s)" (as defined herein, not taking into account possible CDRs and any C-terminal extension) from the sequence of SEQ ID NO:18 and / or 19.

[0029] Serum albumin binders according to different aspects of the invention generally preferably have a binding affinity of 10 to human serum albumin as determined using ProteOn (see Example 1). -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, and more preferably 10 -8 ~10 -12 Dissociation constant (KD) in moles / liter and / or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , e.g. at least 10 12 M -1 Preferably, the serum albumin binders of the present invention bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 500 pM, again as determined using ProteOn (again, see Example 1).

[0030] The serum albumin binders according to the different aspects of the invention are preferably also serum albumin binders which compete with the amino acid sequence of SEQ ID NO:18 for binding to (human) serum albumin and / or which "cross-block" (as defined herein) the binding of the amino acid sequence of SEQ ID NO:18 to (human) serum albumin.

[0031] Preferably, the albumin binders of the present invention are such that they bind to essentially the same amino acid residues and / or epitopes on human serum albumin as SEQ ID NO: 18, and even more preferably, that they share essentially the same amino acid interactions as SEQ ID NO: 18. To this end, according to specific, but non-limiting embodiments, the albumin binders of the present invention preferably either have the same CDRs as the sequence of SEQ ID NO: 18, or, compared to the sequence of SEQ ID NO: 18, preferably contain only mutations within their CDRs (such as conservative amino acid substitutions) such that they are still capable of undergoing the same or essentially the same amino acid interactions with human serum albumin as SEQ ID NO: 18.

[0032] The serum albumin binder according to the different aspects of the present invention is generally also preferably a serum albumin binder that is cross-reactive between human serum albumin and serum albumin from at least one, preferably from at least two, more preferably from at least three, and up to essentially all of the following mammalian species: dog, rat, mouse, rabbit, guinea pig, pig, sheep, cow and cynomolgus monkey.In particular, the serum albumin binder according to the different aspects of the present invention is (at least) a serum albumin binder that is cross-reactive between human serum albumin and dog serum albumin, and preferably also between human serum albumin and / or dog serum albumin on the one hand and at least one, preferably at least two, more preferably all three of the serum albumin from rat serum albumin, mouse serum albumin and cynomolgus monkey on the other hand. In this regard, the serum albumin binder of the present invention may have improved cross-reactivity (especially between human serum albumin on the one hand and dog serum albumin on the other hand) compared with serum albumin binders having (essentially) the same CDR as Alb-11 and / or Alb-23D.See the data in the experimental section below.

[0033] For reference, Figure 11 gives an alignment of serum albumins from different mammalian species (source: http: / / macromoleculeinsights.com / albumin.php, the amino acid numbering in Figure 11 is the numbering used in said webpage). For convenience, in the sequence of human serum albumin, stretches of amino acids are highlighted that are predicted to be part of the putative epitope of the amino acid sequence of the invention. Without being limited to any particular mechanism or hypothesis, it is predicted that the amino acid sequence of the invention is (essentially) capable of binding to a corresponding stretch of amino acid residues (one or more amino acid residues therein) present in the amino acid sequence of serum albumin of a mammal with which the amino acid sequence of the invention is cross-reactive.

[0034] In general, a serum albumin binder of the invention can be considered to be cross-reactive between human serum albumin and serum albumin from one of these species when it is capable of binding to human serum albumin with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM; and to serum albumin from said species with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, both as determined using ProteOn (again, see Example 1).

[0035] The serum albumin binder according to the different aspects of the present invention preferably also comprises - greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours; e.g., having a serum half-life in humans (expressed as t1 / 2 beta) of about 1 week, 2 weeks, up to the half-life of serum albumin in humans (estimated to be around 19 days); And / or: - a serum albumin binder which, when linked to a therapeutic moiety or entity, confers on the resulting polypeptide of the invention a serum half-life in humans (expressed as t1 / 2 beta) of greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours; for example, about 1 week, 2 weeks, up to the half-life of serum albumin in humans (estimated to be around 19 days).

[0036] The half-life in a mammalian species other than human depends, among other factors, mainly on the binding properties (such as affinity) of the albumin binder of the present invention to serum albumin from said mammalian species, as well as on the half-life of native serum albumin in said species. According to a preferred embodiment of the present invention, when a serum albumin binder of the present invention is cross-reactive (as defined herein) between human serum albumin and serum albumin from another mammalian species, the half-life of the serum albumin binder of the present invention (and / or the compound of the present invention comprising said serum albumin binder) when determined in said species is preferably at least 5%, such as at least 10%, more preferably at least 25%, such as about 50%, and possibly up to 100% of the half-life of native serum albumin in said species.

[0037] Compared to the sequence of SEQ ID NO:18, the serum albumin binder of the invention preferably also has (at least): - one or more humanizing substitutions; and / or - one or more mutations (i.e. amino acid substitutions, deletions or additions, particularly substitutions) that reduce binding by existing antibodies; and optionally contain one or more further mutations as described herein.

[0038] For suitable humanizing substitutions (and suitable combinations thereof), see, for example, WO 09 / 138519 (or prior art cited in WO 09 / 138519) and WO 08 / 020079 (or prior art cited in WO 08 / 020079), as well as Tables A-3 to A-8 from WO 08 / 020079 (which are lists showing possible humanizing substitutions). Some preferred but non-limiting examples of such humanizing substitutions are Q108L and A14P or suitable combinations thereof. Such humanizing substitutions may also be suitably combined with one or more other mutations as described herein (e.g., with one or more mutations that reduce binding by existing antibodies).

[0039] For suitable mutations (and suitable combinations of such mutations) that can reduce binding by existing antibodies, see, for example, WO 2012 / 175741 and WO 2015 / 173325, and also, for example, WO 2013 / 024059 and WO 2016 / 118733. As described herein, such mutations can include one or more amino acid substitutions, deletions or additions (particularly substitutions) (suitable combinations thereof), which mutations are often in the so-called C-terminal region of the ISV. For example, such mutations may include mutations (particularly substitutions) at one or more of positions 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, 108 and / or mutations at one or more positions in the C-terminal VTVSS sequence (i.e. positions 109, 110, 111, 112 and 113), with one or more mutations at positions 11, 89, 110 and / or 112 being particularly preferred. Some preferred, but non-limiting examples of such mutations are suitable substitutions (where necessary) such that after mutation, at the indicated positions, there is one of the following amino acid residues: 11L, 11K, 11V, 14A, 14P, 41A, 41L, 41P, 41S, 41T, 42E, 42G, 87A, 87T, 89A, 89L, 89T, 108L, 110K, 110Q, 112K and / or 112Q (wherein 11L, 89A, 89L, 89T, 110K, 110Q, 112K and 112Q are particularly preferred); or any suitable combination of such substitutions, such as, by way of example and not limitation: a combination of 11V with 89L or 89T; a combination of 11V with 110K or 110Q; or a combination of 11V with 89L and 110K or 110Q. Such mutations that reduce binding by a pre-existing antibody may also be suitably combined with one or more other mutations as described herein (eg, with one or more humanizing substitutions).

[0040] If desired (as further described herein, in particular when the serum albumin binder of the invention is present at and / or forms the C-terminus of the compound of the invention in which it is present), the serum albumin binder of the invention (and, as further described herein, also the compound of the invention) may also comprise a C-terminal extension (such as a C-terminal alanine residue) to reduce binding of pre-existing antibodies. As described in WO 2012 / 175741, such a C-terminal extension reduces binding by pre-existing antibodies. Suitable C-terminal extensions may generally be further described herein, and in particular may be a C-terminal extension of formula (X) n where X can be any naturally occurring amino acid (but preferably is not cysteine) and n can be 1, 2, 3, 4 or 5. Again, see WO 2012 / 175741, and also, for example, WO 2015 / 173325, WO 2013 / 024059 and WO 2016 / 118733. The presence of such a C-terminal extension may also be suitably combined with one or more of the other mutations described herein (e.g., with one or more humanizing substitutions and / or one or more mutations that reduce binding by a pre-existing antibody).

[0041] Other mutations that may be present in the serum albumin binders of the invention include, by way of example and without limitation, one or more mutations (particularly substitutions) that improve expression in a desired host cell or host organism, one or more mutations (particularly substitutions) that improve stability and / or resistance to degradation and / or proteases, and / or one or more other mutations intended to optimize the sequence of the amino acid sequence of the invention (by way of example and without limitation, one or more mutations that (further) reduce any tendency of the albumin binder to form dimers); or any suitable combination of such mutations.

[0042] Some non-limiting examples of such mutations are suitable substitutions (if necessary) such that after mutation, at the indicated positions, there is one of the following amino acid residues: 16G, 49A, 61D, 74S, 75K, 76N, 82M and 83R; or any suitable combination of such substitutions (e.g., to form an SKN motif at positions 74-76). Also, if necessary (as further described herein), the serum albumin binders of the invention may have a D at position 1 (i.e., an E1D mutation compared to the sequences of SEQ ID NOs:18 and / or 19), particularly when the serum albumin binder of the invention is present at and / or forms the N-terminus of the compound of the invention in which it is present. Such mutations may again be suitably combined with one or more other mutations as described herein (e.g., with one or more humanizing substitutions and / or one or more mutations that reduce binding by existing antibodies).

[0043] Other mutations that can be present in the amino acid sequences of the present invention will be apparent to those of skill in the art based on the disclosure herein.

[0044] It is also possible that a single mutation (or a suitable combination of mutations) may provide multiple functions or advantages. By way of example and not limitation, the humanized Q108L substitution may also reduce binding by existing antibodies.

[0045] Some preferred, but non-limiting examples of amino acid residues (i.e. mutations compared to the amino acid sequence of SEQ ID NO:18) that may be present in the amino acid sequences of the invention (i.e. by themselves or in suitable combinations) include: 11V (i.e. L11V), 14P (i.e. A14P), 16G (i.e. D16G), 49A (i.e. S49A), 61D (i.e. N61D), 74S (i.e. D74S), 82M (i.e. L82M), 83R (i.e. K83R), 89L (i.e. V89L), 89T (i.e. V89T), 110K (e.g. T110K) or 110Q (e.g. T110Q); and, optionally (as further described herein), 1D (e.g. E1D) and / or a C-terminal extension (X) as defined herein. n (e.g., 114A). See also the sequences and mutations shown in Figures 4A and 4B. For example, some preferred, but non-limiting examples of suitable combinations of such amino acid residues (i.e., mutations compared to the amino acid sequence of SEQ ID NO:18) include the following: -L11V, T14P, D16G, N61D, D74S, K83R, V89L -L11V, T14P, D16G, D74S, L82M, K83R, V89L -L11V, T14P, D16G, N61D, D74S, L82M, K83R, V89L -L11V, T14P, D16G, S49A, D74S, K83R, V89L -L11V, T14P, D16G, S49A, N61D, D74S, K83R, V89L -L11V, T14P, D16G, S49A, D74S, L82M, K83R, V89L -L11V, T14P, D16G, S49A, N61D, D74S, L82M, K83R, V89L -L11V, T14P, D74S, K83R, V89L and other suitable combinations will be apparent to one of skill in the art based on the disclosure herein.

[0046] Some preferred, but non-limiting, amino acid sequences of the present invention are: (i) V at position 11 and L at position 89; (ii) T at position 89; (iii) positions 74-76 forming an SKN motif; (iv) V at position 11, L at position 89 and T at positions 74-76 forming an SKN motif; and (iv) T at position 89 and T at positions 74-76 forming an SKN motif; which amino acid sequences of the present invention are as further described herein (and therefore may suitably contain one or more further amino acid mutations as described herein).

[0047] Some preferred, but non-limiting examples of amino acid sequences of the present invention are given in FIG. 2 as follows: - SEQ ID NOs: 18 to 26, which are examples of amino acid sequences of the invention without a C-terminal alanine extension; - SEQ ID NOs: 27 to 35, which are examples of amino acid sequences of the invention with C-terminal extensions (in each case exemplified by a C-terminal alanine extension, which is a generally preferred C-terminal extension); and - SEQ ID NOs:36-44, which are examples of amino acid sequences of the invention having N-terminal E1D mutations.

[0048] Based on the further disclosure herein, it will be apparent to one of ordinary skill in the art that: - albumin binders of the invention having a C-terminal extension (e.g. those of SEQ ID NOs: 27-35) are often used as the polypeptides of the invention (as defined herein) in which they are present / at their C-terminus; -Albumin binders of the invention having E1D mutations (e.g., those of SEQ ID NOs: 36-44) are often used as the polypeptides of the invention in which they are present / at their N-terminus; Albumin binders of the invention that do not have a -C-terminal extension or an E1D mutation (eg those of SEQ ID NOs:18-26) will often be located somewhere in the "middle" of a polypeptide of the invention.

[0049] Each of the amino acid sequences of SEQ ID NOs:18-44, and proteins, polypeptides and other compounds and constructs comprising same (as further described herein), form further aspects of the present invention.

[0050] In a further aspect, the present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and / or SEQ ID and each of these amino acid sequences of the invention (as well as the polypeptides of the invention as defined herein which comprise such amino acid sequences of the invention) form further aspects of the invention.

[0051] As further described herein, the amino acid sequences provided by the present invention are proteins that can bind, in particular specifically (as described herein) bind, to human serum albumin.Therefore, they can be used as binding units or binding domains for binding to (human) serum albumin, for example, to confer an increase in half-life (as defined herein) to therapeutic compounds, moieties or entities.For the use of serum albumin binding domains to increase the half-life of therapeutic compounds, moieties or entities, see, for example, WO 2004 / 041865, WO 2006 / 122787, EP 2 139 918, WO 2011 / 006915, WO 2012 / 175400 and / or WO 2014 / 111550.The serum albumin binders of the present invention can generally be used in the same manner and for the same purposes as the serum albumin binders described in these references.

[0052] In some further non-limiting embodiments, the present invention also relates to: - proteins, polypeptides and other constructs, molecules or chemical entities that comprise or consist essentially of at least one serum albumin binder of the invention as described herein (also referred to herein as "compounds of the invention" or "polypeptides of the invention"); - a method for expressing / producing the serum albumin binders of the invention and / or the compounds of the invention; - a host cell, host organism or other (expression) system capable of expressing or producing the serum albumin binders of the invention and / or the compounds of the invention; - compositions and products (such as pharmaceutical compositions and products) comprising the serum albumin binders of the invention and / or the compounds of the invention; - nucleotide sequences and nucleic acids, e.g. (expression) vectors, encoding the serum albumin binders of the invention and / or the compounds of the invention; - Compounds of the invention and / or uses of the compounds of the invention, e.g. use of the compounds of the invention to increase the (serum) half-life of therapeutic compounds, moieties or entities, as well as therapeutic and / or prophylactic uses of the compounds of the invention.

[0053] These and further aspects, embodiments, advantages, applications and uses of the present invention will become apparent from the further description herein.

[0054] In this specification: - The term "immunoglobulin single variable domain" (also referred to as "ISV" or "ISVD") is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that is capable of forming a functional antigen-binding site without interaction with another variable domain (e.g. without VH / VL interactions as required between the VH and VL domains of a conventional 4-chain monoclonal antibody). Examples of ISVDs will be clear to the skilled person and include, for example, nanobodies (including VHH, humanized VHH and / or camelized VH, e.g. camelized human VH), IgNAR domains, VH domain or derived (single domain) antibodies (such as dAbs™), and VL domain or derived (single domain) antibodies (such as dAbs™). Unless expressly stated otherwise herein, ISVDs based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. Most preferably, unless expressly indicated otherwise herein, the ISVD is a nanobody. - the term "nanobody" is generally as defined in WO 2008 / 020079 or WO 2009 / 138519, and thus in specific embodiments generally refers to a VHH, a humanized VHH or a camelized VH (such as a camelized human VH), or generally a sequence-optimized VHH (e.g. optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximum expression). Note that the term nanobody (Nanobody or Nanobodies) is a registered trademark of Ablynx NV, and therefore may also be referred to as Nanobody® and / or Nanobodies®; - In general, unless otherwise indicated herein, the ISVDs, nanobodies, polypeptides, proteins and other compounds and constructs mentioned herein are intended for use in the prevention or treatment of diseases or disorders in humans (and / or, optionally, also in warm-blooded animals, in particular mammals). Thus, in general, the ISVDs, nanobodies, polypeptides, proteins and other compounds and constructs described herein are preferably such that they can be used as and / or can suitably be part of (biological) drugs or other pharmacologic or therapeutically active compounds and / or pharmaceutical products or compositions. Such drugs, compounds or products are preferably such that they are suitable for administration to humans, for example for the prevention or treatment of subjects in need of such prevention or treatment or as part of, for example, clinical trials. As further described herein, for this purpose such drugs or compounds may contain, in addition to the ISVD provided by the invention, other moieties, entities or binding units (which may also be, as described herein, for example, one or more other further therapeutic moieties and / or one or more other moieties that affect the pharmacokinetic or pharmacodynamic properties of the ISVD-based or nanobody-based biologic, such as its half-life). Suitable examples of such further therapeutic or other moieties will be clear to the skilled artisan and may, for example, generally include any therapeutically active protein, polypeptide or other binding domain or binding unit, as well as modifications such as, for example, those described on pages 149-152 of WO 2009 / 138159. The ISVD-based or nanobody-based biologic is preferably a therapeutic or is intended to be used as a therapeutic (including prophylactic and diagnostic) and for this purpose preferably contains at least one ISVD against a therapeutically relevant target (such as, for example, RANK-L, vWF, IgE, RSV, CXCR4, IL-23 or other interleukins).For some specific, but non-limiting examples of such ISVD-based or nanobody-based biological agents, see Examples 8-18, and also various applications, for example, by Ablynx NV (e.g., by way of example and without limitation, WO 2004 / 062551, WO 2006 / 122825, WO 2008 / 020079 and WO 2009 / 068627), as well as applications such as (and without limitation) WO 2006 / 038027, WO 2006 / 059108, WO 2007 / 063308, WO 2007 / 063311, WO 2007 / 066016 and WO 2007 / 085814. Also, as further described herein, the further moiety may be an ISVD or nanobody as described herein directed against a (human) serum protein, such as (human) serum albumin, which may also be useful for therapeutic use, in particular in and / or for extending the half-life of the TNF-binding agents described herein. See, for example, WO 2004 / 041865, WO 2006 / 122787 and WO 2012 / 175400, which generally describe the use of serum-albumin binding nanobodies for half-life extension. Also, in this specification, unless expressly stated otherwise in this specification, all terms referred to in this specification have the meaning given in WO 2009 / 138519 (or the prior art cited in WO 2009 / 138519) or WO 2008 / 020079 (or the prior art cited in WO 2008 / 020079). Also, if a method or technique is not specifically described in this specification, it can be performed as described in WO 2009 / 138519 (or the prior art cited in WO 2009 / 138519) or WO 2008 / 020079 (or the prior art cited in WO 2008 / 020079).Also, as described herein, any pharmaceutical product or composition comprising any ISVD or compound of the invention may also comprise one or more further ingredients known per se for use in pharmaceutical products or compositions (i.e. depending on the intended pharmaceutical form), and / or one or more other compounds or active ingredients intended, for example, for therapeutic use (i.e. to provide a combination product).

[0055] Also, when used in the specification or claims, the following terms have the same meaning as given on pages 62 to 75 of WO 2009 / 138519 and / or can be determined in the manner described therein, where applicable: "agonist", "antagonist", "inverse agonist", "non-polar uncharged amino acid residues", "polar uncharged amino acid residues", "polar charged amino acid residues", "sequence identity", "exactly the same" and "amino acid difference" (when referring to a sequence comparison of two amino acid sequences), "essentially isolated", "domain", "binding domain", "antigenic determinant", "epitope", "against" or "directed against", "specificity" and "half-life". In addition, the terms "modulating" and "to modulate," "interaction site," "specific for," "cross-blocking," "cross-blocked," and "cross-blocking," and "essentially independent of pH" are as defined (and / or can be determined as described) in WO 2010 / 130832 to Ablynx NV, pages 74-79. Also, when referring to constructs, compounds, proteins, or polypeptides of the invention, terms such as "monovalent," "bivalent" (or "multivalent"), "bispecific" (or "multispecific"), and "biparatopic" (or "multiparatopic") may have the meanings given in WO 2009 / 138519, WO 2010 / 130832, or WO 2008 / 020079.

[0056] The term "half-life", as used herein with respect to an ISVD, a Nanobody, an ISVD-based biological preparation, a Nanobody-based biological preparation or any other amino acid sequence, compound or polypeptide referred to herein, may generally be defined as described in paragraph o) on page 57 of WO 2008 / 020079 and as referred to herein, and refers to the time at which the serum concentration of an amino acid sequence, compound or polypeptide is reduced by 50% in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration by natural mechanisms of the sequence or compound. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention may be determined in any manner known per se, for example by pharmacokinetic analysis. Suitable techniques will be clear to the skilled person and may, for example, generally be as described in paragraph o) on page 57 of WO 2008 / 020079. Also as mentioned in paragraph o) on page 57 of WO 2008 / 020079, half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta and area under the curve (AUC). In this regard, it should be noted that the term "half-life" as used herein refers in particular to t1 / 2-beta or terminal half-life (t1 / 2-alpha and / or AUC or both may not be taken into account). See, for example, the experimental section below and standard textbooks such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). See also "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).Similarly, the term "increased half-life" or "increased half-life" is also as defined in paragraph o) on page 57 of WO 2008 / 020079 and specifically refers to an increase in t1 / 2-beta, regardless of an increase in t1 / 2-alpha and / or AUC or both.

[0057] When a term is not specifically defined herein, the term has its ordinary meaning in the art that would be apparent to one of ordinary skill in the art. For example, Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd.Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, New York, NY, (1985); Old et al. al., “Principles of Gene Manipulation: An Introduction to Genetic Engineering”, 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., “Immunology” (6th. Ed.), Mosby / Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al. al., “Immunobiology” (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), as well as the general background art cited therein.

[0058] Also, as already indicated herein, the amino acid residues of Nanobodies are numbered according to the conventional numbering for VH given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to the VHH domain from camel in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195; or as referred to herein. According to this numbering, FR1 of the Nanobody comprises amino acid residues at positions 1 to 30, CDR1 of the Nanobody comprises amino acid residues at positions 31 to 35, FR2 of the Nanobody comprises amino acids at positions 36 to 49, CDR2 of the Nanobody comprises amino acid residues at positions 50 to 65, FR3 of the Nanobody comprises amino acid residues at positions 66 to 94, CDR3 of the Nanobody comprises amino acid residues at positions 95 to 102, and FR4 of the Nanobody comprises amino acid residues at positions 103 to 113. [In this regard, it should be noted - as is well known in the art for VH domains and for VHH domains - that the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e. one or more positions according to the Kabat numbering may be unoccupied in the actual sequence or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.However, in general, according to Kabat numbering, and regardless of the number of amino acid residues in the CDR, it can be said that position 1 according to Kabat numbering corresponds to the start of FR1 (and vice versa), position 36 according to Kabat numbering corresponds to the start of FR2 (and vice versa), position 66 according to Kabat numbering corresponds to the start of FR3 (and vice versa), and position 103 according to Kabat numbering corresponds to the start of FR4 (and vice versa).

[0059] An alternative method for numbering the amino acid residues of VH domains, which is equally applicable to VHH domains from camelids and to nanobodies, is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, embodiments and figures, unless otherwise indicated, we will follow the numbering according to Kabat as applied to VHH domains by Riechmann and Muyldermans.

[0060] It should also be noted that unless otherwise expressly indicated herein, the figures, any sequence listing and the experimental sections / examples are provided only to further illustrate the present invention and should not be construed or interpreted in any way as limiting the scope of the present invention and / or the appended claims.

[0061] As further described herein, the serum albumin binders of the present invention can be advantageously used as moieties, binding units or fusion partners to increase the half-life of therapeutic compounds, moieties or entities, such as polypeptides, proteins, compounds (including but not limited to small molecules) or other therapeutic entities.

[0062] Thus, in another aspect, the present invention provides a polypeptide, protein, construct, compound or other chemical entity comprising or consisting essentially of a serum albumin binder of the invention and one or more other amino acid sequences, (binding) domains, binding units or other moieties or chemical entities.

[0063] In particular, the present invention provides a polypeptide, protein, construct, compound or other chemical entity comprising a serum albumin binder of the present invention and one or more (e.g. one or two) therapeutic moieties (which may be the same or different, e.g. directed against the same target or different targets, and when directed against the same target, directed against the same or different epitopes, parts, domains or subunits of said target), which are suitably linked to each other either directly or via one or more suitable linkers or spacers. Such a polypeptide, protein or construct may, by way of example and without limitation, be a fusion protein, as further described herein.

[0064] The present invention further relates to therapeutic uses of such polypeptides, proteins, constructs or compounds, and to pharmaceutical compositions comprising such polypeptides, proteins, constructs or compounds.

[0065] In one embodiment, at least one therapeutic moiety comprises or essentially consists of a therapeutic protein, polypeptide, compound, factor or other entity.In a preferred embodiment, the therapeutic moiety is directed against a desired antigen or target, can bind to a desired antigen (particularly, can specifically bind to a desired antigen), and / or can interact with a desired target.In another embodiment, at least one therapeutic moiety comprises or essentially consists of a therapeutic protein or polypeptide. In further embodiments, at least one therapeutic moiety comprises or essentially consists of a binding domain or binding unit, such as an immunoglobulin or immunoglobulin sequence (including but not limited to a fragment of an immunoglobulin), such as an antibody or antibody fragment (including but not limited to an ScFv fragment), or another suitable protein scaffold, such as a protein A domain (such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binzetal., Nat. Biotech 2005, Vol 23:1257), as well as DNA or RNA based binding moieties, including but not limited to DNA or RNA aptamers (Ulrichetal., Comb Chem High Throughput Screen 2006 9(8):619-32).

[0066] In yet another embodiment, at least one therapeutic moiety comprises or consists essentially of an antibody variable domain, such as a heavy chain variable domain or a light chain variable domain.

[0067] In a preferred embodiment, at least one therapeutic moiety comprises or essentially consists of at least one immunoglobulin single variable domain, such as a domain antibody, a single domain antibody, a "dAb" or a nanobody (such as a VHH, humanized VHH or camelized VH) or an IgNAR domain.

[0068] In specific embodiments, at least one therapeutic moiety comprises or consists essentially of at least one monovalent Nanobody, or a bivalent, multivalent, bispecific or multispecific Nanobody construct.

[0069] Polypeptides, (fusion) proteins, constructs or compounds comprising a serum albumin binder of the invention and one or more therapeutic moieties can generally be (prepared and used as) described in the above cited prior art (such as WO 04 / 041865, WO 06 / 122787, WO 2012 / 175400 and WO 2015 / 173325; see also, for example, WO 2004 / 003019, EP 2 139 918, WO 2011 / 006915 and WO 2014 / 111550), with the proviso that a serum albumin binder of the invention is used instead of the half-life increasing moieties described previously.

[0070] Polypeptides, (fusion) proteins, constructs or compounds comprising a serum albumin binder of the invention and one or more therapeutic moieties will generally and preferably have an increased half-life (as described herein, preferably expressed as t1 / 2-beta) compared to the therapeutic moiety or moieties themselves.

[0071] In general, a compound, polypeptide, construct or fusion protein described herein preferably has a half-life (again, as described herein and preferably expressed as t1 / 2-beta) 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 corresponding therapeutic moiety itself (when measured either in humans or in a suitable animal such as mouse or cynomolgus monkey).

[0072] Also, preferably, any such compound, polypeptide, fusion protein or construct has a half-life in humans (again, as described herein, preferably expressed as t1 / 2-beta) that is increased by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example more than 12 hours, compared to the half-life of the corresponding therapeutic moiety itself.

[0073] Also, preferably, the compound or polypeptide of the invention has a half-life in humans (again as described herein, preferably expressed as t1 / 2-beta) of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, for example about 1 day, 2 days, 1 week, 2 weeks, up to the half-life of serum albumin in humans (estimated to be around 19 days).

[0074] As mentioned, in one embodiment the serum albumin binding agents of the invention are used to increase the half-life of (one or more) immunoglobulin single variable domains, such as domain antibodies, single domain antibodies, "dAbs", VHHs or nanobodies (e.g. VHHs, humanised VHHs or camelised VHs, e.g. camelised human VHs).

[0075] Thus, one embodiment of the present invention relates to a polypeptide, construct or fusion protein comprising a serum albumin binder of the invention and one or more (e.g. one or two) immunoglobulin single variable domain sequences, which are suitably linked to each other directly or, optionally, via one or more suitable linkers or spacers. As mentioned herein, such immunoglobulin single variable domains present in such a polypeptide, construct or fusion protein may each independently be a domain antibody, a single domain antibody, a "dAb" or a nanobody (e.g. a VHH, a humanized VHH or a camelized VH, e.g. a camelized human VH); and according to one specific, but non-limiting embodiment, at least one (and up to all) of these immunoglobulin single variable domains comprises two or three disulfide bridges. Preferably, all ISVDs present in such a compound of the present invention are nanobodies.

[0076] When a compound of the invention has an ISVD at its C-terminus (e.g. an ISVD directed against a serum albumin binder of the invention or a therapeutic target), said C-terminal ISVD (and thus, by extension, the entire compound of the invention) preferably has a C-terminal extension at its C-terminus, which is directly linked to the last C-terminal amino acid residue of the ISVD (usually the amino acid residue at position 113 according to Kabat) (unless the ISVD contains one or more amino acid deletions such that the sequence of the ISVD ends before position 113). Thus, in general, the C-terminal extension is directly linked to the C-terminal VTVSS sequence (SEQ ID NO:15) of the C-terminal ISV (and thus, by extension, to the C-terminal TVTSS sequence of the compound of the invention) or to the C-terminal sequence of the C-terminal ISVD that corresponds to the C-terminal ISVD sequence (e.g., where said C-terminal sequence of the C-terminal ISVD contains one or more substitutions or deletions compared to the normal VTVSS sequence, such as T110K, T110Q, S112K or S112K).

[0077] It will also be clear to one skilled in the art that when a compound of the invention has a serum albumin binder of the invention at its C-terminus, said serum albumin binder of the invention will carry said C-terminal extension.

[0078] Generally, any C-terminal extension used herein (i.e., at the C-terminus of the compound of the invention and / or the serum albumin binder of the invention) can generally be as described in WO 2012 / 174741 or WO 2015 / 173325 (see also, for example, WO 2103 / 024059 and WO 2016 / 118733). In particular, the C-terminal extension can be represented by the formula (X): n wherein n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (preferably 1 or 2, such as 1); and each X is independently selected from naturally occurring amino acid residues (but according to a preferred embodiment it does not contain any cysteine ​​residues), preferably a (preferably naturally occurring) amino acid residue independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0079] Such a C-terminal extension X (n) According to some preferred but non-limiting embodiments, X and n can be as follows: (a) n=1 and X=Ala; (b) n = 2 and each X = Ala; (c) n = 3 and each X = Ala; (d) n=2 and at least one X=Ala, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; (e) n=3 and at least one X=Ala, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; (f) n=3 and at least two X=Ala, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; (g) n=1 and X=Gly; (h) n = 2 and each X = Gly; (i) n = 3 and each X = Gly; (j) n=2 and at least one X=Gly, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; (k) n=3 and at least one X=Gly, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; (l) n=3 and at least two X=Gly, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; (m) n = 2 and each X = Ala or Gly; (n) n = 3 and each X = Ala or Gly; (o) n=3 and at least one X=Ala or Gly, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; or (p) n=3 and at least two X=Ala or Gly, where the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably are independently selected from Val, Leu and / or Ile; Here, embodiments (a), (b), (c), (g), (h), (i), (m) and (n) are particularly preferred, embodiments where n=1 or 2 are preferred, and embodiments where n=1 are particularly preferred.

[0080] It should also be noted that preferably any C-terminal extension present in a serum albumin binder of the invention does not contain a (free) cysteine ​​residue (unless said cysteine ​​residue is used or intended for further functionalization, e.g. PEGylation).

[0081] Some specific, but non-limiting examples of useful C-terminal extensions are the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA or GAG.

[0082] Also preferably, when a compound of the invention has an ISVD at its C-terminus (e.g. an ISVD directed against a serum albumin binder of the invention or a therapeutic target), said C-terminal ISVD (at least) preferably contains one or more mutations that reduce binding by existing antibodies (i.e. as described herein for the serum albumin binder of the invention and generally as described in WO 2012 / 175741 and WO 2015 / 173325, and also e.g. in WO 2013 / 024059 and WO 2016 / 118733), even more preferably in addition to the C-terminal extension as described herein. In this regard, it will be clear to the skilled person that when a compound of the invention has a serum albumin binder of the invention at its C-terminus, said serum albumin binder of the invention (at least) preferably contains such mutations (i.e. preferably in addition to the C-terminal extension).

[0083] More generally, according to a specific embodiment of the invention, when a compound of the invention contains two or more ISVDs (e.g. a serum albumin binder of the invention and one or more ISVDs against a therapeutic target), preferably all of these ISVDs contain mutations that reduce binding to pre-existing antibodies (again, preferably in addition to a C-terminal extension linked to the C-terminal ISVD, if the compound of the invention has an ISVD at its C-terminus).

[0084] When a compound of the invention has an ISVD at its N-terminus (e.g. an ISVD directed against a serum albumin binder of the invention or a therapeutic target), said N-terminal ISVD (and thus, by extension, the entire compound of the invention) preferably contains a D at position 1. In this regard, again, it will be clear to the skilled person that when a compound of the invention has a serum albumin binder of the invention at its N-terminus, said serum albumin binder of the invention preferably has a D at position 1 (e.g. an E1D mutation compared to the sequences of SEQ ID NO:18 and / or 19, e.g. an amino acid sequence of the invention of SEQ ID NO:36-44).

[0085] In some further embodiments, the present invention relates to a protein, polypeptide or other compound or construct comprising or consisting essentially of at least one (preferably only one) serum albumin binder of the invention and at least one (e.g. one, two or three) therapeutic moieties or entities, said serum albumin binder and one or more therapeutic moieties or entities being suitably linked, optionally via one or more suitable linkers, which protein, polypeptide, compound, construct is as follows: - When the protein, polypeptide, compound, construct has an ISVD at its C-terminus, the protein, polypeptide, compound, construct (the C-terminal ISVD) has a C-terminal extension (X) at its C-terminus n (as further described herein); and / or - when it has an ISVD at its C-terminus, at least said C-terminal ISVD contains one or more mutations (as further described herein) that reduce binding of pre-existing antibodies; - when having an ISVD at its N-terminus, said protein, polypeptide, compound, construct (its N-terminal ISVD) preferably contains a D at position 1; and / or - in said ISVD, the protein, polypeptide or other compound may have an ISVD at its N-terminus, in which case said N-terminal ISVD preferably has D or E1D at position 1; -Preferably, essentially all of the ISVDs present in said protein, polypeptide, compound or construct contain one or more mutations (as further described herein) that reduce binding of pre-existing antibodies.

[0086] According to a particular aspect of the invention, all therapeutic moieties present in the compounds of the invention are ISVDs (i.e. ISVDs against a therapeutic target), in particular heavy chain ISVDs, and more particularly nanobodies (i.e. nanobodies against a therapeutic target).

[0087] By way of example and not limitation, such compounds of the invention may include: - 1 copy of a serum albumin binder of the invention and one ISVD (preferably a nanobody) against a therapeutic target; or - one copy of a serum albumin binder of the invention and two ISVDs (preferably two nanobodies) against a therapeutic target (the ISVDs may be the same or different and, when different, may be directed against the same target, against different epitopes on the same target or against different therapeutic targets); or -1 copy of a serum albumin binder of the invention and 3 ISVDs (preferably 3 nanobodies) against a therapeutic target (the ISVDs may be the same or different and, when different, may be directed against the same target, against different epitopes on the same target or against different therapeutic targets).

[0088] Some non-limiting examples of constructs, fusion proteins or polypeptides of the invention can be illustrated as follows, where "[Alb]" represents a serum albumin binder of the invention, "[therapeutic moiety 1]" and "[therapeutic moiety 2]" represent therapeutic moieties (which, as mentioned, can each independently be an immunoglobulin single variable domain), "-" represents a suitable linker (optional; suitable examples are 9GS and 35GS linkers), and the N-terminus is on the left and the C-terminus is on the right: [Alb]-[Treatment part 1] [Treatment part 1]-[Alb]-X (n) [Alb]-[Treatment part 1]-[Treatment part 1] [Treatment part 1]-[Treatment part 1]-[Alb]-X (n) [Treatment part 1]-[Alb]-[Treatment part 1] [Alb]-[Treatment part 1]-[Treatment part 2] [Treatment part 1]-[Treatment part 2]-[Alb]-X (n) [Treatment part 1]-[Alb]-[Treatment part 2]

[0089] When the therapeutic moiety is an ISVD (preferably a nanobody) against a therapeutic target, a preferred, but non-limiting, construct, fusion protein or polypeptide of the invention can be illustrated as follows, where "[Alb]" represents a serum albumin binder of the invention, "[Therapeutic ISVD1]" and "[Therapeutic ISVD2]" represent ISVDs against the therapeutic target (which ISVDs may be the same or different and, when different, may be directed against the same target, against different epitopes on the same target or against different therapeutic targets), "-" represents a suitable linker (which is optional), X(n) represents a C-terminal extension as described herein, and the N-terminus is on the left and the C-terminus is on the right: [Alb]-[Therapeutic ISVD1]-X (n) [Therapeutic ISVD1]-[Alb]-X (n) [Alb]-[Therapeutic ISVD1]-[Therapeutic ISVD1]-X (n) [Therapeutic ISVD1]-[Therapeutic ISVD1]-[Alb]-X (n) [Therapeutic ISVD1]-[Alb]-[Therapeutic ISVD1]-X (n) [Alb]-[Therapeutic ISVD1]-[Therapeutic ISVD2]-X (n) [Therapeutic ISVD1]-[Therapeutic ISVD2]-[Alb]-X (n) [Therapeutic ISVD1]-[Alb]-[Therapeutic ISVD2]-X (n)

[0090] Thus, in another aspect, the present invention relates to a multispecific (in particular bispecific) Nanobody construct comprising a serum albumin binder of the invention and at least one other Nanobody (e.g. one or two other Nanobodies, which may be the same or different), preferably directed against a desired target (which is preferably a therapeutic target) and / or another Nanobody useful or suitable for therapeutic, prophylactic and / or diagnostic purposes. Again, the serum albumin binder of the invention and the other Nanobody may be suitably linked to each other directly or optionally via one or more suitable linkers or spacers.

[0091] For a general description of multivalent and multispecific polypeptides containing one or more Nanobodies and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; and for example WO 96 / 34103, WO 99 / 23221, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0092] By way of example, some examples of compounds of the invention are given in SEQ ID NOs: 46-52, where the anti-HER2-nanobody of SEQ ID NO: 45 is used as a representative example of an anti-targeting nanobody and where the constituent nanobodies are in different positions of the compounds of the invention. The compounds of SEQ ID NOs: 46-49 are illustrative of bivalent bispecific compounds of the invention, and the compounds of SEQ ID NOs: 50-52 are illustrative of trivalent bispecific compounds of the invention. In each case, the compounds contain an E1D mutation and a C-terminal alanine residue, and contain the use of representative but non-limiting examples of suitable linkers (i.e., the 15GS linker in SEQ ID NOs: 47 and 48 and / or the 35GS linker in SEQ ID NOs: 46 and 49-52).

[0093] Some other examples of some specific multispecific and / or multivalent polypeptides of the invention can be found in the applications by Ablynx NV mentioned herein. In particular, for a general description of multivalent and multispecific constructs comprising at least one Nanobody against a serum protein for increasing half-life, nucleic acids encoding same, compositions comprising same, preparations as mentioned above, and uses as mentioned above, please refer to the above-mentioned International Applications WO 04 / 041865 and WO 06 / 122787 (the serum albumin binders of the invention described herein can generally be used similarly to the half-life-extending Nanobodies described herein, such as Alb-8), as well as the general descriptions and specific examples of such constructs given, for example, in WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0094] The present invention also relates to a nucleotide sequence or nucleic acid encoding the albumin binder, compound or polypeptide of the present invention.The present invention further includes a genetic construct comprising the aforementioned nucleotide sequence or nucleic acid and one or more elements for a genetic construct known per se.The genetic construct may be in the form of a plasmid or vector.Again, such a construct may be generally as described in the published patent applications of Ablynx NV, such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0095] The present invention also relates to a host or host cell containing such a nucleotide sequence or nucleic acid and / or expressing (or capable of expressing) an albumin binder, compound or polypeptide of the present invention. Again, such a host cell may generally be as described in Ablynx NV's published patent applications, such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0096] The present invention also relates to a method for preparing an albumin binder, compound or polypeptide of the invention, comprising culturing or maintaining a host cell as described herein under conditions such that said host cell produces or expresses the albumin binder, compound or polypeptide of the invention, and optionally further comprising isolating the albumin binder, compound or polypeptide of the invention so produced. Again, such methods may generally be performed as described in published patent applications of Ablynx NV, such as, for example, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0097] The present invention also relates to pharmaceutical compositions comprising at least one compound or polypeptide of the present invention and optionally at least one pharma- ceutically acceptable carrier, diluent or excipient. Such preparations, carriers, excipients and diluents may generally be as described in published patent applications of Ablynx NV, such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0098] However, since the compounds or polypeptides of the present invention have an increased half-life, they are preferably administered into the blood circulation.Thus, they can be administered in any suitable manner that allows the compounds or polypeptides of the present invention to enter the blood circulation, for example, intravenously, via injection or infusion, or in any other suitable manner, including oral administration, subcutaneous administration, intramuscular administration, administration through the skin, intranasal administration, administration through the lungs, etc.Suitable methods and routes of administration will be clear to those skilled in the art from the teachings of, for example, Ablynx NV's published patent applications, such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0099] Thus, in another aspect, the present 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 a compound or polypeptide of the present invention, comprising administering to a subject in need thereof a pharma- ceutical active amount of a compound or polypeptide of the present invention and / or a pharmaceutical composition comprising same. The diseases and disorders that can be prevented or treated by the use of a compound or polypeptide of the present invention as described herein are generally the same as the diseases and disorders that can be prevented or treated by the use of a therapeutic moiety or moieties present in a compound or polypeptide of the present invention.

[0100] In the context of the present invention, the term "prophylaxis and / or treatment" not only includes preventing and / or treating a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or delaying 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 duration of a disease and / or any symptoms associated therewith and / or preventing a further increase in the severity of a disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by a disease, and generally any pharmacological effect that is beneficial to the patient being treated.

[0101] The subject to be treated may be any warm-blooded animal, but in particular a mammal, and more particularly a human. As will be apparent to those skilled in the art, the subject to be treated is in particular a person suffering from or at risk of the diseases and disorders mentioned herein.

[0102] In another embodiment, the present invention relates to a method for immunotherapy, in particular a method for passive immunotherapy, comprising administering to a subject suffering from or at risk of the diseases and disorders mentioned herein a pharma- ceutical active amount of a compound or polypeptide of the present invention and / or a pharmaceutical composition comprising same.

[0103] The compound or polypeptide of the present invention and / or the composition comprising it are administered according to a treatment regimen suitable for preventing and / or treating the disease or disorder to be prevented or treated.The clinician can generally determine the appropriate 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 its symptoms, the specific polypeptide of the present invention to be used, the specific route of administration and the pharmaceutical formulation or composition to be used, the age, sex, weight, diet, general condition of the patient, and similar factors well known to the clinician.

[0104] In general, a treatment regimen comprises the administration of one or more pharma- ceutically effective amounts or doses of one or more compounds or polypeptides of the present invention, or one or more compositions comprising the same. The specific amount or dose to be administered can be determined by the clinician, again based on the factors mentioned above.

[0105] Generally, for the prevention and / or treatment of the diseases and disorders mentioned herein, and depending on the specific disease or disorder to be treated, the potency and / or half-life of the compound or polypeptide of the present invention to be used, the specific route of administration, and the specific pharmaceutical formulation or composition used, the compound or polypeptide of the present invention is generally administered in an amount of 1 gram to 0.01 micrograms / kg body weight / day, preferably 0.1 gram to 0.1 micrograms / kg body weight / day, for example about 1, 10, 100 or 1000 micrograms / kg body weight / day, continuously (e.g., by infusion), either as a single daily dose or as multiple divided doses during the day. The clinician can generally determine the appropriate daily dose depending on the factors mentioned herein. It will also be apparent that in certain cases, the clinician may choose to deviate from these amounts, for example based on the factors mentioned above and the clinician's professional judgment. Generally, some guidance as to the amount to be administered can be obtained from the amount normally administered for a comparable conventional antibody or antibody fragment against the same target administered via essentially the same route, but taking into account differences in affinity / avidity, efficacy, biodistribution, half-life and similar factors well known to those of skill in the art.

[0106] Also, since the compounds of the present invention contain the half-life extended serum albumin binders of the present invention, they do not necessarily have to be administered essentially continuously (e.g., by infusion), but can be administered at suitable intervals (to be determined by one of ordinary skill in the art). For example, they can be administered (at a suitable dosage) once every two days, once every four days, once a week, once every two weeks, in some cases once every four days or even less frequently, e.g., by injection or infusion.

[0107] One aspect of the present invention relates to a pharmaceutical composition comprising at least one compound or polypeptide of the invention, which is intended to be administered at intervals of once per week to once every 4 weeks, in particular once every 7 days to once every 21 days, for example once every 7 or 14 days.

[0108] Typically, a single polypeptide of the invention will be used in the above methods, however, it is also within the scope of the invention to use two or more polypeptides of the invention in combination.

[0109] The polypeptides of the present invention may also be used in combination with one or more additional pharmacologic active compounds or components, i.e., as a combination treatment regimen, which may or may not produce synergistic effects.Again, the clinician can select such additional compounds or components and the appropriate combination treatment regimen based on the above factors and the clinician's professional judgment.

[0110] In particular, the polypeptides of the invention may be used in combination with other pharma- ceutically active compounds or ingredients that are or can be used in the prevention and / or treatment of diseases and disorders that can be prevented or treated with the fusion proteins or constructs of the invention, which may or may not result in a synergistic effect.

[0111] The effectiveness of the treatment regimen used according to the present invention may be determined and / or tracked in any manner known per se for the disease or disorder involved, as will be apparent to the clinician.The clinician may also, as necessary and / or depending on the individual case, modify or alter a particular treatment regimen to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side effects, and / or to achieve a suitable balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesirable side effects on the other hand.

[0112] Generally, the treatment regimen is followed until the desired therapeutic effect is achieved and / or for as long as the desired therapeutic effect is maintained, which again can be determined by the clinician.

[0113] Other aspects, embodiments, advantages and applications of the present invention will become apparent from the further description herein.

[0114] The present invention will now be further illustrated by the following non-limiting preferred embodiments, examples and figures: [Brief description of the drawings]

[0115] [Figure 1] 1 is a table listing several amino acid positions specifically referred to herein and their numbering according to several alternative numbering systems (such as Aho and IMGT); [Figure 2-1] The amino acid sequences referred to herein are listed below; [Figure 2-2] The amino acid sequences referred to herein are listed below; [Figure 2-3] The amino acid sequences referred to herein are listed below; [Figure 2-4] The amino acid sequences referred to herein are listed below; [Figure 2-5] The amino acid sequences referred to herein are listed below; [Figure 2-6] The amino acid sequences referred to herein are listed below; [Diagram 2-7] The amino acid sequences referred to herein are listed below; [Figure 3A] Shows an alignment of the sequences of SEQ ID NOs:18 and 19 (the present invention) with the prior art sequences of SEQ ID NOs:1 and 2; [Figure 3B] Shows an alignment of the sequences of SEQ ID NOs:18 and 19 (the present invention) with the prior art sequences of SEQ ID NOs:1 and 2; [Figure 4] Showing alignment of SEQ ID NO:18-44; [Figure 5-1] An alignment of serum albumins from different mammalian species is provided. [Figure 5-2] An alignment of serum albumins from different mammalian species is provided. [Figure 5-3]An alignment of serum albumins from different mammalian species is provided. [Figure 6] 1 is a graph showing the binding of SEQ ID NO:1 (reference)-cMycHis6 to coated HSA in the presence of serum albumin binder SEQ ID NO:19 (invention). 1.5 nM SEQ ID NO:1-cMycHis6 and a range of concentrations of His6Flag3-SEQ ID NO:1 (reference), Flag3His6-SEQ ID NO:19 (invention) or cAblys3-Flag3His6 (reference) were incubated on coated HSA. Bound SEQ ID NO:1-cMycHis6 was detected with goat anti-cMyc and rabbit anti-goat antibodies labeled with HRP. [Figure 7] Graph showing binding of HSA to FcRn in the presence of serum albumin binder of SEQ ID NO:19 (invention). Human FcRn-human β2 microglobulin heterodimer was immobilized on a CM5 chip. Binding of 1 μM HSA in the absence or presence of 2 μM nanobody in 50 mM NaPO4+150 mM NaCl+0.05% Tween-20 pH 6.0 was monitored on a Biacore T100 instrument.

[0116] The entire contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated by reference, particularly for the teachings referred to herein above. EXAMPLES

[0117] Experimental section Example 1 Affinity for serum albumin The affinity of the serum albumin binder of SEQ ID NO:19 (the present invention) for human (Sigma-Aldrich A3782), cynomolgus monkey (produced in-house), mouse (Albumin Bioscience 2601), rat (Sigma-Aldrich A4538), rabbit (Sigma-Aldrich A0764), guinea pig (Gentaur GPSA62), pig (Sigma-Aldrich A4414), sheep (Sigma-Aldrich A3264), dog (Abcam 119814) and bovine (Sigma-Aldrich A3059) serum albumin (SA) was measured via surface plasmon resonance (SPR) on a ProteOn XPR36 (BioRad) instrument. Serum albumin was immobilized on a GLC ProteOn chip via amine coupling using the ProteOn Amine Coupling Kit (BioRad). Different concentrations (300nM, 100nM, 33.3nM, 11.1nM, 3.7nM and 1.23nM) of serum albumin binder of SEQ ID NO:19 (invention) were injected in HBS-P+pH7.4 buffer (GE Healthcare) at 45μL / min for 120 seconds, followed by dissociation for 900 seconds. No or very little binding of serum albumin binder of SEQ ID NO:19 (invention) on rabbit, sheep and bovine SA was observed. The affinity of serum albumin binder of SEQ ID NO:19 (invention) for human, cynomolgus monkey, rat, mouse and guinea pig SA was higher compared to the respective affinity of albumin binder of SEQ ID NO:1. The results are given in Table 1 below, showing that serum albumin binder of SEQ ID NO:19 (invention) is cross-reactive with dog and pig SA.

[0118] [Table 3]

[0119] The long serum half-life of albumin is driven mainly by two features: (i) the large size of albumin (65 kDa) limits its glomerular filtration, and (ii) albumin binds to FcRn at low pH (pH 6), preventing albumin degradation in lysosomes after passive endocytosis in endothelial and epithelial cells by recycling from early endosomes to the extracellular environment. For albumin-binding nanobodies to provide a long serum half-life through albumin binding and subsequent recycling, they need to remain bound to albumin in the pH range of 5.0 to 7.4. The dissociation rates of serum albumin binders of SEQ ID NO:19 (invention) from HSA at pH 5, pH 6 and pH 7.4 were measured with a ProteOn device as described above, including SEQ ID NO:1 (reference) as a reference. The serum albumin binder of SEQ ID NO:19 (invention) and SEQ ID NO:1 (reference) were injected at 500 nM and 300 nM, respectively, in HBS-P+pH 7.4 buffer. The dissociation buffers were 50 mM NaOAc / HOAc+150 mM NaCl+0.05% Tween-20 pH 5.0, 50 mM NaOAc / HOAc+150 mM NaCl+0.05% Tween-20 pH 6.0 and HBS-P+pH 7.4, respectively. Dissociation was analyzed for 2700 seconds. The dissociation rate for the serum albumin binder of SEQ ID NO:19 (invention) is not significantly different over the pH range of 5.0 to 7.4. The results are shown in Table 2.

[0120] [Table 4]

[0121] Example 2 Epitope Epitope binning was analyzed by competitive ELISA. Human serum albumin was coated at 125ng / ml in PBS overnight at 4°C. After blocking with PBS+1% casein, 1.5nM SEQ ID NO:1(reference)-cMycHis6 and a series of concentrations of competitors (His6Flag3-SEQ ID NO:19 serum albumin binder (invention), His6Flag3-SEQ ID NO:1(reference) as positive control, or chicken egg white lysozyme binding single domain antibody cAblys3-Flag3His6 as negative control) were added. Bound SEQ ID NO:1(reference)-cMycHis6 was detected with goat anti-cMyc (Abcam ab19234) and HRP-labeled rabbit anti-goat (Genway 18-511-244226) antibodies. The serum albumin binder of SEQ ID NO:19 (invention) and SEQ ID NO:1 (reference) do not bind to the same epitope on HSA (FIG. 6).

[0122] Example 3 Interference with the interaction between SA and FcRn For the serum albumin binder of SEQ ID NO:19 (the present invention) to provide a long half-life through albumin binding and subsequent recycling, it should not interfere with the binding of albumin to FcRn. This was analyzed by SPR on a Biacore T100 (GE Healthcare) instrument. Human FcRn-human β2 microglobulin heterodimer (Sino Biological CT009-H08H) was immobilized on a CM5 chip via standard amine coupling (Biacore amine coupling kit). A mixture of 1 μM HSA and 2 μM nanobody (serum albumin binder of His6Flag3-SEQ ID NO:19 (invention), His6Flag3-SEQ ID NO:1 (reference) or cAblys3-Flag3His6) in 50 mM NaPO4+150 mM NaCl+0.05% Tween-20 pH 6.0 was injected at 10 μl / min for 120 s, followed by dissociation for 600 s. Binding curves were qualitatively compared with those of 1 μM HSA in the absence of nanobody. Serum albumin binder of SEQ ID NO:19 (invention) did not interfere with the binding of HSA to FcRn (Figure 7).

[0123] Example 4 PK profile in dogs The pharmacokinetics of a trivalent nanobody construct (SEQ ID NO:53) after a single intravenous (iv) dose is studied in dogs. The trivalent construct was produced in Pichia pastoris and contains two nanobodies against respiratory syncytial virus (RSV) with the albumin-binding nanobody of SEQ ID NO:21 at the C-terminus (the three nanobodies are linked together using a (Gly4Ser)7 linker).

[0124] The pharmacokinetic profile of the construct will be evaluated in a pharmacokinetic study (non-crossover, single dose) in 12 fasted healthy male beagle dogs (health determined by pre-study health examination). The construct will be administered intravenously.

[0125] Animals are fasted overnight (but allowed continued access to water) prior to dosing and blood samples are taken pre-dose, 24, 168, and 336 hours post-dose (total fasting time not to exceed 24 hours). Food is returned 4 hours post-dose or immediately following the 24, 168, and 336 hour blood draws. Body weights are also recorded and tracked throughout the study.

[0126] 1 ml of whole blood for plasma (or 2-3 ml before dosing and at 24, 168 and 336 hours) is collected from a peripheral vein before dosing and at 5, 15, 30 minutes and 1, 4, 8, 12, 24, 72, 168, 240, 336, 504 and 672 hours after initial dosing. Collected samples for serum processing are kept at room temperature and allowed to clot. Blood is centrifuged at 10,000 rpm for 2 minutes using a refrigerated centrifuge (set to maintain 4 °C). Samples are frozen within 30 minutes of collection until they are subjected to analysis (including measurement of the concentration of the nanobody construct involved).

Claims

1. An amino acid sequence which is an immunoglobulin single variable domain capable of binding to (human) serum albumin: - CDR1 (according to Abm) which is the amino acid sequence GGTFSTYVMG (SEQ ID NO: 12) or an amino acid sequence which has two or one amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO: 12; and - CDR2 (according to Abm) which is the amino acid sequence AISQNSIHTY (SEQ ID NO: 13) or an amino acid sequence which has 3, 2 or 1 amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO: 13; and - a CDR3 (according to Abm) which is the amino acid sequence SRFTSWYTADYEYDY (SEQ ID NO:14) or an amino acid sequence which has 3, 2 or 1 amino acid difference (as defined herein) from the amino acid sequence of SEQ ID NO:

14.

2. 2. The amino acid sequence of claim 1, having: - CDR1 (according to Abm) with the amino acid sequence GGTFSTYVMG (SEQ ID NO: 12); and - CDR2 (according to Abm) with the amino acid sequence AISQNSIHTY (SEQ ID NO: 13); and - CDR3 (according to Abm), which has the amino acid sequence SRFTSWYTADYEYDY (SEQ ID NO: 14).

3. 2. The amino acid sequence according to claim 1, which is capable of binding to human serum albumin with an affinity better than 100 nM, preferably better than 50 nM, as determined using ProteOn.

4. 10. The amino acid sequence according to any one of the preceding claims, which is a heavy chain immunoglobulin single variable domain.

5. 2. An amino acid sequence according to any one of the preceding claims which is a VHH, a humanised VHH or a camelised VH (in particular a camelised human VH).

6. such that it has a serum half-life in humans (expressed as t1 / 2 beta) of greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, and even more preferably greater than 72 hours; and / or:

2. An amino acid sequence according to any one of the preceding claims, which when linked to a therapeutic moiety or entity confers on the resulting polypeptide of the invention a serum half-life in humans (expressed as t1 / 2 beta) of greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours.

7. having a degree of sequence identity with the sequence of SEQ ID NO: 18 and / or 19 of at least 85%, preferably at least 90%, more preferably at least 95% (the CDRs and any C-terminal extensions which may be present are not taken into account when determining the degree of sequence identity); and / or:

13. An amino acid sequence according to any one of the preceding claims, having no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid difference(s)" (as defined herein, not taking into account possible CDRs and any C-terminal extension) from the sequence of SEQ ID NO: 18 and / or 19.

8. having a degree of sequence identity with the sequence of SEQ ID NO: 18 of at least 85%, preferably at least 90%, more preferably at least 95% (the possible CDRs and any C-terminal extensions are not taken into account when determining the degree of sequence identity); and / or:

13. An amino acid sequence according to any one of the preceding claims, having no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid difference(s)" (as defined herein, not taking into account possible CDRs and any C-terminal extension) from the sequence of SEQ ID NO:

18.

9. having a degree of sequence identity with the sequence of SEQ ID NO: 19 of at least 85%, preferably at least 90%, more preferably at least 95% (the CDRs and any C-terminal extensions which may be present are not taken into account when determining the degree of sequence identity); and / or:

8. An amino acid sequence according to any one of claims 1 to 7, which has no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid difference(s)" (as defined herein, not taking into account possible CDRs and any C-terminal extension) from the sequence of SEQ ID NO:

19.

10. 2. An amino acid sequence according to any one of the preceding claims, which contains one or more mutations that reduce binding by existing antibodies compared to the sequence of SEQ ID NO:

18.

11. 10. Amino acid sequence according to any one of the preceding claims, which is a VHH and which contains one or more humanizing substitutions compared to the sequence of SEQ ID NO:

18.

12. An amino acid sequence which is an immunoglobulin single variable domain capable of binding to (human) serum albumin and which is selected from SEQ ID NO: 18-44.

13. A protein, polypeptide or other construct, compound, molecule or chemical entity comprising at least one amino acid sequence according to any one of claims 1 to 12.

14. 14. The protein, polypeptide or other construct, compound, molecule or chemical entity of claim 13, comprising at least one therapeutic moiety or entity.

15. 15. The protein, polypeptide or other construct, compound, molecule or chemical entity of claim 13 or 14 which is a fusion protein.

16. 16. A protein, polypeptide or other construct, compound, molecule or chemical entity according to any one of claims 13 to 15, having a serum half-life in humans (expressed as t1 / 2 beta) of more than 6 hours, preferably more than 12 hours, more preferably more than 24 hours, even more preferably more than 72 hours.

17. A protein, polypeptide or other construct, compound, molecule or chemical entity according to any one of claims 13 to 16, When the protein, polypeptide, compound, construct has an ISVD at its C-terminus, the protein, polypeptide, compound, construct (the C-terminal ISVD) has a C-terminal extension (X ｎ (as further described herein); and / or When it has an ISVD at its C-terminus, at least said C-terminal ISVD contains one or more mutations (as further described herein) that reduce binding of pre-existing antibodies; and / or said protein, polypeptide, compound, construct, when it has an ISVD at its N-terminus, preferably contains a D at position 1; and / or In said ISVD, the protein, polypeptide or other compound may have an ISVD at its N-terminus, in which case said N-terminal ISVD preferably has D or E1D at position 1; and / or Preferably, a protein, polypeptide or other construct, compound, molecule or chemical entity, wherein essentially all of the ISVDs present in said protein, polypeptide, compound, construct contain one or more mutations that reduce binding of a pre-existing antibody.

18. A pharmaceutical composition comprising a protein, polypeptide or other construct, compound, molecule or chemical entity according to any one of claims 13 to 17.

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