Improved Serum Albumin Binding Agent

Abstract

The present invention relates to amino acid sequences that bind serum albumin. Specifically, the present invention relates to improved immunoglobulin single variable domains (also referred to herein as "ISV" or "ISVD"), and more specifically to improved heavy chain immunoglobulin single variable domains that bind serum albumin (also referred to herein as "ISV" or "ISVD"), and to proteins, polypeptides and other constructs, compounds, molecules or chemical entities comprising such improved serum albumin binders.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680079196.5, with an application date of November 17, 2016 and an invention title of "Improved Serum Albumin Binding Agents".

[0002] The present invention relates to amino acid sequences that bind to serum albumin.

[0003] Specifically, the present invention relates to improved immunoglobulin single variable domains (also referred to herein as "ISV" or "ISVD"), and more specifically to improved heavy chain immunoglobulin single variable domains that bind to serum albumin (also referred to herein as "ISV" or "ISVD"), as well as proteins, polypeptides, and other constructs, compounds, molecules, or chemical entities that comprise such improved serum albumin binding agents.

[0004] Specifically, the present invention relates to improved nanobodies that bind to serum albumin, as well as proteins, polypeptides, and other constructs, compounds, molecules, or chemical entities that comprise such improved nanobodies that bind to serum albumin.

[0005] The improved ISVD that binds to serum albumin provided by the present invention is also referred to herein as "the amino acid sequence of the present invention", "the serum albumin binding agent of the present invention", "the albumin binding agent of the present invention", or "serum albumin binding agent" or "albumin binding agent". Further, proteins, polypeptides, and other constructs, compounds, molecules, or chemical entities that comprise at least one serum albumin binding agent of the present invention are also referred to herein as "the compounds of the present invention" or "the polypeptides of the present invention".

[0006] Preferably, the polypeptide of the present invention is a fusion protein.

[0007] Those skilled in the art will appreciate other aspects, embodiments, features, uses, and advantages of the present invention based on the disclosure herein.

[0008] In the present application, amino acid residues / positions in the immunoglobulin heavy chain variable domain will be indicated using the Kabat numbering. For convenience, Figure 1 the table provided lists some of the amino acid positions that will be specifically referred to herein and their numbering according to some alternative numbering systems (such as Aho and IMGT. Note: Unless otherwise specifically indicated, for this specification and the claims, the Kabat numbering is decisive; other numbering systems are for reference only).

[0009] Regarding CDRs, as is well known in the art, there are multiple conventions for defining and describing CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the positions of structural loop regions). See, for example, the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and the claims, although CDRs according to Kabat may also be referred to, most preferably the CDRs are defined based on the Abm definition (which is based on Oxford Molecular's AbM antibody modeling software), as this is regarded as the best compromise between the Kabat and Chothia definitions. See again the website http: / / www.bioinf.org.uk / abs / ).

[0010] Accordingly, in this specification and the claims, all CDRs are defined according to the Abm convention, unless expressly stated otherwise herein.

[0011] ISVDs (and in particular nanobodies) that can bind serum albumin and their uses are well known in the art, for example from WO 2004 / 041865, WO 2006 / 122787, EP 2 139 918, WO 2011 / 006915, WO2012 / 175400 and WO 2014 / 111550, which describe ISVDs that bind serum albumin and their use for extending the serum half-life of therapeutic compounds, moieties and entities (as defined in these applications).

[0012] WO 2006 / 122787 discloses a humanized serum albumin-binding nanobody (referred to as Alb-8, see SEQ ID NO:50 herein) as SEQ ID NO:62. WO 2012 / 175400 discloses a humanized serum albumin-binding nanobody (referred to as Alb-23D, see SEQ ID NO:1 herein) as SEQ ID NO:6, and also discloses that some amino acid differences between Alb-8 and Alb-23D (for example, specifically, the presence of the SKN motif at positions 74 - 76) may have a beneficial effect on the physical properties of the albumin binder. 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 NO:50, SEQ ID NO:1 and SEQ ID NO:2 - 7, respectively. Table A also gives the sequence of "Reference A" (a reference compound used in the experimental section) as SEQ ID NO:119. Figures 3A to 3CShows different alignments of SEQ ID NO: 1, 50, and 119. Figure 3D The figure of Figure 3D shows the binding of pre-existing antibodies from 6 sera depleted of serum-albumin to Alb-23D, to Alb-23 (SEQ ID NO: 1 in WO 2006 / 122787, which is identical to Alb-23D but without the C-terminal alanine), to reference A with a C-terminal alanine, to reference A, to Alb-8 with a C-terminal alanine, and to Alb-8. The nanobodies were immobilized using a HIS6-FLAG3 tag, and the binding of the pre-existing antibodies in the samples was determined using a ProteOn, in particular using the protocol used in the experimental section below. Figures 3A - 3D The results in Figures 3A - 3D show that for the three nanobodies without a C-terminal extension (i.e., Alb23, reference A, and Alb-8) and for the three nanobodies with a C-terminal extension (i.e., Alb23D, reference A-Ala, and Alb-8-Ala), the binding of pre-existing antibodies is comparable. It can also be seen that for each of the three nanobodies tested, the addition of a C-terminal alanine results in a comparable decrease in the binding of pre-existing antibodies.

[0013] Table A: Alb-8, Alb-23D, and their CDRs

[0014]

[0015]

[0016] The object of the present invention is to provide improved serum albumin binders, in particular relative to the serum albumin binders disclosed in WO2006 / 122787 and WO 2012 / 175400.

[0017] More specifically, the object of the present invention is to provide improved nanobodies that bind serum albumin, which are variants of the nanobodies that bind serum albumin described in WO2006 / 122787 and WO 2012 / 175400, and which have reduced binding to interfering factors (commonly referred to as "pre-existing antibodies") that may be present in sera from some healthy human subjects as well as from patients. Reference is made to WO 12 / 175741, WO 2013 / 024059, and also to, for example, Holland et al. (J. Clin. Immunol. 2013, 33(7):1192-203) and the co-pending non-prepublished PCT application PCT / EP2015 / 060643 of the assignee, filed on May 13, 2015 and entitled "Improved immunoglobulin variable domains".

[0018] The improved serum albumin binder provided by the present invention is also referred to herein as "the serum albumin binder of the present invention".

[0019] Among the serum-albumin binders listed in Table A, Alb-23D has a C-terminal alanine extension, i.e., an alanine residue at the C-terminal end (sometimes also referred to as "position 114") of the ISVD-sequence compared to the normal C-terminal sequence VTVSS (SEQ ID NO:116, as present in Alb-8). As described in WO 12 / 175741, this C-terminal alanine extension can prevent the binding of so-called "pre-existing antibodies" (presumably IgG's) to a putative epitope located in the C-terminal region of the ISV. In addition to other residues, this epitope is presumed to include the amino acid residues of the surface-exposed C-terminal sequence VTVSS and the amino acid residue at position 14 (and amino acid residues adjacent / close to it in said amino acid sequence, such as positions 11, 13, and 15), and may also include the amino acid residue at position 83 (and amino acid residues adjacent / close to it in said amino acid sequence, such as positions 82, 82a, 82b, and 84) and / or the amino acid residue at position 108 (and amino acid residues adjacent / close to it in said amino acid sequence, such as position 107).

[0020] Although the presence of such a C-terminal alanine (or generally a C-terminal extension) can greatly reduce (and in a large number of cases, even substantially completely prevent) the binding of "pre-existing antibodies" that can be found in the sera from some subjects (healthy subjects and patients), it has been found that sera from some subjects (such as sera from patients with some immune diseases such as SLE) can contain pre-existing antibodies that can bind to the C-terminal region of the ISV (when such a region is exposed), even when the ISV contains such a C-terminal alanine (or more generally, such a C-terminal extension). Reference is again made to the co-pending and un-prepublished PCT application PCT / EP2015 / 060643 of the assignee, filed on May 13, 2015 and entitled "Improved immunoglobulin variable domains".

[0021] Accordingly, a specific object of the present invention is to provide serum-albumin binders which are variants of the improved serum-albumin-binding nanobodies listed in Table A and which have reduced binding of the so-called "pre-existing antibodies", in particular antibodies of the kind described in PCT / EP2015 / 060643 (i.e., those pre-existing antibodies that can bind to the exposed C-terminal region of the ISV, even in the presence of a C-terminal extension), and which also exhibit a low frequency and / or height of T-cell response in a dendritic cell-T cell proliferation assay (and / or a lower frequency and / or height of T-cell response compared to SEQ ID NO:1 and / or SEQ ID NO:50 in a dendritic cell / T cell proliferation assay). This assay for measuring the frequency and height of T-cell response is designed to identify such proteins that cause helper T-cell proliferation and thus potentially lead to the development of a helper T-cell immune response.

[0022] Generally, the present invention achieves this object as follows: providing amino acid sequences which are nanobodies that bind serum albumin, said nanobodies being variants of the amino acid sequence of SEQ ID NO:1 (as further described herein), and as further described herein (and, as will be understood by the person skilled in the art based on the alignments given in the figures and based on the other disclosures herein, since the amino acid sequence of SEQ ID NO:1 is a variant of the amino acid sequence of SEQ ID NO:50 (and vice versa), the serum-albumin-binding nanobodies disclosed herein are also variants of the sequence of SEQ ID NO:50).

[0023] These amino acid sequences are also referred to herein as "the amino acid sequences of the present invention" or "the serum-albumin binders of the present invention". Some preferred, but non-limiting, examples of the amino acid sequences of the present invention are in Figure 2are given as SEQ ID NO:8-49 (sequences without C-terminal alanine extension) and SEQ ID NO:61-102 (which give the amino acid sequences corresponding to the sequences of SEQ ID NO 8-49, but each has a C-terminal alanine extension); see also Figure 4A and Figure 4B for the alignments in, and Figure 23and 24. From these alignments and the other disclosures herein, it can be seen that, compared to the sequence of SEQ ID NO: 1 and / or SEQ ID NO: 50, the albumin binder of the present invention has: (i) a V at position 5 and a V at position 11; and / or (ii) one or more of the following amino acid residues (i.e. in a suitable combination): 29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G and / or 104T (i.e., such that, compared to the sequence of SEQ ID NO: 1, it contains at least one amino acid mutation selected from F29A, F29H, R30T, S31D, S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T). They preferably have a V at position 5 and a V at position 11 and one or more of the aforementioned amino acid residues / mutations. And, as will be apparent from these alignments and the other disclosure herein, the amino acid residue at position 89 is preferably selected from T, A or L. And, in general, the albumin binding amino acid sequences described herein preferably have a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO: 1 (wherein any C-terminal extension that may be present and L5V and L11V mutations are not taken into account when determining the degree of sequence identity); and / or have no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and without taking into account any C-terminal extension that may be present and without taking into account L5V and L11V mutations) with the sequence of SEQ ID NO: 1. Also, for some aspects of the invention, the amino acid sequences disclosed herein will be defined with reference to the sequence of SEQ ID NO:50, in which case the albumin binding amino acid sequences described herein preferably have a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:50 (wherein any C-terminal extension that may be present and the L11V mutation relative to SEQ ID NO:50 are not taken into account when determining the degree of sequence identity; and / or have no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid difference(s)" (as defined herein and without taking into account any C-terminal extension that may be present and without taking into account the L11V mutation) with the sequence of SEQ ID NO:50.Also, when defining an aspect of the invention by reference to the sequence identity and / or amount of amino acid differences relative to SEQ ID NO:1 (and / or SEQ ID NO:50, as appropriate), and by reference to the presence of specific mutations (i.e., L5V, L11V, and any other specific mutations expressly mentioned for this aspect of the invention), and / or by reference to the presence of one or more specific CDRs, these expressly mentioned mutations and / or CDRs should not be considered when determining the degree of sequence identity and / or the number of amino acid differences, respectively.

[0024] Some preferred amino acid residues / mutations that may be present in the amino acid sequences of the invention are: 29H (i.e., F29H relative to SEQ ID NO:1), 101D (i.e., S101D relative to SEQ ID NO:1), and 104T (i.e., S104T relative to SEQ ID NO:1), or any suitable combination of any two of these, such as all three of these. Also, these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1), or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1), or alternatively L at position 89 (i.e., V89L relative to SEQ ID NO:1).

[0025] Some other preferred amino acid residues / mutations that may be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. Also, these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1), or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1), or alternatively L at position 89 (i.e., V89L relative to SEQ ID NO:1).

[0026] Some other preferred amino acid residues / mutations that can exist are: 30S (i.e., R30S relative to SEQ ID NO:1) in combination with T, A, or L (and preferably L), and having G or T at position 104 (and preferably further in combination with some or all of the further preferred mutations described herein with respect to this aspect of the invention, such as L at position 89 and N at position 16). The amino acid sequence according to this aspect can thus have the same CDR (according to Abm) as the CDR of SEQ ID NO:50 (which also contains S at position 30); however, compared to the sequence of SEQ ID NO:50, the amino acid sequence according to this aspect of the invention has at least the following amino acid differences relative to the sequence of SEQ ID NO:50: (i) L11V; (ii) V89T, V89A, or V89L (and especially V89L) and (iii) S104G or S104T. Another preferred mutation is 31D: specifically, this can result in an amino acid sequence of the invention having a CDR1 that is the sequence GFTFRDFGMS (SEQ ID NO:54).

[0027] Some further preferred amino acid sequences of the invention (which contain other combinations of some of the specific mutations mentioned herein with respect to the serum albumin binders of the invention) are given in Figure 2 as SEQ ID NO:121 - 144 [Note: The sequences of SEQ ID NO:121 and 122 and the sequences of SEQ ID NO:127 and 128 are the same]. Among these, SEQ ID NO:121 - 126 have E at position 1 and a C-terminal alanine, and SEQ ID NO:127 - 132 have D at position 1 and a C-terminal alanine. SEQ ID NO:133 - 138 and SEQ ID NO:139 - 143 correspond to SEQ ID NO:121 - 126 and SEQ ID NO:127 - 132 respectively, but without the C-terminal alanine. Figure 23Also shown is the alignment of the sequences of SEQ ID NOs: 121 - 132 with SEQ ID NOs: 1, 50, and 119. Based on the disclosure herein, one of ordinary skill in the art will appreciate that the albumin binders of SEQ ID NOs: 121 - 132, and particularly those of SEQ ID NOs: 121 - 126, will often be used as / present at the C - terminal end of the constructs in which they are found, and the albumin binders of SEQ ID NOs: 139 - 144 will often be used as / present at the N - terminal end of the constructs in which they are found (similarly, the albumin binders of SEQ ID NOs: 133 - 138 are often present in the "center of such constructs"). Also, the serum albumin binders of SEQ ID NOs: 127 - 132 are particularly suitable for use in a monovalent form, e.g., for research purposes). Each of the amino acid sequences of SEQ ID NOs: 121 - 144, and the proteins, polypeptides, and other compounds and constructs that contain them (as further described herein) form other aspects of the present invention.

[0028] Some further preferred amino acid sequences of the present invention (which contain other combinations of some of the specific mutations mentioned herein with respect to the serum albumin binders of the present invention) are given in Figure 2 as SEQ ID NOs: 145 - 184, SEQ ID NOs: 185 - 208, and SEQ ID NOs: 209 - 244. Figure 24A To C also shows the alignment of the sequences of SEQ ID NOs: 145 - 184, SEQ ID NOs: 185 to 208, and SEQ ID NOs: 209 - 244, in each case aligned with the sequences of SEQ ID NOs: 1, 50, and 119. Like the other albumin binders of the present invention described herein, those sequences from SEQ ID NOs: 145 - 184, SEQ ID NOs: 185 - 208, and SEQ ID NOs: 209 - 244 that have a C - terminal extension are often present at the C - terminal end of the (fusion) proteins or constructs of the present invention in which they are found. Similarly, those that have a D at position 1 (but no C - terminal extension) are preferably present at the N - terminal end of the (fusion) proteins or constructs of the present invention in which they are found, and those that have an E at position 1 (but no C - terminal extension) can be present at the C - terminal end of the (fusion) proteins or constructs of the present invention in which they are found or (in the case of even higher valency trivalent constructs) the "center". Each of the amino acid sequences of SEQ ID NOs: 145 - 184, SEQ ID NOs: 185 - 208, and SEQ ID NOs: 209 - 244, and the proteins, polypeptides, and other compounds and constructs that contain them (as further described herein) form other aspects of the present invention.

[0029] In addition to having a (strongly) reduced or substantially zero tendency to be bound by existing antibodies as described herein, some of the serum albumin binders disclosed herein (the mutations therein) may also have other favorable properties (i.e., compared to albumin binders disclosed in the prior art such as SEQ ID NO:1 and / or SEQ ID NO:50, and / or compared to other serum albumin binders of the present invention described herein). For example, but not limited to, some combinations of mutations present in the serum albumin binders disclosed herein can result in increased expression levels and / or a reduced tendency to form dimers (see, e.g., WO 2010 / 100135) or reduced immunogenicity in a desired host or expression system such as Escherichia coli, Pichia pastoris, or mammalian cells. For example, some of the mutations described herein can remove immunogenic epitopes (especially T-cell epitopes) with little or no substantial impact on other properties of the albumin binder such as affinity. For example, but not limited to, it is anticipated (based on standard computer environment predictions) that the serum albumin binders of the present invention having a T at position 30 (as further described herein) (such as the sequences of SEQ ID NO:25, 44, 45, 78, 97, and 98, and especially those of SEQ ID NO:121-144 and SEQ ID NO:145-184) can have reduced immunogenicity (i.e., because potential T-cell epitopes have been removed), especially relative to the prior art sequences of SEQ ID NO 1 and 50, but (potentially) also relative to other albumin binders of the present invention that do not contain a T at position 30. (Again based on standard computer environment predictions) it is also anticipated that the serum albumin binders of the present invention of SEQ ID NO:185-208 (which contain an S at position 30 and a G or T at position 104) can have reduced immunogenicity (i.e., because potential T-cell epitopes have been removed), especially relative to the prior art sequences of SEQ ID NO 1 and 50, but (potentially) also relative to other albumin binders of the present invention. And, it is anticipated that some of the mutations described herein (such as 31D, see, e.g., the amino acid sequences of SEQID NO:209-244) can improve the expression and / or manufacturability of the amino acid sequences of the present invention (e.g., in Pichia pastoris or similar yeasts used as hosts), again relative to the prior art sequences of SEQ ID NO:1 and / or 50 and / or relative to other amino acid sequences of the present invention that do not contain the 31D mutation).

[0030] The serum albumin binder of the present invention has CDRs (according to Abm) as described herein. Some preferred combinations of CDR, CDR2, and CDR3 that can be present in the serum albumin binder of the present invention are listed in Table B below. Particularly preferred combinations are indicated by Bold / Underline Indication.

[0031] Table B. Preferred combinations of CDRs (defined according to Abm).

[0032]

[0033]

[0034] Table B (continued).

[0035] <![CDATA CDR1 > <![CDATA CDR2 > <![CDATA CDR3 > <![CDATA GFTFTSFGMS(SEQ ID NO:53) > <![CDATA SISGSGSDTL(SEQ ID NO:6 )]]> <![CDATA GGSLSR(SEQ ID NO:7) > <![CDATA GFTFTSFGMS(SEQ ID NO:53) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGGLSR(SEQ ID NO:55) > <![CDATA GFTFTSFGMS(SEQ ID NO:53) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGSLDR(SEQ ID NO:56) > <![CDATA GFTFTSFGMS(SEQ ID NO:53) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGSLER(SEQ ID NO:57) > <![CDATA GFTFTSFGMS(SEQ ID NO:53) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGSLGR(SEQ ID NO:58), > <![CDATA GFTFTSFGMS(SEQ ID NO:53) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGSLHR(SEQ ID NO:59) > <![CDATA GFTFTSFGMS(SEQ ID NO:53) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGSLSD(SEQ ID NO:60) > GFTFRDFGMS(SEQ ID NO:54) SISGSGSDTL(SEQ ID NO:6) GGSLSR(SEQ ID NO:7) GFTFRDFGMS(SEQ ID NO:54) SISGSGSDTL(SEQ ID NO:6) GGGLSR(SEQ ID NO:55) <![CDATA GFTFRDFGMS(SEQ ID NO:54) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGSLDR(SEQ ID NO:56) > <![CDATA GFTFRDFGMS(SEQ ID NO:54) > <![CDATA SISGSGSDTL(SEQ ID NO:6) > <![CDATA GGSLER(SEQ ID NO:57) > GFTFRDFGMS(SEQ ID NO:54) SISGSGSDTL(SEQ ID NO:6) GGSLGR(SEQ ID NO:58), GFTFRDFGMS(SEQ ID NO:54) SISGSGSDTL(SEQ ID NO:6) GGSLHR(SEQ ID NO:59) GFTFRDFGMS(SEQ ID NO:54) SISGSGSDTL(SEQ ID NO:6) GGSLSD(SEQ ID NO:60)

[0036] One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLER (SEQ ID NO:57).

[0037] Another particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLSR (SEQ ID NO:7).

[0038] According to a non-limiting aspect of the present invention, when CDR1 in the serum albumin binder of the present invention is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa).

[0039] Another particularly preferred combination of CDRs is GFTFRDFGMS (SEQ ID NO:54), SISGSGSDTL (SEQ ID NO:6), and GGSLSR (SEQ ID NO:7). Another preferred combination of CDRs is GFTFRDFGMS (SEQ ID NO:54), SISGSGSDTL (SEQ ID NO:6), and GGSLSR (SEQ ID NO:57).

[0040] According to another aspect of the present invention, in the albumin binders described herein, CDR1 is GFTFSSFGMS (SEQ ID NO: 120), CDR2 is SISGSGSDTL (SEQ ID NO: 6), CDR3 is GGSLSR (SEQ ID NO: 7), and the amino acid residue at position 104 is G or T. In these albumin binders: (i) the amino acid residue at position 16 is G or N and preferably N; (ii) the amino acid residue at position 45 is P or L, and preferably is L; (iii) the amino acid residues at positions 74-76 form an SKN or AKT motif, and preferably form an AKT motif; and (iv) the amino acid residue at position 89 is L, A or T, and preferably is L. More preferably, in these albumin binders of the present invention: (i) the amino acid residue at position 16 is N; (ii) the amino acid residue at position 45 is L; (iii) the amino acid residues at positions 74-76 form an AKT motif, and (iv) the amino acid residue at position 89 is L, A or T, and preferably is L.

[0041] As further described herein, when the serum albumin binder of the present invention is present in a compound or polypeptide of the present invention (as described herein) and forms and / or is present at the C-terminal end of said compound or polypeptide of the present invention, then the serum albumin binder of the present invention (and, by extension, the compound or polypeptide of the present invention) preferably has a C-terminal extension X(n) as further described herein. SEQ ID NOs: 61 to 102 give some non-limiting examples of the albumin binders of the present invention having a C-terminal extension (in this case, a C-terminal alanine).

[0042] When the serum albumin binder of the present invention forms and / or is present at the N-terminal end of a compound or polypeptide of the present invention (and, by extension, the compound or polypeptide of the present invention), then the serum albumin binder of the present invention preferably has a D at position 1 (i.e., an E1D mutation relative to the sequences of SEQ ID NOs: 8 to 49).

[0043] The amino acid sequences of the present invention preferably bind (human) serum albumin with an affinity better than 100 nM, preferably better than 50 nM. For example, the albumin binders of the present invention can have an affinity for (human) serum albumin that is of the same order of magnitude as the affinity of the human serum albumin of SEQ ID NO: 1 and / or SEQ ID NO: 50. For example, refer to the kinetic data given in Example 1.

[0044] Furthermore, the albumin binders provided by the present invention and the compounds and polypeptides of the present invention comprising them (as further described herein) preferably have a half-life (defined as t1 / 2β) in humans of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, and for example about 1 day, 2 days, 1 week, 2 weeks and up to the half-life of serum albumin in humans (estimated to be about 19 days), although the latter may be less critical. For example, the albumin binders of the present invention may have a half-life comparable (and preferably about the same) to that of SEQ ID NO:1 and / or SEQ ID NO:50 in humans. Also, the compounds or polypeptides of the present invention comprising the albumin binders of the present invention may have a half-life comparable (and preferably about the same) to that of the same compounds or polypeptides comprising SEQ ID NO:1 and / or SEQ ID NO:50 (rather than the albumin binders of the present invention) in humans.

[0045] In a first aspect, the present invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds serum albumin and in particular a nanobody that binds serum albumin), wherein:

[0046] - The amino acid residue at position 5 (according to Kabat) is V;

[0047] - The amino acid residue at position 11 (according to Kabat) is V;

[0048] - The amino acid residues at positions 74-76 are the motif SKN;

[0049] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, may specifically be A);

[0050] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0051] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0052] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0053] The amino acid sequence optionally contains:

[0054] - At least one amino acid residue selected from 29A, 29H, 30T, and / or 31D (such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); and / or

[0055] - At least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T (such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G, and / or S104T),

[0056] and the amino acid sequence has:

[0057] - A degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (where any possible C-terminal extension and the L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0058] - No more than 7, preferably no more than 5, such as only 3, 2, or 1 "amino acid differences" (as defined herein and not considering any possible C-terminal extension and not considering the L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0059] According to one specific but non-limiting aspect, the serum albumin binder of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific but non-limiting aspect, the serum albumin binder of the present invention is the amino acid sequence as described herein, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific but non-limiting aspect, the serum albumin binder of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific but non-limiting aspect, the serum albumin binder of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific but non-limiting aspect, the serum albumin binder of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific but non-limiting aspect, the serum albumin binder of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T, and the amino acid residue at position 101 (according to Kabat) is D.

[0060] As mentioned, some preferred amino acid residues / mutations that can be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. And these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6) and GGSLER (SEQ ID NO:57).

[0061] Regarding this first aspect and any other (specific and / or preferred) aspect of the present invention, it should be noted that when it is said that the amino acid sequence (i) of the present invention has a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and the L5V and L11V mutations are not considered when determining the degree of sequence identity) and / or (ii) has no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and without considering any possible C-terminal extension and without considering the L5V and L11V mutations) with the sequence of SEQ ID NO:1, this also includes sequences that do not have amino acid differences from the sequence of SEQ ID NO:1 other than: (i) the L5V and L11V mutations; (ii) any possible C-terminal extension; and (iii) any specific amino acid residue / mutation and / or CDR (i.e., those that need to be present according to this aspect) mentioned in connection with said aspect. It should also be noted that the amino acid sequence of the present invention according to any aspect described herein does not have amino acid differences from SEQ ID NO:1 other than: (i) the L5V and L11V mutations; (ii) any possible C-terminal extension; and (iii) any specific amino acid residue / mutation and / or CDR (i.e., those that need to be present according to this aspect) mentioned in connection with said aspect. As mentioned herein, this applies, mutatis mutandis, to sequences defined herein in terms of their degree of sequence identity and / or the number of amino acid differences with SEQ ID NO:50 (instead of SEQ ID NO:1).

[0062] Moreover, when the amino acid sequence of the invention according to any aspect of the invention has one or more amino acid differences from the sequence of SEQ ID NO:1 (except for the L5V and L11V mutations and the specific amino acid residues / mutations that may be present and / or required to be present according to this aspect, as described herein), then some specific, but non-limiting, examples of such mutations / amino acid differences that may be present (i.e., relative to the sequence of SEQ ID NO:1) are, for example, E1D (i.e., when the serum albumin binder is at the N-terminal end of the polypeptide of the invention) and, for example, one or more suitable "humanized" substitutions (suitable combinations thereof); for example, reference is made to WO 09 / 138519 (or the prior art cited in WO 09 / 138519) and WO 08 / 020079 (or the 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 humanized substitutions). As mentioned herein, this applies, mutatis mutandis, to the sequences defined herein with reference to their degree of sequence identity and / or the number of amino acid differences from the sequence of SEQ ID NO:50 (instead of SEQ ID NO:1).

[0063] In another aspect, the invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds serum albumin and in particular a nanobody that binds serum albumin), wherein:

[0064] - the amino acid residue at position 5 (according to Kabat) is V;

[0065] - the amino acid residue at position 11 (according to Kabat) is V;

[0066] - the amino acid residues at positions 74 - 76 are the motif SKN;

[0067] - the amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, may specifically be A);

[0068] - the amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0069] - the amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0070] - the amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0071] The amino acid sequence:

[0072] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54); and

[0073] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0074] - CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60);

[0075] and the amino acid sequence has:

[0076] - a sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0077] - no more than 7, preferably no more than 5, such as only 3, 2, or 1 "amino acid differences" with the sequence of SEQ ID NO:1 (as defined herein, and any possible C-terminal extension is not considered and L5V and L11V mutations are not considered).

[0078] According to a specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T and the amino acid residue at position 101 (according to Kabat) is D.

[0079] As mentioned, a particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6) and GGSLER (SEQ ID NO:57).

[0080] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, 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, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49,or one of the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102. Each of these amino acid sequences of the present invention forms a further aspect of the present invention.,

[0081] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 or SEQ ID NO:144. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0082] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167 or SEQ ID NO:168. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0083] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183 or SEQ ID NO:184. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0084] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207 and SEQ ID NO:208. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0085] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243 and SEQ ID NO:244. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0086] In a specific, but non-limiting, aspect, the present invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds serum albumin and in particular a nanobody that binds serum albumin), wherein:

[0087] - the amino acid residue at position 5 (according to Kabat) is V;

[0088] - the amino acid residue at position 11 (according to Kabat) is V;

[0089] - the amino acid residues at positions 74 - 76 are the motif SKN;

[0090] - the amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, may specifically be A);

[0091] - the amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0092] - the amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0093] - the amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0094] wherein the amino acid sequence:

[0095] - CDR1 is the amino acid sequence GFTFRSFGMS (SEQ ID NO:5); and

[0096] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0097] - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7);

[0098] and the amino acid sequence has:

[0099] - a sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0100] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" from the sequence of SEQ ID NO:1 (as defined herein, without considering any possible C-terminal extension and without considering the L5V and L11V mutations).

[0101] According to a specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T.

[0102] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, or one of the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0103] In another aspect, the present invention relates to an amino acid sequence which is the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:61.

[0104] In another aspect, the present invention relates to an amino acid sequence which is the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:62.

[0105] In another aspect, the present invention relates to an amino acid sequence which is the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:63.

[0106] In another aspect, the present invention relates to an amino acid sequence which is the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:64.

[0107] In another aspect, the present invention relates to an amino acid sequence which is the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:65.

[0108] In another aspect, the present invention relates to an amino acid sequence which is the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:66.

[0109] In another specific, but non-limiting, aspect, the present invention relates to an amino acid sequence (said amino acid sequence being an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0110] - The amino acid residue at position 5 (according to Kabat) is V;

[0111] - The amino acid residue at position 11 (according to Kabat) is V;

[0112] - The amino acid residues at positions 74 - 76 are the motif SKN;

[0113] - The amino acid residue at position 89 (according to Kabat) is selected from L, T or V;

[0114] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0115] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0116] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0117] wherein the amino acid sequence:

[0118] - CDR1 is the amino acid sequence GFTFRSFGMS (SEQ ID NO:5); and

[0119] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0120] - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7);

[0121] and the amino acid sequence has:

[0122] - A sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0123] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and without considering any possible C-terminal extension and without considering L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0124] According to a specific, but non-limiting, aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific, but non-limiting, aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific, but non-limiting, aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific, but non-limiting, aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T.

[0125] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, or one of the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0126] In another specific, but non-limiting, aspect, the present invention relates to an amino acid sequence (the amino acid sequence is an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0127] - the amino acid residue at position 5 (according to Kabat) is V;

[0128] - the amino acid residue at position 11 (according to Kabat) is V;

[0129] - the amino acid residues at positions 74-76 are the motif SKN;

[0130] - the amino acid residue at position 89 (according to Kabat) is selected from A, N or S;

[0131] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0132] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0133] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0134] wherein the amino acid sequence:

[0135] - CDR1 is the amino acid sequence GFTFRSFGMS (SEQ ID NO:5); and

[0136] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0137] - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7);

[0138] and the amino acid sequence has:

[0139] - A sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0140] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" with the sequence of SEQ ID NO:1 (as defined herein, and any possible C-terminal extension is not considered and L5V and L11V mutations are not considered).

[0141] According to a specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T.

[0142] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, or one of the amino acid sequences of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0143] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0144] - the amino acid residue at position 5 (according to Kabat) is V;

[0145] - the amino acid residue at position 11 (according to Kabat) is V;

[0146] - the amino acid residues at positions 74-76 are the motif SKN;

[0147] - the amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, may specifically be A);

[0148] - the amino acid residue at position 104 (according to Kabat) is selected from A, G or T;

[0149] - the amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0150] - the amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0151] wherein the amino acid sequence:

[0152] - CDR1 is the amino acid sequence GFTFRSFGMS (SEQ ID NO:5); and

[0153] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0154] - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7);

[0155] and the amino acid sequence has:

[0156] - a sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and the L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0157] - no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and without considering any possible C-terminal extension and without considering the L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0158] According to a specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is A.

[0159] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21, or one of the amino acid sequences of SEQ ID NO:72, SEQ ID NO:73 or SEQ ID NO:74. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0160] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence is an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0161] - the amino acid residue at position 5 (according to Kabat) is V;

[0162] - The amino acid residue at position 11 (according to Kabat) is V;

[0163] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, may specifically be A);

[0164] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0165] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0166] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0167] The amino acid sequence contains:

[0168] - At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T or S31D); and / or

[0169] - At least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H or 102D (such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H or R102D),

[0170] And the amino acid sequence has:

[0171] - A sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (where any possible C-terminal extension and L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0172] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" with the sequence of SEQ ID NO:1 (as defined herein, and not considering any possible C-terminal extension and not considering L5V and L11V mutations).

[0173] In the amino acid sequence of the invention described in the above paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN (but can also be, for example, AKT, as exemplified by the sequences of SEQ ID NOs: 124-126 and 130-132). According to a specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T, and the amino acid residue at position 101 (according to Kabat) is D.

[0174] As mentioned, some preferred amino acid residues / mutations that may be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. And these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs (wherein the CDRs contain 30T and 101E residues) is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLER (SEQ ID NO:57). Some preferred but non-limiting examples of such serum albumin binders are given in SEQ ID NOs: 169 - 184.

[0175] Another particularly preferred combination of CDRs (wherein the CDRs contain 30T residue but do not contain 101E residue) is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLSR (SEQ ID NO:7). When the amino acid sequences of the present invention contain these CDRs, the amino acid sequences may further contain G or N at position 16, P or L at position 45, SKN or AKT motif at positions 74 - 76, L, T or A at position 89, and preferably contain G at position 104 (and for others, these amino acid sequences of the present invention may be further defined as herein). Some specific but non-limiting examples of such amino acid sequences of the present invention are given as SEQ ID NOs: 121 - 144, and SEQ ID NOs: 145 - 168.

[0176] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence is an ISVD that binds serum albumin and in particular a nanobody that binds serum albumin), wherein:

[0177] - the amino acid residue at position 5 (according to Kabat) is V;

[0178] - the amino acid residue at position 11 (according to Kabat) is V;

[0179] - the amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, it may specifically be A);

[0180] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0181] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0182] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0183] The amino acid sequence contains:

[0184] - At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T or S31D);

[0185] And the amino acid sequence contains:

[0186] - At least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H or 102D (such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H or R102D),

[0187] And the amino acid sequence has:

[0188] - A sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (where any possible C-terminal extension and the L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0189] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and not considering any possible C-terminal extension and not considering the L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0190] In the amino acid sequence of the invention described in the above paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN. According to a specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T, and the amino acid residue at position 101 (according to Kabat) is D.

[0191] As mentioned, some preferred amino acid residues / mutations that may be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. And these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6) and GGSLER (SEQ ID NO:57).

[0192] In another specific, but non-limiting, aspect, the present invention relates to an amino acid sequence (said amino acid sequence being an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0193] - The amino acid residue at position 5 (according to Kabat) is V;

[0194] - The amino acid residue at position 11 (according to Kabat) is V;

[0195] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, may specifically be A);

[0196] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, may specifically be T);

[0197] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0198] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0199] wherein the amino acid sequence:

[0200] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54); and

[0201] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0202] - CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60);

[0203] such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR2 is not GGSLSR (SEQ ID NO:7) and vice versa, and the amino acid sequence has:

[0204] - a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (where any possible C-terminal extension and the L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0205] - no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and not considering any possible C-terminal extension and not considering the L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0206] In the amino acid sequence of the invention described in the above paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN (but can also be, for example, AKT, as exemplified by the sequences of SEQ ID NOs: 124-126 and 130-132). According to a specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T and the amino acid residue at position 101 (according to Kabat) is D.

[0207] As mentioned, a particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6) and GGSLER (SEQ ID NO: 57).

[0208] Another particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLSR (SEQ ID NO:7). When the amino acid sequences of the present invention contain these CDRs, the amino acid sequences may further contain G or N at position 16, P or L at position 45, the SKN or AKT motif at positions 74 - 76, L, T or A at position 89, and preferably contain G at position 104 (and for others, these amino acid sequences of the present invention may be further defined as herein). Some specific but non - limiting examples of such amino acid sequences of the present invention are given as SEQ ID NO:121 - 132.

[0209] In another specific but non - limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence is an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0210] - The amino acid residue at position 5 (according to Kabat) is V;

[0211] - The amino acid residue at position 11 (according to Kabat) is V;

[0212] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, it may specifically be A);

[0213] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, it may specifically be T);

[0214] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0215] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0216] wherein the amino acid sequence:

[0217] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54); and

[0218] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0219] - CDR3 is an amino acid sequence selected from the following amino acid sequences: GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60);

[0220] The amino acid sequence has:

[0221] - a sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and the L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0222] - no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and without considering any possible C-terminal extension and without considering the L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0223] In the amino acid sequence of the invention described in the above paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN. According to one specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T and the amino acid residue at position 101 (according to Kabat) is D.

[0224] As mentioned, a particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6) and GGSLER (SEQ ID NO:57).

[0225] In another specific, but non-limiting, aspect, the invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds serum albumin and in particular a nanobody that binds serum albumin), wherein:

[0226] - the amino acid residue at position 5 (according to Kabat) is V;

[0227] - the amino acid residue at position 11 (according to Kabat) is V;

[0228] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, it may specifically be A);

[0229] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and may specifically be S or selected from A, G or T (and when selected from A, G and T, it may specifically be T);

[0230] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0231] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0232] The amino acid sequence contains:

[0233] - At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T or S31D); and / or

[0234] - At least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H or 102D (i.e., such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H or R102D,

[0235] and in the amino acid sequence:

[0236] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54); and

[0237] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0238] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60);

[0239] and the amino acid sequence has:

[0240] - a sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and the L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0241] - no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" (as defined herein and without considering any possible C-terminal extension and without considering the L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0242] In the amino acid sequence of the invention described in the above paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN (but can also be, for example, AKT, as exemplified by the sequences of SEQ ID NOs: 124-126 and 130-132). According to a specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific but non-limiting aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T and the amino acid residue at position 101 (according to Kabat) is D. It should also be noted that according to this aspect, the amino acid sequence of the invention should contain at least one mutation as described herein at positions 29, 30, 31, 99, 101 or 102 (relative to the sequence of SEQ ID NO: 1), such that in the amino acid sequence of the invention according to this aspect, if CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 cannot be GGSLSR (SEQ ID NO: 7), and vice versa.

[0243] As mentioned, some preferred amino acid residues / mutations that can be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. And these preferred mutations are preferably combined with T at position 89 (i.e., L89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLER (SEQ ID NO:57). Another particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLSR (SEQ ID NO:7). When the amino acid sequence of the present invention contains these CDRs, the amino acid sequence may further contain G or N at position 16, P or L at position 45, the SKN or AKT motif at positions 74 - 76, L, T or A at position 89, and preferably contains G at position 104 (and for others, these amino acid sequences of the present invention may be further defined as herein). Some specific but non-limiting examples of such amino acid sequences of the present invention are given as SEQ ID NO:121 - 132.

[0244] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of 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, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49, or one of the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0245] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0246] - the amino acid residue at position 5 (according to Kabat) is V;

[0247] - the amino acid residue at position 11 (according to Kabat) is V;

[0248] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and can specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, it can specifically be A);

[0249] - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S or T, and can specifically be S or selected from A, G or T (and when selected from A, G and T, it can specifically be T);

[0250] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0251] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0252] The amino acid sequence contains:

[0253] - At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T or S31D);

[0254] And the amino acid sequence contains:

[0255] - At least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H or 102D (i.e., such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H or R102D,

[0256] And in the amino acid sequence:

[0257] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54); and

[0258] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0259] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60);

[0260] and the amino acid sequence has:

[0261] - A sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (where any possible C-terminal extension and L5V and L11V mutations are not considered when determining the degree of sequence identity); and / or

[0262] - No more than 7, preferably no more than 5, such as only 3, 2, or 1 "amino acid differences" (as defined herein and without considering any possible C-terminal extension and without considering L5V and L11V mutations) with the sequence of SEQ ID NO:1.

[0263] In the amino acid sequence of the invention described in the above paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN. According to a specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T or V. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S or N. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. According to another specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. According to a more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. According to an even more specific, but non-limiting, aspect, the amino acid sequence of the invention is the amino acid sequence described in the above paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T and the amino acid residue at position 101 (according to Kabat) is D.

[0264] As mentioned, some preferred amino acid residues / mutations that can be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. And these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6) and GGSLER (SEQ ID NO:57).

[0265] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of 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:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:48, or one of the amino acid sequences of SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0266] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0267] - the amino acid residue at position 5 (according to Kabat) is V;

[0268] - the amino acid residue at position 11 (according to Kabat) is V;

[0269] - the amino acid residue at position 16 (according to Kabat) is G or N;

[0270] - the amino acid residue at position 45 (according to Kabat) is P or L;

[0271] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be A, L or T;

[0272] - The amino acid residue at position 104 (according to Kabat) is G;

[0273] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0274] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0275] The amino acid sequence contains:

[0276] - CDR1 which is GFTFTSFGMS (SEQ ID NO:53), CDR2 which is SISGSGSDTL (SEQ ID NO:6) and CDR3 which is GGSLSR (SEQ ID NO:7);

[0277] And the amino acid sequence has:

[0278] - A sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1 (wherein any possible C-terminal extension and L5V, L11V, R30T and S104G mutations are not considered when determining the degree of sequence identity); and / or

[0279] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" with the sequence of SEQ ID NO:1 (as defined herein, and any possible C-terminal extension is not considered and L5V, L11V, R30T and S104G mutations are not considered).

[0280] In the amino acid sequence of the present invention described in the above paragraph, the amino acid residues at positions 74 - 76 are preferably the motif SKN or the motif AKT.

[0281] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence is an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), wherein:

[0282] - The amino acid residue at position 5 (according to Kabat) is V;

[0283] - The amino acid residue at position 11 (according to Kabat) is V;

[0284] - The amino acid residue at position 16 (according to Kabat) is G or N;

[0285] - The amino acid residue at position 45 (according to Kabat) is P or L;

[0286] - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and may specifically be A, L or T

[0287] - The amino acid residue at position 104 (according to Kabat) is G;

[0288] - The amino acid residue at position 110 (according to Kabat) is selected from T, K or Q;

[0289] - The amino acid residue at position 112 (according to Kabat) is selected from S, K or Q;

[0290] The amino acid sequence contains:

[0291] - CDR1 which is GFTFTSFGMS (SEQ ID NO:53), CDR2 which is SISGSGSDTL (SEQ ID NO:6), and CDR3 which is GGSLSR (SEQ ID NO:7);

[0292] And the amino acid sequence has:

[0293] - A sequence identity degree of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:50 (where any possible C-terminal extension and L5V, L11V, R30T and S104G mutations are not considered when determining the degree of sequence identity); and / or

[0294] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" with the sequence of SEQ ID NO:50 (as defined herein, and any possible C-terminal extension is not considered and L5V, L11V, R30T and S104G mutations are not considered).

[0295] In the amino acid sequence of the present invention described in the above paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN or the motif AKT.

[0296] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 or SEQ ID NO:144. Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0297] In another specific, but non-limiting, aspect, the present invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds to serum albumin and in particular a nanobody that binds to serum albumin), the amino acid sequence having:

[0298] - a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO:1; and / or

[0299] - no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" from the sequence of SEQ ID NO:1;

[0300] Among them, in the amino acid sequence, the amino acid residue at position 89 (according to Kabat) is selected from A, N, and S. And, preferably, in an amino acid sequence according to this aspect: (i) the amino acid residue at position 5 (according to Kabat) is L or V (and more preferably V); (ii) the amino acid residue at position 11 (according to Kabat) is L or V (and more preferably V); (iii) the amino acid residue at position 104 (according to Kabat) is selected from A, G, S, or T, and can specifically be S or selected from A, G, or T (and when selected from A, G, and T, it can specifically be T); (iv) the amino acid residue at position 110 (according to Kabat) is selected from T, K, or Q; and (v) the amino acid residue at position 112 (according to Kabat) is selected from S, K, or Q. And, an amino acid sequence according to this aspect optionally contains: (i) at least one amino acid residue selected from 29A, 29H, 30T, and / or 31D (such that, compared with the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); and / or (ii) at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T (such that, compared with the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G, and / or S104T). Most preferably, in an amino acid sequence according to this aspect: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54); and (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60).In a specific aspect, CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7). Also, the amino acid sequence according to this aspect in which the amino acid residue at position 89 is A (or alternatively T) is particularly preferred.

[0301] As mentioned, some preferred amino acid residues / mutations that may be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. Also, these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLER (SEQ ID NO:57).

[0302] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49, or one of the amino acid sequences of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102 (and in particular one of the amino acid sequences of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49, or one of the amino acid sequences of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102). Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0303] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being an ISVD that binds serum albumin and in particular a nanobody that binds serum albumin), the amino acid sequence having:

[0304] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the sequence of SEQ ID NO:1; and / or

[0305] - No more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" from the sequence of SEQ ID NO:1;

[0306] Among them, in the amino acid sequence, the amino acid residue at position 104 (according to Kabat) is selected from G, T or A. And, preferably, in an amino acid sequence according to this aspect: (i) the amino acid residue at position 5 (according to Kabat) is L or V (and more preferably V); (ii) the amino acid residue at position 11 (according to Kabat) is L or V (and more preferably V); (iii) the amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T or V, and can specifically be selected from L, V or T or from A, S or N (and when selected from A, S or N, it can specifically be A); (iv) the amino acid residue at position 110 (according to Kabat) is selected from T, K or Q; and (v) the amino acid residue at position 112 (according to Kabat) is selected from S, K or Q. And, in an amino acid sequence according to this aspect, the amino acid sequence optionally contains: (i) at least one amino acid residue selected from 29A, 29H, 30T and / or 31D (such that, compared with the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T or S31D); and / or (ii) at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G and / or 104T (such that, compared with the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T). Specifically, compared with SEQ ID NO:1, they can contain the S101D mutation. Most preferably, in an amino acid sequence according to this aspect: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54); and (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60).In one specific aspect, CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7). Further, the amino acid sequence according to this aspect in which the amino acid residue at position 89 is T (or alternatively A) is particularly preferred.

[0307] As mentioned, some preferred amino acid residues / mutations that may be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. Further, these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLER (SEQ ID NO:57).

[0308] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, 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, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49, or one of the amino acid sequences of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102 (and in particular one of the amino acid sequences of SEQ ID NO:21, 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, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47, or one of the amino acid sequences of SEQ ID NO:74, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 or SEQ ID NO:100). Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0309] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (said amino acid sequence being an ISVD that binds serum albumin and in particular a nanobody that binds serum albumin), said amino acid sequence having:

[0310] - a degree of sequence identity of at least 85%, preferably at least 90%, more preferably at least 95% with the sequence of SEQ ID NO: 1; and / or

[0311] - no more than 7, preferably no more than 5, such as only 3, 2 or 1 "amino acid differences" with the sequence of SEQ ID NO: 1;

[0312] Among them, in the amino acid sequence, the amino acid residue at position 89 (according to Kabat) is selected from A, N, and S, and preferably is A, and the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A, and preferably is T. And, preferably, in an amino acid sequence according to this aspect: (i) the amino acid residue at position 5 (according to Kabat) is L or V (and more preferably is V); (ii) the amino acid residue at position 11 (according to Kabat) is L or V (and more preferably is V); (iii) the amino acid residue at position 110 (according to Kabat) is selected from T, K, or Q; and (iv) the amino acid residue at position 112 (according to Kabat) is selected from S, K, or Q. And, in an amino acid sequence according to this aspect, the amino acid sequence optionally contains: (i) at least one amino acid residue selected from 29A, 29H, 30T, and / or 31D (such that, compared with the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); and / or (ii) at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T (such that, compared with the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G, and / or S104T). Specifically, compared with SEQ ID NO:1, they may contain the S101D mutation. Most preferably, in an amino acid sequence according to this aspect: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54); and (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60).In a specific aspect, CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7). Further, amino acid sequences according to this aspect in which the amino acid residue at position 89 is A or the amino acid residue at position 89 is T are particularly preferred.

[0313] As mentioned, some preferred amino acid residues / mutations that may be present are: 30T (i.e., R30T relative to SEQ ID NO:1) and 101E (i.e., S101E relative to SEQ ID NO:1), and preferably both of them. Further, these preferred mutations are preferably combined with T at position 89 (i.e., V89T relative to SEQ ID NO:1) or alternatively A at position 89 (i.e., V89A relative to SEQ ID NO:1). A particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO:53), SISGSGSDTL (SEQ ID NO:6), and GGSLER (SEQ ID NO:57).

[0314] In another aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49, or one of the amino acid sequences of SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 or SEQ ID NO:102 (and in particular one of the amino acid sequences of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47, or one of the amino acid sequences of SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 or SEQ ID NO:100). Each of these amino acid sequences of the present invention forms another aspect of the present invention.

[0315] In another aspect, the present invention relates to an immunoglobulin single variable domain that is capable of binding (human) serum albumin, wherein:

[0316] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54); and

[0317] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0318] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60);

[0319] such that, when the CDR1 is GFTFRSFGMS (SEQ ID NO:5), the CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa); the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences in the CDRs).

[0320] The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the invention).

[0321] In the serum albumin binders according to this aspect, position 89 is preferably T. And, position 104 is preferably T (especially when the CDR3 is GGSLDR (SEQ ID NO:56)). Most preferably, position 89 is T and position 104 is T (again, especially when the CDR3 is GGSLDR (SEQ ID NO:56)). And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0322] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0323] - The CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54); and

[0324] - The CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0325] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60);

[0326] such that, when CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa); the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences in the CDRs).

[0327] The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the invention).

[0328] In the serum albumin binders according to this aspect: position 89 is preferably T. And, position 104 is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO:56)). Most preferably, position 89 is T and position 104 is T (again, especially when CDR3 is GGSLDR (SEQ ID NO:56)). And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0329] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0330] - The CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); and

[0331] - The CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0332] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56);

[0333] such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa); the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences in the CDRs).

[0334] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the invention).

[0335] In serum albumin binders according to this aspect: position 89 is preferably T. And, position 104 is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO:56)). Most preferably, position 89 is T and position 104 is T (again, especially when CDR3 is GGSLDR (SEQ ID NO:56)). And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0336] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0337] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); and

[0338] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0339] - CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56);

[0340] such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa); the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences in the CDRs).

[0341] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0342] In serum albumin binders according to this aspect: position 89 is preferably T. And, position 104 is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO:56)). Most preferably, position 89 is T and position 104 is T (again, especially when CDR3 is GGSLDR (SEQ ID NO:56)). And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0343] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0344] - CDR1 is the amino acid sequence GFTHRSFGMS (SEQ ID NO:52); and

[0345] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0346] - CDR3 is the amino acid sequence GGSLDR (SEQ ID NO:56);

[0347] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences in the CDRs).

[0348] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0349] In serum albumin binders according to this aspect: position 89 is preferably T. And, position 104 is preferably T. Most preferably, position 89 is T and position 104 is T. And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0350] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0351] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0352] - CDR1 is the amino acid sequence GFTHRSFGMS (SEQ ID NO:52); and

[0353] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0354] - CDR3 is the amino acid sequence GGSLDR (SEQ ID NO:56);

[0355] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences in the CDRs).

[0356] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0357] In serum albumin binders according to this aspect: position 89 is preferably T. And, position 104 is preferably T. Most preferably, position 89 is T and position 104 is T. And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0358] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0359] - CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54); and

[0360] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0361] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60);

[0362] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences in the CDR).

[0363] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptide of the present invention).

[0364] In the serum albumin binder according to this aspect: position 89 is preferably T. And, position 104 is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO:56)). Most preferably, position 89 is T and position 104 is T (again, especially when CDR3 is GGSLDR (SEQ ID NO:56)). And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0365] In another aspect, the present invention relates to an amino acid sequence that is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0366] - The CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54); and

[0367] - The CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0368] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60);

[0369] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences in the CDR).

[0370] The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptide of the present invention).

[0371] In the serum albumin binders according to this aspect: position 89 is preferably T. And, position 104 is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO:56)). Most preferably, position 89 is T and position 104 is T (again, especially when CDR3 is GGSLDR (SEQ ID NO:56)). And, preferably, position 5 is V and / or position 11 is V, and preferably position 5 is V and position 11 is V.

[0372] In another aspect, the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0373] - position 5 is V; and

[0374] - position 11 is V; and

[0375] - CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); and

[0376] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0377] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and preferably is GGSLSR (SEQ ID NO:7).

[0378] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences at positions 5 and 11 and disregarding amino acid differences in the CDRs).

[0379] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the invention).

[0380] In the serum albumin binders according to this aspect, position 89 is preferably L, A, or T, and position 104 is preferably G. Position 16 is also preferably G or N; position 45 is preferably P or L, and positions 74 - 76 are preferably the SKN or AKT motif.

[0381] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0382] - Position 5 is V; and

[0383] - Position 11 is V; and

[0384] - CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); and

[0385] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0386] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and preferably is GGSLSR (SEQ ID NO:7).

[0387] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences at positions 5 and 11 and disregarding amino acid differences in the CDRs).

[0388] The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of the polypeptides of the present invention).

[0389] In the serum albumin binders according to this aspect, position 89 is preferably L, A, or T, and position 104 is preferably G. Position 16 is also preferably G or N; position 45 is preferably P or L, and positions 74 - 76 are preferably an SKN or AKT motif.

[0390] In another aspect, the present invention relates to an amino acid sequence that is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0391] - Position 5 is V; and

[0392] - Position 11 is V; and

[0393] - CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); and

[0394] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0395] - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7).

[0396] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences at positions 5 and 11 and disregarding amino acid differences in the CDRs).

[0397] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (as such or as part of a polypeptide of the invention).

[0398] In serum albumin binders according to this aspect, position 89 is preferably L, A or T, and position 104 is preferably G. Position 16 is also preferably G or N; position 45 is preferably P or L, and positions 74-76 are preferably an SKN or AKT motif.

[0399] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0400] - position 5 is V; and

[0401] - position 11 is V; and

[0402] - CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); and

[0403] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0404] - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7);

[0405] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences at positions 5 and 11 and disregarding amino acid differences in the CDRs).

[0406] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (as such or as part of a polypeptide of the invention).

[0407] In serum albumin binders according to this aspect, position 89 is preferably L, A or T, and position 104 is preferably G. Position 16 is also preferably G or N; position 45 is preferably P or L, and positions 74-76 are preferably an SKN or AKT motif.

[0408] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0409] - Position 5 is V; and

[0410] - Position 11 is V; and

[0411] - CDR1 is the amino acid sequence GFTFRDFGMS (SEQ ID NO:54); and

[0412] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0413] - CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and preferably is GGSLSR (SEQ ID NO:7) or GGSLER (SEQ ID NO:57);

[0414] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences at positions 5 and 11 and disregarding amino acid differences in the CDRs).

[0415] The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the present invention).

[0416] In the serum albumin binders according to this aspect, position 89 is preferably L, A or T, and position 104 is preferably G. Position 16 is also preferably G or N; position 45 is preferably P or L, and positions 74 - 76 are preferably the SKN or AKT motif.

[0417] In another aspect, the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0418] - Position 5 is V; and

[0419] - Position 11 is V; and

[0420] - CDR1 is the amino acid sequence GFTFRDFGMS (SEQ ID NO:54); and

[0421] - The CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0422] - The CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and preferably is GGSLSR (SEQ ID NO:7) or GGSLER (SEQ ID NO:57);

[0423] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences at positions 5 and 11 and disregarding amino acid differences in the CDRs).

[0424] The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the present invention).

[0425] In the serum albumin binders according to this aspect, position 89 is preferably L, A or T, and position 104 is preferably G. Position 16 is also preferably G or N; position 45 is preferably P or L, and positions 74 - 76 are preferably the SKN or AKT motif.

[0426] Some preferred but non-limiting examples of the amino acid sequences of the present invention (wherein CDR1 is SEQ ID NO:54) are the amino acid sequences of SEQ ID NO:26, 46, 47, 79, 99, 100 and 209 - 244, and especially those of SEQ ID NO:209 - 244.

[0427] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein the amino acid residue at position 5 is V and the amino acid residue at position 11 is V, and the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1. The amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54), and preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and preferably selected from GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56). In a specific but non-limiting aspect of these amino acid sequences (having V at position 11 or L at position 11) according to the invention, when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa).

[0428] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptide of the invention).

[0429] In another aspect, the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein the amino acid residue at position 5 is V and the amino acid residue at position 11 is V, and the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50. The amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54), and is preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and is preferably selected from GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56). In a specific but non-limiting aspect of these amino acid sequences (having V at position 11 or L at position 11) according to the present invention, when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa). It should also be noted that the albumin binder according to this aspect may contain S at position 30, as present in CDR1 of SEQ ID NO:50 (according to Abm).

[0430] The albumin binders according to this aspect are preferably further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the present invention).

[0431] In another aspect, the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin and which contains at least one of the following amino acid residues or a suitable combination of two or more of the following amino acid residues (i.e., in a suitable combination): 29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G and / or 104T (such that, compared to the sequence of SEQ ID NO:1, it contains at least one amino acid mutation selected from F29A, F29H, R30T, S31D, S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T); said amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences compared to the sequence of SEQ ID NO:1.

[0432] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the present invention).

[0433] In the amino acid sequence according to this aspect, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Moreover, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54), and is preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and is preferably selected from GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56), such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa).

[0434] In another aspect, the present invention relates to an amino acid sequence that is an immunoglobulin single variable domain capable of binding (human) serum albumin and contains at least one of the following amino acid residues or a suitable combination of two or more of the following amino acid residues (i.e., in a suitable combination): 29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T (such that, relative to the sequence of SEQ ID NO:50, they contain at least one amino acid mutation selected from F29A, F29H, R30T, S31D, S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G, and / or S104T); the amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:50.

[0435] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0436] In the amino acid sequence according to this aspect, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Also, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54), and is preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and is preferably selected from GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56), such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa). It should also be noted that the albumin binder according to this aspect may contain an S at position 30, as present in CDR1 (according to Abm) of SEQ ID NO:50.

[0437] In another aspect, the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding to (human) serum albumin and which contains at least one or more of the following amino acid residues (in a suitable combination): 29H, 101D and 104T (i.e., such that, compared to the sequence of SEQ ID NO:1, they contain at least the amino acid mutations F29H, S101D and S104T); said amino acid sequence having (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences compared to the sequence of SEQ ID NO:1.

[0438] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptide of the present invention).

[0439] In the amino acid sequence according to this aspect, the amino acid residue at position 5 is V and the amino acid residue at position 11 is V. Also, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53) and GFTFRDFGMS (SEQ ID NO:54), and preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and preferably selected from GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56), such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa).

[0440] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin and which contains at least one or more of the following amino acid residues (in a suitable combination): 29H, 101D and 104T (such that, relative to the sequence of SEQ ID NO: 50, they contain at least the amino acid mutations F29H, S101D and S104T); said amino acid sequence having (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO: 50.

[0441] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the invention).

[0442] In the amino acid sequence according to this aspect, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Further, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54), and is preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTHRSFGMS (SEQ ID NO:52); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and is preferably selected from GGSLSR (SEQ ID NO:7) and GGSLDR (SEQ ID NO:56), such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa). It should also be noted that the albumin binder according to this aspect may contain S at position 30, as present in CDR1 (according to Abm) of SEQ ID NO:50.

[0443] Another specific, but non-limiting, aspect of the present invention relates to a serum albumin binder as defined herein, wherein: (i) CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7), and wherein (ii) the amino acid residue at position 5 is preferably V and / or the amino acid at position 11 is preferably V (and preferably position 5 is V and position 11 is V). The albumin binders according to this aspect preferably have no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1, and / or no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50. The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of a polypeptide of the present invention).

[0444] Another specific, but non-limiting, aspect of the present invention relates to a serum albumin binder as defined herein, wherein: (i) CDR1 is GFTFSSFGMS (SEQ ID NO:120), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7), and wherein (ii) the amino acid residue at position 5 is preferably V and / or the amino acid at position 11 is preferably V (and preferably position 5 is V and position 11 is V). The albumin binders according to this aspect preferably have no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1, and / or no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50. The albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of a polypeptide of the present invention). Some preferred, but non-limiting, examples of this group of serum albumin binders of the present invention (i.e., having V at positions 5 and 11 and based on CDR1 of SEQ ID NO:120) are represented by SEQ ID NOs: 185 - 208.

[0445] Specifically, in the serum albumin binders according to this aspect, position 16 can be G or N, and preferably is N; position 45 can be P or L, and preferably is L; and positions 74-76 are preferably the SKN or AKT motif, and preferably the AKT motif; position 89 can be L, A or T, and preferably is L; and position 104 is preferably G or T.

[0446] Again, some preferred but non-limiting examples of this group of serum albumin binders of the present invention (i.e., having V at positions 5 and 11 and based on CDR1 of SEQ ID NO: 120) are represented by SEQ ID NOs: 185-208.

[0447] Accordingly, another specific but non-limiting aspect of the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein: (i) CDR1 is GFTFSSFGMS (SEQ ID NO: 120), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7), and wherein:

[0448] - The amino acid residue at position 5 is V;

[0449] - The amino acid residue at position 11 is V;

[0450] - The amino acid residue at position 16 is G or N, and preferably is N;

[0451] - The amino acid residue at position 45 is P or L, and preferably is L;

[0452] - The amino acid residues at positions 74-76 form the SKN or AKT motif, and preferably form the AKT motif;

[0453] - The amino acid residue at position 89 is L, A or T, and preferably is L; and

[0454] - The amino acid residue at position 104 is G or T;

[0455] The amino acid sequence preferably has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO: 1 (disregarding the CDRs and the mutations at the positions specifically mentioned above).

[0456] An even more specific, but non-limiting, aspect of the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein: (i) CDR1 is GFTFSSFGMS (SEQ ID NO: 120), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7), and wherein:

[0457] - The amino acid residue at position 5 is V;

[0458] - The amino acid residue at position 11 is V;

[0459] - The amino acid residue at position 16 is N;

[0460] - The amino acid residue at position 45 is L;

[0461] - The amino acid residues at positions 74 - 76 form an AKT motif;

[0462] - The amino acid residue at position 89 is L, A or T, and preferably is L; and

[0463] - The amino acid residue at position 104 is G or T;

[0464] The amino acid sequence preferably has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO: 1 (disregarding the CDRs and the mutations at the positions specifically mentioned above).

[0465] Another specific, but non-limiting, aspect of the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein: (i) CDR1 is GFTFSSFGMS (SEQ ID NO: 120), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7), and wherein:

[0466] - The amino acid residue at position 5 is V;

[0467] - The amino acid residue at position 11 is V;

[0468] - The amino acid residue at position 16 is G or N, and preferably is N;

[0469] - The amino acid residue at position 45 is P or L, and preferably is L;

[0470] - The amino acid residues at positions 74 - 76 form an SKN or AKT motif, and preferably form an AKT motif;

[0471] - The amino acid residue at position 89 is L, A or T, and preferably is L; and

[0472] - The amino acid residue at position 104 is G or T;

[0473] The amino acid sequence preferably has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding the CDRs and mutations at the positions specifically mentioned above).

[0474] An even more specific but non - limiting aspect of the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein: (i) CDR1 is GFTFSSFGMS (SEQ ID NO:120), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7), and wherein:

[0475] - The amino acid residue at position 5 is V;

[0476] - The amino acid residue at position 11 is V;

[0477] - The amino acid residue at position 16 is N;

[0478] - The amino acid residue at position 45 is L;

[0479] - The amino acid residues at positions 74 - 76 form an AKT motif;

[0480] - The amino acid residue at position 89 is L, A or T, and preferably is L; and

[0481] - The amino acid residue at position 104 is G or T;

[0482] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding the CDRs and mutations at the positions specifically mentioned above).

[0483] In another aspect, the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0484] -CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5) and GFTFTSFGMS (SEQ ID NO:53); and

[0485] -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0486] -CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7) and GGSLER (SEQ ID NO:57);

[0487] such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa); the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences in the CDRs).

[0488] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptide of the invention).

[0489] In the serum albumin binders according to this aspect: position 89 is preferably T (or alternatively A).

[0490] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0491] -CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); and

[0492] -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0493] -CDR3 is the amino acid sequence GGSLER (SEQ ID NO:57);

[0494] the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding amino acid differences in the CDRs).

[0495] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0496] In a serum albumin binder according to this aspect: position 89 is preferably T (or alternatively A).

[0497] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0498] - CDR1 is an amino acid sequence selected from the amino acid sequences: GFTFRSFGMS (SEQ ID NO:5) and GFTFTSFGMS (SEQ ID NO:53); and

[0499] - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0500] - CDR3 is an amino acid sequence selected from the amino acid sequences: GGSLSR (SEQ ID NO:7) and GGSLER (SEQ ID NO:57);

[0501] such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa); the amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences in said CDRs).

[0502] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0503] In a serum albumin binder according to this aspect: position 89 is preferably T (or alternatively A).

[0504] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0505] - CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); and

[0506] -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and

[0507] -CDR3 is the amino acid sequence GGSLER (SEQ ID NO:57);

[0508] The amino acid sequence has no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding amino acid differences in the CDRs).

[0509] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (as such or as part of a polypeptide of the invention).

[0510] In serum albumin binders according to this aspect: position 89 is preferably T (or alternatively A).

[0511] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin and which contains at least one or more of the following amino acid residues (in a suitable combination): 30T and / or 101E (such that, compared to the sequence of SEQ ID NO:1, they contain at least the amino acid mutations R30T and / or S101E, and preferably both); the amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1. In the amino acid sequence according to this aspect, the amino acid residue at position 5 is V and the amino acid residue at position 11 is V. Also, position 89 is preferably T or A, most preferably T.

[0512] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (as such or as part of a polypeptide of the invention).

[0513] Also, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54), and preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTFTSFGMS (SEQ ID NO:53); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and preferably selected from GGSLSR (SEQ ID NO:7) and GGSLER (SEQ ID NO:57), such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa). Most preferably, CDR1 is GFTFTSFGMS (SEQ ID NO:53), CDR2 is SIGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLER (SEQ ID NO:57).

[0514] In another aspect, the present invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin and contains at least one or more of the following amino acid residues (in a suitable combination): 30T and / or 101E (such that, relative to the sequence of SEQ ID NO:50, they contain at least the amino acid mutations S30T and / or S101E, and preferably both); the amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1, or zero amino acid differences from the sequence of SEQ ID NO:50. In the amino acid sequence according to this aspect, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Also, position 89 is preferably T or A, most preferably T.

[0515] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of the polypeptides of the invention).

[0516] Also, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO:5), GFTARSFGMS (SEQ ID NO:51), GFTHRSFGMS (SEQ ID NO:52), GFTFTSFGMS (SEQ ID NO:53), and GFTFRDFGMS (SEQ ID NO:54), and is preferably selected from GFTFRSFGMS (SEQ ID NO:5) and GFTFTSFGMS (SEQ ID NO:53); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and is preferably selected from GGSLSR (SEQ ID NO:7) and GGSLER (SEQ ID NO:57), such that when CDR1 is GFTFRSFGMS (SEQ ID NO:5), then CDR3 is not GGSLSR (SEQ ID NO:7) (and vice versa). Most preferably, CDR1 is GFTFTSFGMS (SEQ ID NO:53), CDR2 is SIGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLER (SEQ ID NO:57).

[0517] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0518] - position 5 is V;

[0519] - position 11 is V; and

[0520] - position 30 is T;

[0521] The amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding mutations at positions 5, 11 and 30).

[0522] The serum albumin binder according to this aspect preferably further has N or P at position 16; P or L at position 45; the SKN or AKT motif at positions 74 - 76; A, L or T at position 89; and G at position 104.

[0523] The albumin binders according to this aspect are preferably further described herein. For example, they preferably have the affinity for serum albumin as described herein, and / or they have the serum half-life as further described herein (as such or as part of the polypeptides of the invention).

[0524] Also, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and is preferably GGSLSR (SEQ ID NO:7).

[0525] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0526] - position 5 is V; and

[0527] - position 11 is V; and

[0528] - position 30 is T; and

[0529] The amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding mutations at positions 5, 11 and 30).

[0530] The serum albumin binders according to this aspect preferably further have an N or P at position 16; a P or L at position 45; an SKN or AKT motif at positions 74-76; an A, L or T at position 89; and a G at position 104.

[0531] The albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (as such or as part of the polypeptides of the invention).

[0532] Also, the amino acid sequences according to this aspect preferably have the following CDRs: (i) CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and is preferably GGSLSR (SEQ ID NO:7).

[0533] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0534] - position 5 is V; and

[0535] - position 11 is V; and

[0536] - position 30 is T; and

[0537] - position 89 is A, L or T; and

[0538] - position 104 is G; and

[0539] The amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:1 (disregarding mutations at positions 5, 11, 30, 89 and 104).

[0540] The serum albumin binders according to this aspect preferably further have an N or P at position 16; a P or L at position 45; and an SKN or AKT motif at positions 74-76.

[0541] Albumin binders according to this aspect are preferably as further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of the polypeptides of the invention).

[0542] Furthermore, the amino acid sequence according to this aspect preferably has the following CDRs: (i) CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59) and GGSLSD (SEQ ID NO:60), and is preferably GGSLSR (SEQ ID NO:7).

[0543] In another aspect, the invention relates to an amino acid sequence which is an immunoglobulin single variable domain capable of binding (human) serum albumin, wherein:

[0544] - position 5 is V; and

[0545] - position 11 is V; and

[0546] - position 30 is T; and

[0547] - position 89 is A, L or T; and

[0548] - position 104 is G; and

[0549] the amino acid sequence has (i) no more than 7, preferably no more than 5, such as 5, 4, 3, 2, 1 or zero amino acid differences from the sequence of SEQ ID NO:50 (disregarding mutations at positions 5, 11, 30, 89 and 104).

[0550] Serum albumin binders according to this aspect preferably also have N or P at position 16; P or L at position 45; and the SKN or AKT motif at positions 74 - 76.

[0551] Albumin binders according to this aspect are preferably further described herein. For example, they preferably have an affinity for serum albumin as described herein, and / or they have a serum half-life as further described herein (either as such or as part of a polypeptide of the invention).

[0552] Furthermore, the amino acid sequences according to this aspect preferably have the following CDRs: (i) CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); (ii) CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and (iii) CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO:7), GGGLSR (SEQ ID NO:55), GGSLDR (SEQ ID NO:56), GGSLER (SEQ ID NO:57), GGSLGR (SEQ ID NO:58), GGSLHR (SEQ ID NO:59), and GGSLSD (SEQ ID NO:60), and is preferably GGSLSR (SEQ ID NO:7).

[0553] When they are present at the C-terminal end of a compound or polypeptide of the invention and / or form the C-terminal end of a compound or polypeptide of the invention (or when they otherwise have an "exposed" C-terminal end in a protein, polypeptide, or other compound or construct, which generally means that the C-terminal end of the ISV is not associated or linked to a constant domain such as the CH1 domain; again referring to WO 12 / 175741 and PCT / EP2015 / 06043), the serum albumin binders of the invention preferably also have the formula (X) n of a C-terminal extension, where n is 1 - 10, preferably 1 - 5, such as 1, 2, 3, 4, or 5 (and preferably 1 or 2, such as 1); and each X is (preferably a naturally occurring) amino acid residue, which is independently selected from naturally occurring amino acid residues (although according to a preferred aspect, it does not contain any cysteine residues), and is preferably independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I).

[0554] According to some preferred, but non-limiting, aspects of such a C-terminal extension X (n) X and n can be as follows:

[0555] (a) n = 1 and X = Ala;

[0556] (b) n = 2 and each X = Ala;

[0557] (c) n = 3 and each X = Ala;

[0558] (d) n = 2 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile);

[0559] (e) n = 3 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile);

[0560] (f) n = 3 and at least two X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile);

[0561] (g) n = 1 and X = Gly;

[0562] (h) n = 2 and each X = Gly;

[0563] (i) n = 3 and each X = Gly;

[0564] (j) n = 2 and at least one X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile);

[0565] (k) n = 3 and at least one X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile);

[0566] (l) n = 3 and at least two X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile);

[0567] (m) n = 2 and each X = Ala or Gly;

[0568] (n) n = 3 and each X = Ala or Gly;

[0569] (o) n = 3 and at least one X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile); or

[0570] (p) n = 3 and at least two X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu, and / or Ile);

[0571] Aspects (a), (b), (c), (g), (h), (i), (m) and (n) are particularly preferred, aspects where n = 1 or 2 are preferred, and aspects where n = 1 are particularly preferred.

[0572] It should also be noted that preferably, any C-terminal extension present in the serum albumin binder of the present invention does not contain (free) cysteine residues (unless the cysteine residue is used or intended for further functionalization, such as PEGylation).

[0573] 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.

[0574] When the serum albumin binder of the present invention contains a mutation at position 110 or 112 (optionally in combination with a mutation at position 11 and / or 89 as described herein), the C-terminal amino acid residues of framework 4 (starting from position 109) can be as follows: (i) if no C-terminal extension is present: VTVKS (SEQ ID NO:104), VTVQS (SEQ ID NO:105), VKVSS (SEQ ID NO:106) or VQVSS (SEQ ID NO:107); or (ii) if a C-terminal extension is present: VTVKSX (n) (SEQ ID NO:108), VTVQSX(n) (SEQ ID NO:109), VKVSSX(n) (SEQ ID NO:110) or VQVSSX (n) (SEQ ID NO:111), such as VTVKSA (SEQ ID NO:112), VTVQSA (SEQ ID NO:113), VKVSSA (SEQ IDNO:114) or VQVSSA (SEQ ID NO:115). When the serum albumin binder of the present invention does not contain a mutation at position 110 or 112 (but only contains a mutation at position 11 and / or 89 as described herein), the C-terminal amino acid residues of framework 4 (starting from position 109) will often be: (i) when no C-terminal extension is present: VTVSS (SEQ ID NO:116), as in the sequences of SEQ ID NOs: 8 to 49; or (ii) when a C-terminal extension is present: VTVSSX (n) (SEQ ID NO:117) such as VTVSSA (SEQ ID NO:118) (as in the sequences of SEQ ID NOs: 61 to 102). In these C-terminal sequences, X and n are as defined herein for C-terminal extensions.

[0575] Furthermore, when the serum albumin binder of the present invention is present at the N-terminal end of the compound or polypeptide of the present invention and / or forms the N-terminal end of the compound or polypeptide of the present invention, then the serum albumin binder preferably has a D at position 1 (i.e., an E1D mutation relative to the sequences given in SEQ ID NOs: 1, 8-50, and 61-102).

[0576] Furthermore, generally, when the compound or polypeptide of the present invention has a heavy chain ISVD at its C-terminal end (which can be the serum albumin binder of the present invention, but can also be, for example, an ISVD that binds to a therapeutic target), then the C-terminal ISVD (and by extension, the compound or polypeptide of the present invention) preferably has a C-terminal extension X(n) as described herein. Similarly, when the compound or polypeptide of the present invention has a heavy chain ISVD at its N-terminal end (which can be the serum albumin binder of the present invention, but can also be, for example, an ISVD that binds to a therapeutic target), then the N-terminal ISVD (and by extension, the compound or polypeptide of the present invention) preferably has a D at position 1.

[0577] Furthermore, preferably, when the compound or polypeptide of the present invention contains one or more other ISVDs in addition to the albumin binder of the present invention (the other ISVDs can be, for example, one or more ISVDs that bind to therapeutic targets), then all ISVDs preferably present in the compound or polypeptide contain one or more framework mutations in their sequences that reduce the binding of pre-existing antibodies. Specifically, when these other ISVDs are nanobodies or (single) domain antibodies (i.e., consist essentially of a VH domain and / or are derived from a VH domain), they can contain amino acid residues / mutations (suitable combinations thereof) at positions 11, 89, 110, and / or 112, which are substantially as described in PCT / EP2015 / 060643.

[0578] As mentioned, the amino acid sequences provided by the present invention are proteins that can bind and can specifically (as described herein) bind human serum albumin. Thus, they can be used as such binding units or binding domains for binding (human) serum albumin, for example, to confer an increased half-life (as defined herein) to a therapeutic compound, moiety or entity. Regarding the use of serum albumin-binding domains for increasing the half-life of a therapeutic compound, moiety or entity, reference is generally made to WO 2004 / 041865, WO 2006 / 122787, EP 2 139 918, WO 2011 / 006915, WO 2012 / 175400 and / or WO 2014 / 111550. The 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.

[0579] The present invention also relates to proteins, polypeptides and other constructs, molecules or chemical entities comprising one or more of the serum albumin binders of the present invention as described herein or consisting essentially thereof; to methods for expressing / producing the improved heavy chain immunoglobulin variable domains of the present invention and / or for expressing / producing proteins, polypeptides and other constructs, molecules or chemical entities comprising them; to compositions and products (such as pharmaceutical compositions and products) containing the improved heavy chain immunoglobulin variable domains of the present invention and / or proteins, polypeptides and other constructs, molecules or chemical entities comprising them; to nucleotide sequences and nucleic acids encoding the improved heavy chain immunoglobulin variable domains of the present invention and / or encoding proteins or polypeptides comprising them; and to the use (and in particular therapeutic, prophylactic and diagnostic use) of the improved heavy chain immunoglobulin variable domains of the present invention and proteins, polypeptides and other constructs, molecules or chemical entities comprising them.

[0580] Other aspects, embodiments, advantages, applications and uses of the present invention will be apparent from the other descriptions herein.

[0581] In this specification:

[0582] - The term "immunoglobulin single variable domain" (also referred to as "ISV" or "ISVD") is generally used to denote an immunoglobulin variable domain (which can be a heavy or light chain domain, including VH, VHH or VL domains) that can form a functional antigen-binding site without interacting with another variable domain (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional 4-chain monoclonal antibody). Examples of ISVDs will be apparent to the person skilled in the art and include, for example, nanobodies (including VHH, humanized VHH and / or camelized VH such as camelized human VH), IgNAR domains, (single domain) antibodies that are or are derived from VH domains (such as dAb TM ) and (single domain) antibodies that are or are derived from VL domains (such as dAb TM ). 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 stated otherwise herein, the ISVD will be a nanobody.

[0583] - The term "nanobody" is generally defined as in WO 2008 / 020079 or WO 2009 / 138519 and thus generally denotes a VHH, humanized VHH or camelized VH (such as camelized human VH), or generally denotes a sequence-optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with a known human framework region and maximum expression). It should be noted that the term nanobody is a registered trademark of Ablynx N.V. and can thus also be referred to as and / or );

[0584] - Generally, unless otherwise indicated herein, the ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs mentioned herein are intended for use in preventing or treating diseases or disorders in humans (and / or optionally also in warm-blooded animals and especially mammals). Thus, generally, the ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs described herein are preferably such that they can be used as (biological) drugs or other pharmaceutically or therapeutically active compounds and / or drug products or compositions and / or can suitably be part thereof. Such a drug, compound, or product is preferably such that it is suitable for administration to humans, for example for preventing or treating a subject in need of such prevention or treatment, or for example as part of a clinical trial. As further described herein, for this purpose, such a drug or compound may contain other moieties, entities, or binding units in addition to the ISVDs provided by the present invention (which may also be, for example, one or more other additional therapeutic moieties and / or one or more other moieties that affect the pharmacokinetic or pharmacodynamic properties (such as its half-life) of an ISVD-based or nanobody-based biological reagent). Suitable examples of such other therapeutic moieties or other moieties will be apparent to the person skilled in the art and may generally include, for example, any therapeutically active protein, polypeptide, or other binding domain or binding unit, and also modifications such as those described on pages 149 to 152 of WO 2009 / 138159. The ISVD-based or nanobody-based biological reagents are preferably therapeutic agents or intended to be used as therapeutic agents (which include prophylactic and diagnostic agents), and for this purpose preferably contain at least one ISVD directed against a therapeutically relevant target (such as RANK-L, vWF, IgE, RSV, CXCR4, IL-23, or other interleukins, etc.). For some specific, but non-limiting, examples of such ISVD-based or nanobody-based biological reagents, reference is made to Examples 8-18, and also to various applications of Evelo Biosciences, Inc. (such as, for example, but not limited to WO2004 / 062551, WO 2006 / 122825, WO 2008 / 020079, and WO 2009 / 068627), and also to applications such as (but not limited to) WO 2006 / 038027, WO 2006 / 059108, WO 2007 / 063308, WO 2007 / 063311, WO2007 / 066016, and WO 2007 / 085814. And, as further described herein, the other moiety may be an ISVD or nanobody directed against a (human) serum protein such as (human) serum albumin as described herein, and such an ISVD or nanobody may also be used for therapeutic purposes, especially for neutralizing the TNF binder described herein and / or for extending its half-life.Reference is made, for example, to WO 2004 / 041865, WO 2006 / 122787 and WO 2012 / 175400, which generally describe the use of nanobodies that bind serum albumin for half-life extension. Also, in the present specification, unless otherwise explicitly mentioned herein, all terms mentioned herein 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, in the absence of a specific description of a method or technique herein, it may be carried out 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 other components known per se for use in a pharmaceutical product or composition (i.e., depending on the intended pharmaceutical form) and / or, for example, one or more other compounds or active ingredients intended for therapeutic use (i.e., providing a combination product).

[0585] Also, when used in this specification or claims, the following terms have the same meaning as given on pages 62 - 75 of WO 2009 / 138519, and / or may be determined in the manner described on pages 62 - 75 of WO 2009 / 138519 when applicable: "agonist", "antagonist", "inverse agonist", "non - polar, uncharged amino acid residue", "polar, uncharged amino acid residue", "polar, charged amino acid residue", "sequence identity", "identical", and "amino acid difference" (when representing sequence alignment of two amino acid sequences), "(in) substantially isolated (form)", "domain", "binding domain", "epitope", "antigenic determinant", "epitope", "anti" or "against" (antigen), "specificity", and "half - life". Additionally, the terms "modulate" and "regulate", "interaction site", "specific for", "cross - blocking", "cross - blocked", and "cross - blocking" and "substantially independent of pH" are described on pages 74 - 79 of WO2010 / 130832 of Eiger BioPharmaceuticals, Inc. (and / or may be determined as described above). Also, when referring to constructs, compounds, proteins, or polypeptides of the present invention, terms such as "monovalent", "divalent" (or "multivalent"), "bispecific" (or "multispecific"), and "biparatopic" (or "multiparatopic") may have the meaning given in WO 2009 / 138519, WO 2010 / 130832, or WO 2008 / 020079.

[0586] The term "half-life" as used herein in connection with the ISVD, nanobody, ISVD-based bioreagent, nanobody-based bioreagent, or any other amino acid sequence, compound, or polypeptide mentioned herein can generally be defined as described in paragraph o) on page 57 of WO2008 / 020079, and as mentioned therein, represents the time required to reduce the serum concentration of the amino acid sequence, compound, or polypeptide by 50% in vivo, for example due to degradation of the sequence or compound by natural mechanisms and / or clearance or sequestration of the sequence or compound. The in vivo half-life of the amino acid sequences, compounds, or polypeptides of the present invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and can be, for example, generally as described in paragraph o) on page 57 of WO 2008 / 020079. Also as mentioned in paragraph o) on page 57 of WO2008 / 020079, using parameters such as t1 / 2-α, t1 / 2-β, and area under the curve (AUC), the half-life can be expressed. In this regard, it should be noted that the term "half-life" as used herein specifically refers to t1 / 2-β or the terminal half-life (where t1 / 2-α and / or AUC or both may be disregarded). For example, refer to the experimental section below, as well as to standard manuals such as Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Also refer to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd revised edition (1982). Similarly, the term "increase in half-life" or "increased half-life" is also defined as described in paragraph o) on page 57 of WO 2008 / 020079, and in particular represents an increase in t1 / 2-β, whether or not there is an increase in t1 / 2-α and / or AUC or both.

[0587] When a term is not specifically defined herein, it has its ordinary meaning in the art, which will be apparent to those skilled in the art. For example, reference may be made to standard manuals such as 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, N.Y., (1985); Old et al., “Principles of Gene Manipulation: An Introduction to Genetic Engineering”, 2nd ed., 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., “Immunobiology” (6th ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), as well as the general background art cited herein.

[0588] Also, as already noted herein, the amino acid residues of the nanobody are numbered according to the general numbering of VH given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, publication No. 91), which is applicable to the VHH domains from Camelidae in the article by Riechmann and Muyldermans, J. Immunol. Methods, June 23, 2000; 240(1-2):185-195; or as mentioned herein. According to this numbering, the FR1 of the nanobody contains the amino acid residues at positions 1-30, the CDR1 of the nanobody contains the amino acid residues at positions 31-35, the FR2 of the nanobody contains the amino acids at positions 36-49, the CDR2 of the nanobody contains the amino acid residues at positions 50-65, the FR3 of the nanobody contains the amino acid residues at positions 66-94, the CDR3 of the nanobody contains the amino acid residues at positions 95-102, and the FR4 of the nanobody contains the amino acid residues at positions 103-113. [In this regard, it should be noted - as is well known in the art with respect to VH domains and VHH domains - that the total number of amino acid residues in each CDR can 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 not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. However, generally speaking, according to the Kabat numbering and regardless of the numbering of the amino acid residues in the CDR, position 1 according to the Kabat numbering corresponds to the start of FR1, and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2, and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3, and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4, and vice versa].

[0589] An alternative method of numbering the amino acid residues of a VH domain (which method can also be applied in a similar manner to VHH domains and nanobodies from camelids) 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 this specification, aspects, and figures, unless otherwise indicated, the Kabat numbering as used by Riechmann and Muyldermans for VHH domains will be followed.

[0590] It should also be noted that the figures, any sequence listing, and the experimental section / Examples are provided solely for further illustration of the invention and should not be construed or interpreted as limiting the invention and / or the scope of the appended claims in any way, unless expressly stated otherwise herein.

[0591] As further described herein, the serum albumin binders of the invention can advantageously be used as a moiety, binding unit, or fusion partner to increase the half-life of a therapeutic moiety such as a polypeptide, protein, compound (including, but not limited to, small molecules), or other therapeutic entity.

[0592] Thus, in another aspect, the invention provides a polypeptide, protein, construct, compound, or other chemical entity comprising the serum albumin binder of the invention and one or more other amino acid sequences, (binding) domains, binding units, or other moieties or chemical entities or consisting essentially thereof.

[0593] Specifically, the invention provides a polypeptide, protein, construct, compound, or other chemical entity comprising the serum albumin binder of the invention and one or more (such as one or two) therapeutic moieties (which can be the same or different and can, for example, target the same or different targets, and when they target the same target, can target the same or different epitopes, moieties, domains, or subunits of the target), which are suitably linked to each other directly or via one or more suitable linkers or spacers. Such a polypeptide, protein, or construct can be, for example, but not limited to, a fusion protein, as further described herein.

[0594] The invention also relates to the therapeutic use of such polypeptides, proteins, constructs, or compounds and to pharmaceutical compositions comprising such polypeptides, proteins, constructs, or compounds.

[0595] In one aspect, the at least one therapeutic moiety comprises, consists of, or consists essentially 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, capable of binding to a desired antigen (and in particular capable of specifically binding to a desired antigen), and / or capable of interacting with a desired target. In another embodiment, the at least one therapeutic moiety comprises, or consists essentially of, a therapeutic protein or polypeptide. In another embodiment, the at least one therapeutic moiety comprises, or consists essentially of, a binding domain or binding unit, such as an immunoglobulin or immunoglobulin sequence (including, but not limited to, fragments of immunoglobulins), such as an antibody or antibody fragment (including, but not limited to, ScFv fragments), or another suitable protein scaffold, such as a protein A domain (such as Affibodies TM ), amylastatin, fibronectin, lipocalin, CTLA-4, T-cell receptor, engineered ankyrin repeats, avimer, and PDZ domain (Binz et al., Nat. Biotech 2005, Vol 23:1257) and DNA- or RNA-based binding moieties, including, but not limited to, DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

[0596] In another aspect, the 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.

[0597] In a preferred aspect, the at least one therapeutic moiety comprises, or consists essentially of, at least one immunoglobulin single variable domain, such as a domain antibody, single domain antibody, "dAb", or nanobody (such as VHH, humanized VHH, or camelized VH), or IgNAR domain.

[0598] In a specific embodiment, the at least one therapeutic moiety comprises, or consists essentially of, at least one monovalent nanobody or a bivalent, multivalent, bispecific, or multispecific nanobody construct.

[0599] Polypeptides, (fusion) proteins, constructs, or compounds comprising the serum albumin binder of the present invention and one or more therapeutic moieties can generally be prepared and used as described in the prior art cited above (such as WO04 / 041865 and WO 06 / 122787), but with the serum albumin binder of the present invention replacing the half-life increasing moiety described in the prior art.

[0600] Polypeptides, (fusion) proteins, constructs or compounds comprising a serum albumin binder of the invention and one or more therapeutic moieties generally and preferably have an increased half-life compared to the therapeutic moiety itself.

[0601] Generally, the compounds, polypeptides, constructs or fusion proteins described herein preferably have a half-life that is at least 1.5-fold, preferably at least 2-fold, such as at least 5-fold, for example at least 10-fold or more than 20-fold greater than the half-life of the corresponding therapeutic moiety itself (as measured in humans or a suitable animal such as a mouse or a cynomolgus monkey).

[0602] And, preferably, compared to the half-life of the corresponding therapeutic moiety itself, any such compound, polypeptide, fusion protein or construct has an increased half-life in humans of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours.

[0603] And, preferably, the half-life (preferably defined as t1 / 2β) of the compounds or polypeptides of the invention in humans exceeds 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours and for example about 1 day, 2 days, 1 week, 2 weeks and up to the half-life of serum albumin in humans (estimated to be about 19 days).

[0604] The half-life can generally be defined as the time required to reduce the serum concentration of the polypeptide by 50% in vivo, for example due to degradation of the ligand and / or clearance or sequestration of the ligand by natural mechanisms. Specifically, the half-life can be defined as in WO2009 / 068627.

[0605] Methods for pharmacokinetic analysis and half-life determination are well known to those skilled in the art. Details can be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). Also refer to "Pharmacokinetics", M Gibaldi & D Perron, Marcel Dekker publication, 2nd revised edition (1982).

[0606] As mentioned, in one aspect, the serum albumin binder of the invention can be 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 (such as VHHs, humanized VHHs or camelized VHs such as camelized human VHs).

[0607] Accordingly, one embodiment of the present invention relates to a polypeptide, construct or fusion protein comprising a serum albumin binder of the present invention and one or more (such as one or two) immunoglobulin single variable domain sequences, which are directly or optionally appropriately connected to each other via one or more suitable linkers or spacers. As mentioned herein, each such immunoglobulin single variable domain present in such a polypeptide, construct or fusion protein can independently be a domain antibody, single domain antibody, "dAb'", or nanobody (such as VHH, humanized VHH or camelized VH, such as camelized human VH); and according to a specific but non-limiting aspect, at least one (and up to all) of these immunoglobulin single variable domains contains two or three disulfide bridges.

[0608] As mentioned, when a polypeptide, construct or fusion protein has a heavy chain ISVD at its C-terminal end (the ISVD can be a serum albumin binder of the present invention or an ISVD against a therapeutic target, such as a nanobody against a therapeutic target), then the polypeptide, construct or fusion protein (the ISVD present at the C-terminal end) preferably has a C-terminal extension at its C-terminal end. Again, the C-terminal extension will have the formula (X) n , where n is 1-10, preferably 1-5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is (preferably a naturally occurring) amino acid residue, which is independently selected from naturally occurring amino acid residues (although according to a preferred aspect, it does not contain any cysteine residues), and is preferably independently selected from alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0609] And, as mentioned, when a polypeptide, construct or fusion protein has a heavy chain ISVD at its N-terminal end (the ISVD can be a serum albumin binder of the present invention or an ISVD against a therapeutic target, such as a nanobody against a therapeutic target), then the polypeptide, construct or fusion protein (the ISVD present at the C-terminal end) preferably has a D or E1D mutation at position 1.

[0610] Accordingly, in another aspect, the present invention relates to a protein, polypeptide or other compound, which:

[0611] - comprises or consists essentially of: at least one (and preferably only one) serum albumin binder of the present invention and at least one (such as one, two or three) therapeutic moiety or entity (wherein the serum albumin binder and the one or more therapeutic moieties or entities are optionally appropriately connected via one or more suitable linkers);

[0612] - having a heavy chain ISVD at its C-terminal end, wherein the ISVD at the C-terminal end has a C-terminal extension (X) n (as further described herein);

[0613] - said protein, polypeptide or other compound may also have a heavy chain ISVD at its N-terminal end, in which case the N-terminal ISVD end preferably has a D or E1D at position 1.

[0614] And, in a preferred aspect, when one or more other ISVDs are present in addition to the serum albumin binder of the present invention (i.e., when one or more therapeutic moieties present are ISVDs), then the (one or more or all) said "therapeutic" ISVDs preferably also have an amino acid residue / mutation (combination) that reduces the binding of pre-existing antibodies. When the ISVD is a heavy chain ISVD, then as described in PCT / EP2015 / 060643, these mutations can specifically be one or more mutations (suitable combinations) at positions 11, 89, 110 and 112, and said mutations can be substantially the same kind of mutations (or combinations of mutations) as described herein for the serum albumin binder of the present invention. Preferably, if such other ISVDs are present at the C-terminal end, then at least the therapeutic ISVD contains such mutations at positions 11, 89, 110 and / or 112 (i.e., in addition to the C-terminal extension as described herein).

[0615] According to a specific aspect, all therapeutic moieties present in the construct, fusion protein or polypeptide are ISVDs (i.e., ISVDs directed against a therapeutic target), and in particular heavy chain ISVDs, and more particularly nanobodies (i.e., nanobodies directed against a therapeutic target).

[0616] For example, but not limited to, a construct, fusion protein or polypeptide comprising the serum albumin binder of the present invention may comprise:

[0617] - one copy of the serum albumin binder of the present invention and one ISVD (and preferably a nanobody) directed against a therapeutic target; or

[0618] - one copy of the serum albumin binder of the present invention and two ISVDs (and preferably two nanobodies) directed against a therapeutic target (said 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

[0619] -One copy of the serum albumin binder of the present invention and three ISVDs (and preferably three nanobodies) against a therapeutic target (the ISVDs can be the same or different, and when different, can be against the same target, against different epitopes on the same target, or against different therapeutic targets).

[0620] Some non-limiting examples of the constructs, fusion proteins or polypeptides of the present invention can be schematically represented as follows, where "[Alb]" represents the serum albumin binder of the present 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 (which is optional; suitable examples are 9GS and 35GS linkers), and the N-terminus is on the left hand side and the C-terminus is on the right hand side:

[0621] [Alb]-[Therapeutic moiety 1]

[0622] [Therapeutic moiety 1]-[Alb]-X (n)

[0623] [Alb]-[Therapeutic moiety 1]-[Therapeutic moiety 1]

[0624] [Therapeutic moiety 1]-[Therapeutic moiety 1]-[Alb]-X (n)

[0625] [Therapeutic moiety 1]-[Alb]-[Therapeutic moiety 1]

[0626] [Alb]-[Therapeutic moiety 1]-[Therapeutic moiety 2]

[0627] [Therapeutic moiety 1]-[Therapeutic moiety 2]-[Alb]-X (n)

[0628] [Therapeutic moiety 1]-[Alb]-[Therapeutic moiety 2]

[0629] When the therapeutic moiety is an ISVD (and preferably a nanobody) against a therapeutic target, preferred but non-limiting constructs, fusion proteins or polypeptides of the present invention can be schematically represented as follows, where "[Alb]" represents the serum albumin binder of the present invention, "[Therapeutic ISVD 1]" and "[Therapeutic ISVD 2]" represent ISVDs against a therapeutic target (the ISVDs can be the same or different, and when different, can be 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 hand side and the C-terminus is on the right hand side:

[0630] [Alb]-[Therapeutic ISVD 1]-X (n)

[0631] [Therapeutic ISVD 1]-[Alb]-X (n)

[0632] [Alb]-[Therapeutic ISVD 1]-[Therapeutic ISVD 1]-X (n)

[0633] [Therapeutic ISVD 1]-[Therapeutic ISVD 1]-[Alb]-X (n)

[0634] [Therapeutic ISVD 1]-[Alb]-[Therapeutic ISVD 1]-X (n)

[0635] [Alb]-[Therapeutic ISVD 1]-[Therapeutic ISVD 2]-X (n)

[0636] [Therapeutic ISVD 1]-[Therapeutic ISVD 2]-[Alb]-X (n)

[0637] [Therapeutic ISVD 1]-[Alb]-[Therapeutic ISVD 2]-X (n)

[0638] Thus, in another aspect, the present invention relates to a multispecific (and in particular bispecific) nanobody construct comprising a serum albumin binder of the present invention and at least one other nanobody (such as one or two other nanobodies, which may be the same or different) and / or another nanobody, wherein said at least one other nanobody is preferably directed against a desired target (which is preferably a therapeutic target), and said another nanobody can be used or is suitable for therapeutic, prophylactic and / or diagnostic purposes. Again, the serum albumin binder of the present invention and said other nanobodies may be directly or optionally appropriately linked to each other via one or more suitable linkers or spacers.

[0639] 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.

[0640] Some other examples of specific multispecific and / or multivalent polypeptides of the invention can be found in the applications of Ablynx NV mentioned herein. Specifically, for the general description of multivalent and multispecific constructs containing at least one nanobody against a serum protein (for increasing the half-life), nucleic acids encoding them, compositions containing them, the preparation of the foregoing substances and the use of the foregoing substances, reference is made to the international applications WO 04 / 041865 and WO 06 / 122787 mentioned above (the serum albumin binders of the invention described herein can generally be similarly used for the nanobodies for extending the half-life such as Alb-8 described therein), and reference is also made to the general description and specific examples of such constructs given in for example WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0641] In one aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) comprising a serum albumin binder of the invention and one or more additional heavy chain ISVDs (such as nanobodies or (single) domain antibodies comprising or derived from VH domains), wherein said one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0642] - The amino acid residue at position 11 is preferably selected from L or V; and

[0643] - The amino acid residue at position 89 is preferably suitably selected from T, V or L; and

[0644] - The amino acid residue at position 110 is preferably suitably selected from T, K or Q; and

[0645] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0646] such that (i) position 89 is T; or (ii) position 89 is L and position 11 is V; or (iii) position 89 is L and position 110 is K or Q; or (iv) position 89 is L and position 112 is K or Q; or (v) position 89 is L, position 11 is V, and position 110 is K or Q; or (vi) position 89 is L, position 11 is V, and position 112 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (vii) position 11 is V and position 112 is K or Q.

[0647] In another aspect, the invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) that comprises a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein the one or more other heavy chain ISVDs each contain the following amino acid residues:

[0648] - 89T; or

[0649] - 89L in combination with 11V; or

[0650] - 89L in combination with 110K or 110Q; or

[0651] - 89L in combination with 112K or 112Q; or

[0652] - 89L in combination with 11V and 110K or 110Q; or

[0653] - 89L in combination with 11V and 112K or 112Q; or

[0654] - 11V in combination with 110K or 110Q; or

[0655] - 11V in combination with 112K or 112Q.

[0656] In another aspect, the invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) that comprises a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein the one or more other heavy chain ISVDs each contain the following amino acid residues:

[0657] - The amino acid residue at position 11 is preferably selected from L or V; and

[0658] - The amino acid residue at position 89 is T; and

[0659] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q (and preferably is T); and

[0660] - The amino acid residue at position 112 is preferably suitably selected from S, K, or Q (and preferably is S).

[0661] In another aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) that comprises a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein said one or more other heavy chain ISVDs each contain the following amino acid residues:

[0662] - the amino acid residue at position 11 is V; and

[0663] - the amino acid residue at position 89 is L; and

[0664] - the amino acid residue at position 110 is preferably suitably selected from T, K or Q; and

[0665] - the amino acid residue at position 112 is preferably suitably selected from S, K or Q.

[0666] In another aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) that comprises a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein said one or more other heavy chain ISVDs each contain the following amino acid residues:

[0667] - 11V in combination with 89L; or

[0668] - 11V in combination with 110K or 110Q;

[0669] - 11V in combination with 112K or 112Q;

[0670] - 11V in combination with 89L and 110K or 110Q; or

[0671] - 11V in combination with 89L and 112K or 112Q.

[0672] In another aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) that comprises a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein said one or more other heavy chain ISVDs each contain the following amino acid residues:

[0673] - 89L in combination with 11V; or

[0674] - 89L in combination with 110K or 110Q; or

[0675] - 89L in combination with 112K or 112Q; or

[0676] - 89L in combination with 11V and 110K or 110Q; or

[0677] - 89L in combination with 11V and 112K or 112Q.

[0678] In another aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) comprising a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein said one or more other heavy chain ISVDs each contain the following amino acid residues:

[0679] - 110K or 110Q in combination with 11V; or

[0680] - 110K or 110Q in combination with 89L; or

[0681] - 110K or 110Q in combination with 11V and 89L.

[0682] In another aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) comprising a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein said one or more other heavy chain ISVDs each contain the following amino acid residues:

[0683] - 112K or 112Q in combination with 11V; or

[0684] - 112K or 112Q in combination with 89L; or

[0685] - 112K or 112Q in combination with 11V and 89L.

[0686] In another aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) comprising a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein said one or more other heavy chain ISVDs each contain a T at position 89.

[0687] In another aspect, the invention relates to a protein, polypeptide or other compound or construct (and preferably a fusion protein) comprising a serum albumin binder of the invention and one or more other heavy chain ISVDs, wherein said one or more other heavy chain ISVDs each contain a V at position 11 and an L at position 89.

[0688] Again, when such a protein, polypeptide or other compound or construct has a (heavy chain) ISVD at its C-terminal end, it preferably contains a C-terminal extension X(n) (as described herein), and when such a protein, polypeptide or other compound or construct has a (heavy chain) ISVD at its N-terminal end, it preferably has a D at position 1. Such a protein, polypeptide or other compound or construct also preferably has a half-life as further described herein.

[0689] The present invention also relates to nucleotide sequences or nucleic acids encoding the albumin-binding agents, compounds or polypeptides of the present invention. The present invention also includes genetic constructs comprising said nucleotide sequences or nucleic acids and one or more elements of genetic constructs known per se. Such genetic constructs may be in the form of plasmids or vectors. Again, such constructs can generally be as described in the published patent applications of Ebbelingx B.V. (such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627).

[0690] The present invention also relates to a host or host cell containing such nucleotide sequences or nucleic acids and / or expressing (or capable of expressing) the albumin-binding agents, compounds or polypeptides of the present invention. Again, such host cells can generally be as described in the published patent applications of Ebbelingx B.V. (such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627).

[0691] The present invention also relates to a method for preparing the albumin-binding agents, compounds or polypeptides of the present invention, said method comprising culturing or maintaining a host cell as described herein under conditions such that the host cell produces or expresses the albumin-binding agents, compounds or polypeptides of the present invention, and optionally further comprising isolating the albumin-binding agents, compounds or polypeptides of the present invention so produced. Again, such methods can be carried out generally as described in the published patent applications of Ebbelingx B.V. (such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO2009 / 068627).

[0692] The present invention also relates to a pharmaceutical composition comprising at least one compound or polypeptide of the present invention and optionally at least one pharmaceutically acceptable carrier, diluent or excipient. Such articles, carriers, excipients and diluents can generally be as described in the published patent applications of Ebbelingx B.V. (such as WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627).

[0693] However, since the compounds or polypeptides of the present invention have an increased half-life, they are preferably administered into the circulation. Thus, they can be administered in any suitable manner that allows the compounds or polypeptides of the present invention to enter the circulation, such as intravenously, via injection or infusion, or in any other suitable manner (including oral administration, subcutaneous administration, intramuscular administration, transdermal administration, intranasal administration, administration via the lung, etc.). Suitable methods and routes of administration will be apparent to the person skilled in the art, again for example also from the teachings of the published patent applications of Evox Therapeutics Limited (such as WO04 / 041862, WO2006 / 122786, WO 2008 / 020079, WO 2008 / 142164 or WO2009 / 068627).

[0694] Thus, in another aspect, the present invention relates to a method for preventing and / or treating at least one disease or disorder that can be prevented or treated by using the compounds or polypeptides of the present invention, the method comprising administering to a subject in need thereof a pharmaceutically active amount of the compounds or polypeptides of the present invention and / or a pharmaceutical composition comprising them. The diseases and disorders that can be prevented or treated by using the compounds or polypeptides of the present invention as described herein are generally the same as those that can be prevented or treated by using the therapeutic moieties present in the compounds or polypeptides of the present invention.

[0695] In the context of the present invention, the term "preventing and / or treating" not only encompasses preventing and / or treating a disease, but generally also encompasses preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, alleviating and / or relieving 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, alleviating or reversing any physiological damage caused by a disease, and any pharmacological effects that are generally beneficial to the patient being treated.

[0696] The subject to be treated can be any warm-blooded animal, but specifically a mammal, and more specifically a human. As will be apparent to the person skilled in the art, the subject to be treated will specifically be a person suffering from the diseases and disorders mentioned herein or at risk of the diseases and disorders mentioned herein.

[0697] In another embodiment, the present invention relates to a method for immunotherapy, and in particular for passive immunotherapy, the method comprising administering to a subject suffering from the diseases and disorders mentioned herein or at risk of the diseases and disorders mentioned herein a pharmaceutically active amount of the compounds or polypeptides of the present invention and / or a pharmaceutical composition comprising them.

[0698] The compounds or polypeptides of the present invention and / or compositions comprising them are administered according to a treatment regimen suitable for preventing and / or treating a disease or disorder to be prevented or treated. A clinician is generally 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 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.

[0699] Generally, the treatment regimen will comprise administering one or more compounds or polypeptides of the present invention, or one or more compositions comprising them, in one or more pharmaceutically effective amounts or doses. The specific amount or dose to be administered can be determined by the clinician, again based on the above factors.

[0700] Generally, for preventing and / or treating 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 compounds or polypeptides of the present invention are generally administered continuously (e.g., by infusion), as a single daily dose or as multiple divided doses during the day, in an amount of from 1 gram to 0.01 microgram per kilogram body weight per day, preferably from 0.1 gram to 0.1 microgram per kilogram body weight per day, such as about 1, 10, 100 or 1000 micrograms per kilogram body weight per day. A clinician is generally able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be apparent that in certain circumstances, the clinician may choose to deviate from these amounts, for example based on the above factors and his expert judgment. Generally, some guidance regarding the amount to be administered can be obtained from the usual dosages of comparable conventional antibodies or antibody fragments directed against the same target administered via substantially the same route, but taking into account differences in affinity / avidity, potency, biodistribution, half-life and similar factors well known to the person skilled in the art.

[0701] Moreover, since the compounds of the present invention contain serum albumin binders that extend the half-life of the present invention, they do not need to be administered substantially continuously (e.g., by infusion), but they can be administered at suitable intervals (determined by the person skilled in the art). For example, they can be administered (at a suitable dose) once every 2 days, once every 4 days, once a week, once every 2 weeks and in certain cases once every 4 weeks or even at a lower frequency (e.g., by injection or infusion).

[0702] One aspect of the present invention relates to a pharmaceutical composition comprising at least one compound or polypeptide of the present invention, wherein the composition is intended to be administered regularly between once a week and once every 4 weeks, especially between once every 7 days and once every 21 days, such as once every 7 or 14 days.

[0703] Often, in the above methods, a single polypeptide of the invention will be used. However, two or more polypeptides of the invention are used in combination within the scope of the invention.

[0704] The polypeptides of the invention can also be used in combination with one or more other pharmaceutically active compounds or ingredients, i.e., as a combined treatment regimen, which may or may not result in a synergistic effect. Again, the clinician will be able to select such other compounds or ingredients, as well as the appropriate combined treatment regimen, based on the above factors and his or her expert judgment.

[0705] Specifically, the polypeptides of the invention can be used in combination with other pharmaceutically active compounds or ingredients that are or can be used for the prevention and / or treatment of diseases and disorders that can be prevented or treated with the fusion proteins or constructs of the invention, and as a result, a synergistic effect may or may not be obtained.

[0706] It will be obvious to the clinician that the effectiveness of the treatment regimen used according to the invention can be determined and / or followed for the disease or disorder involved in any manner known per se. In appropriate cases, and / or on a case-by-case basis, the clinician will also be able to change or modify a specific treatment regimen in order to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side effects, and / or to achieve the following appropriate balance: on the one hand, achieving the desired therapeutic effect, and on the other hand, avoiding, limiting or reducing unwanted side effects.

[0707] Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and / or as long as the desired therapeutic effect is maintained. Again, this can be determined by the clinician.

[0708] Other aspects, embodiments, advantages and applications of the invention will be obvious from the other descriptions herein.

[0709] The invention will now be further described by means of the following non-limiting preferred aspects, examples and drawings, in which:

[0710] - Figure 1 The table of lists some amino acid positions that will be specifically mentioned herein and their numbering according to some alternative numbering systems (such as Aho and IMGT);

[0711] - Figure 2 lists the amino acid sequences mentioned herein;

[0712] - Figures 3A - 3C shows the alignment of SEQ ID NO:1, 50 and 119, and Figure 3D shows the binding of existing antibodies from 6 sera depleted of serum-albumin;

[0713] -Figure 4A shows the alignment of SEQ ID NO:1 and 8 - 50, and Figure 4B shows the alignment of SEQ ID NO:1, 50 and 61 - 102;

[0714] - Figure 5 shows two corresponding graphs of the data points obtained in Example 1, in which 96 serum samples were tested for binding to [Reference A], [Reference A + C - terminal alanine] and three variants of the present invention (i.e., [Reference A + L11V + V89T + C - terminal alanine], [Reference A + L11V + V89T + T110K + C - terminal alanine] and [Reference A + L11V + V89T + S104T + C - terminal alanine]) (68 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre - existing antibodies that can bind even in the presence of C - terminal alanine, and 11 from SLE patients). It should be noted that with respect to the prior art sequences of SEQ ID NO:1 and 50, Reference A already contains the L5V mutation such that all three variants contain V at positions 5 and 11 in addition to the indicated specific mutations). Each point represents the binding level of one of the 96 samples tested. The data points shown in the right - hand graph and the left - hand graph are the same; in the right - hand graph, the data points measured for each individual sample of each compound tested are connected by lines (so, the slope of the line gives an indication of the extent to which the binding of pre - existing antibodies is reduced when the mutations and C - terminal alanine of the present invention are introduced);

[0715] - Figure 6 shows in detail the data from Figure 5 obtained in Example 1 regarding 11 samples from SLE patients;

[0716] - Figure 7 's table lists the binding data of the data points compiled in Figure 5 ;

[0717] - Figure 8Shows two corresponding graphs of the data points obtained in Example 2, in which 96 serum samples (68 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre-existing antibodies that are able to bind even in the presence of C-terminal alanine, and 11 from SLE patients) were tested for binding to [Reference A], [Reference A + C-terminal alanine] and three variants of the present invention (i.e., [Reference A + L11V + V89A + C-terminal alanine], [Reference A + L11V + V89A + T110K + C-terminal alanine] and [Reference A + L11V + V89L + S104G + C-terminal alanine], respectively). It should be noted that with respect to the prior art sequences of SEQ ID NO:1 and 50, Reference A already contains the L5V mutation, such that all three variants contain V at positions 5 and 11 in addition to the specific mutations indicated. Each point represents the binding level of one of the 96 samples tested. The data points shown in the right-hand graph and the left-hand graph are the same; in the right-hand graph, the data points measured for each individual sample of each compound tested are connected by lines (so, the slope of the lines gives an indication of the extent to which the binding of pre-existing antibodies is reduced when the mutations and C-terminal alanine of the present invention are introduced);

[0718] - Figure 9 Shows in detail the data from Figure 8 which were obtained in Example 2 regarding 11 samples from SLE patients;

[0719] - Figure 10 The table oflists the binding data of the data points compiled in Figure 8 ;

[0720] - Figure 11Shows two corresponding graphs of the data points obtained in Example 3, in which 96 serum samples were tested for binding to [Reference A], [Reference A + C-terminal alanine], and three variants of the present invention (i.e., [Reference A + L11V + V89L + S101G + C-terminal alanine], [Reference A + L11V + V89L + S104A + C-terminal alanine], and [Reference A + L11V + V89L + S101E + C-terminal alanine] respectively). Of these, 68 were from human healthy subjects, 17 were from healthy human volunteers whose sera contained pre-existing antibodies that were able to bind even in the presence of C-terminal alanine, and 11 were from SLE patients. It should be noted that with respect to the prior art sequences of SEQ ID NO: 1 and 50, Reference A already contains the L5V mutation such that all three variants also contain V at positions 5 and 11 in addition to the specific mutations indicated. Each point represents the binding level of one of the 96 samples tested. The data points shown in the right-hand graph and the left-hand graph are the same; in the right-hand graph, the data points measured for each individual sample of each compound tested are connected by lines (so, the slope of the line gives an indication of the extent to which the binding of the pre-existing antibodies is reduced when the mutations and C-terminal alanine of the present invention are introduced);

[0721] - Figure 12 Details are shown of data from Figure 11 which were obtained in Example 3 for 11 samples from SLE patients;

[0722] - Figure 13 The table of Figure 11 lists the binding data for the data points compiled in

[0723] - Figure 14Shows two corresponding graphs of the data points obtained in Example 4, in which 96 serum samples were tested for binding to [Reference A], [Reference A + C-terminal alanine], and three variants of the present invention (i.e., [Reference A + L11V + R30T + V89L + C-terminal alanine], [Reference A + L11V + S31D + V89L + C-terminal alanine], and [Reference A + L11V + V89S + C-terminal alanine] respectively). 68 of the samples were from human healthy subjects, 17 were from healthy human volunteers whose sera contained pre-existing antibodies that were able to bind even in the presence of C-terminal alanine, and 11 were from SLE patients. It should be noted that with respect to the prior art sequences of SEQ ID NO: 1 and 50, Reference A already contains the L5V mutation, such that all three variants also contain V at positions 5 and 11 in addition to the specific mutations indicated. Each point represents the binding level of one of the 96 samples tested. The data points shown in the right-hand graph and the left-hand graph are the same; in the right-hand graph, the data points measured for each individual sample with each of the compounds tested are connected by lines (so, the slope of the lines gives an indication of the extent to which the binding of the pre-existing antibodies is reduced when the mutations and C-terminal alanine of the present invention are introduced);

[0724] - Figure 15 Shows in detail the data from Figure 14 which was obtained in Example 4 for 11 samples from SLE patients;

[0725] - Figure 16 The table of Figure 14 lists the binding data for the data points compiled in

[0726] - Figure 17Shows two corresponding graphs of the data points obtained in Example 5, where 96 serum samples were tested for binding to [Reference A], [Reference A + C-terminal alanine], and three variants of the present invention (i.e., [Reference A + L11V + V89N + C-terminal alanine], [Reference A + L11V + V89N + T110K + C-terminal alanine], and [Reference A + L11V + V89S + T110K + C-terminal alanine], respectively). Of these, 69 were from human healthy subjects, 17 were from healthy human volunteers whose sera contained pre-existing antibodies that were able to bind even in the presence of C-terminal alanine, and 10 were from SLE patients. It should be noted that with respect to the prior art sequences of SEQ ID NO:1 and 50, Reference A already contains the L5V mutation, such that all three variants also contain V at positions 5 and 11 in addition to the indicated specific mutations). Each point represents the binding level of one of the 96 samples tested. The data points shown in the right-hand graph and the left-hand graph are the same; in the right-hand graph, the data points measured for each individual sample with each of the compounds tested are connected by lines (so, the slope of the lines gives an indication of the extent to which the binding of pre-existing antibodies is reduced when the mutations and C-terminal alanine of the present invention are introduced);

[0727] - Figure 18 Details are shown of data from Figure 17 obtained in Example 5 with respect to 10 samples from SLE patients;

[0728] - Figure 19 The table of lists the binding data for the data points compiled in Figure 17 ;

[0729] - Figure 20Shows two corresponding graphs of the data points obtained in Example 6, where 96 serum samples (69 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre-existing antibodies that are able to bind even in the presence of C-terminal alanine, and 10 from SLE patients) were tested for binding to [Reference A], [Reference A + C-terminal alanine], and three variants of the present invention (i.e., [Reference A + L11V + V89L + S101H + C-terminal alanine], [Reference A + L11V + V89L + R102D + C-terminal alanine], and [Reference A + L11V + C-terminal alanine], respectively). It should be noted that with respect to the prior art sequences of SEQ ID NO:1 and 50, Reference A already contains the L5V mutation such that all three variants also contain V at positions 5 and 11 in addition to the indicated specific mutations). Each point represents the binding level of one of the 96 samples tested. The data points shown in the right-hand graph and the left-hand graph are the same; in the right-hand graph, the data points measured for each individual sample of each compound tested are connected by lines (so, the slope of the lines gives an indication of the extent to which the binding of the pre-existing antibodies is reduced when the mutations and C-terminal alanine of the present invention are introduced);

[0730] - Figure 21 Details are shown of data from Figure 20 obtained in Example 6 regarding 10 samples from SLE patients;

[0731] - Figure 22 The table of... lists the binding data of the data points compiled in Figure 20 ...

[0732] - Figure 23 Shows the alignment of the sequences of SEQ ID NO:1, 50, 119, and 121 - 132.

[0733] - Figure 24A Alignments of sequences A to C are of SEQ ID NO:145 - 184, SEQ ID NO:185 to 208, and SEQ ID NO:209 - 244, respectively, aligned with the sequences of SEQ ID NO:1, 50, and 119 in each case.

[0734] - Figure 25 Shows the binding data of some representative albumin binders having S, R, or T at position 30.

[0735] Experimental section

[0736] The human samples used in the experimental section below were obtained from commercial sources or human volunteers (after obtaining all required consents and approvals) and were used in accordance with applicable laws and regulatory requirements, including, but not limited to, those regarding medical secrecy and patient privacy.

[0737] In the following examples, unless otherwise specifically indicated, the binding of pre-existing antibodies present in the samples used (i.e., from healthy volunteers, rheumatoid arthritis (RA) patients, and SLE patients) to the tested nanobodies was determined using ProteOn as follows:

[0738] The nanobodies were captured on serum albumin or using monoclonal anti-FLAG M2 via the FLAG3 tag.

[0739] In the case of evaluating the binding of pre-existing antibodies to nanobodies captured on human serum albumin (HSA) using ProteOn XPR36 (Bio-Rad Laboratories, Inc.), PBS / Tween (phosphate buffered saline, pH 7.4, 0.005% Tween 20) was used as the running buffer and the experiment was performed at 25 °C. The ligand lane of the ProteOn GLC sensor chip was activated with EDC / NHS (flow rate 30 μl / min) and 10 μg / ml HAS in ProteOn acetate buffer pH 4.5 was injected (flow rate 100 μl / min) to reach an immobilization level of approximately 3200 RU. After immobilization, the surface was inactivated with ethanolamine HCl (flow rate 30 μl / min). The nanobodies were injected onto the HSA surface at 45 μl / min for 2 minutes to reach a nanobody capture level of approximately 200 RU. The samples containing pre-existing antibodies were centrifuged at 14,000 rpm for 2 minutes and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%) and then injected at 45 μl / min for 2 minutes, followed by a subsequent 400 s dissociation step. After each cycle (i.e., before the new nanobody capture and blood sample injection steps), the HSA surface was regenerated with a 2 minute injection of HCl (100 mM) at 45 μl / min. Sensing map processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). After double referencing by subtracting 1) nanobody-HSA dissociation and 2) non-specific binding to the reference ligand lane, a sensing map showing pre-existing antibody binding was obtained. The binding level of pre-existing antibodies was determined by setting the reporting point at 125 s (5 s after the end of binding). The percentage decrease in pre-existing antibody binding was calculated relative to the binding level of the reference nanobody at 125 s.

[0740] In the case of using a ProteOn XPR36 (Bio-Rad Laboratories, Inc.) to evaluate the binding of an existing antibody to a FLAG-tagged nanobody captured on monoclonal anti-FLAG M2 (Sigma), PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) was used as the running buffer and the experiment was carried out at 25 °C. The ligand channel of the ProteOn GLC sensor chip was activated with EDC / NHS (flow rate 30 μl / min), and 10 μg / ml anti-FLAG M2 mAb in ProteOn acetate buffer pH 4.5 was injected (flow rate 100 μl / min) to achieve an immobilization level of approximately 4000 RU. After immobilization, the surface was inactivated with ethanolamine HCl (flow rate 30 μl / min). The nanobody was injected onto the anti-FLAG M2 surface at 45 μl / min for 2 minutes to achieve a nanobody capture level of approximately 100 RU. To reduce non-specific binding of the blood sample to the anti-FLAG M2 surface, 100 nM 3xFLAG peptide (Sigma) was added to the blood sample. The sample containing the existing antibody was centrifuged at 14,000 rpm for 2 minutes, and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%), then injected at 45 μl / min for 2 minutes, followed by a subsequent 600-second dissociation step. After each cycle (i.e., before the new nanobody capture and blood sample injection steps), the anti-FLAG M2 surface was regenerated with a 10-second injection of glycine pH 1.5 (10 mM) at 150 μl / min. Sensing map processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). After double referencing by subtracting 1) the nanobody-anti-FLAG M2 dissociation and 2) the non-specific binding to the reference ligand channel, a sensing map showing the binding of the existing antibody was obtained. The binding level of the existing antibody was determined by setting the reporting point at 125 seconds (5 seconds after the end of binding). The percentage reduction in the binding of the existing antibody was calculated relative to the binding level of the reference nanobody at 125 seconds.

[0741] Example 1:

[0742] Binding of pre - existing antibodies present in 96 serum samples (68 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre - existing antibodies that can bind even in the presence of C - terminal alanine, and 11 from SLE patients) was tested for reference A (SEQ ID NO:119), reference A with C - terminal alanine, and three variants of the present invention (i.e., [reference A + L11V+V89T + C - terminal alanine], [reference A+L11V+V89T+T110K + C - terminal alanine], and [reference A+L11V+V89T+S104T + C - terminal alanine]), all of which have an N - terminal HIS6 tag. Immobilized human serum albumin was used to capture the compounds, and binding was measured using ProteOn according to the protocol given earlier in this experimental section.

[0743] Results are shown in Figure 5 (for all samples) and 6 (for SLE samples only). Figure 7 Listed are the results for each sample that forms one of the Figure 5 data points in.

[0744] Also, for the serum albumin binders tested, kinetic analysis (Langmuir, simultaneous ka / kd model) of the binding interaction with immobilized serum albumin was performed. The results are listed in Table C.

[0745] Table C:

[0746]

[0747] Example 2:

[0748] Binding of pre - existing antibodies present in 96 serum samples (68 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre - existing antibodies that can bind even in the presence of C - terminal alanine, and 11 from SLE patients) was tested for reference A (SEQ ID NO:119), reference A with C - terminal alanine, and three variants of the present invention (i.e., [reference A + L11V+V89A + C - terminal alanine], [reference A+L11V+V89A+T110K + C - terminal alanine], and [reference A+L11V+V89L+S104G + C - terminal alanine]), all of which have an N - terminal HIS6 tag. Immobilized human serum albumin was used to capture the compounds, and binding was measured using ProteOn according to the protocol given earlier in this experimental section.

[0749] Results are shown in Figure 8 (for all samples) and 9 (for SLE samples only). Figure 10 Listed are theFigure 8 The results for each sample of one of the data points in

[0750] Also, for the tested serum albumin binders, a kinetic analysis (Langmuir, simultaneous ka / kd model) of the binding interaction with immobilized serum albumin was performed. The results are listed in Table D.

[0751] Table D:

[0752]

[0753] Example 3:

[0754] Reference A (SEQ ID NO:119), reference A with a C-terminal alanine, and three variants of the present invention (i.e., [reference A+L11V+V89L+S101G+C-terminal alanine], [reference A+L11V+V89L+S104A+C-terminal alanine], and [reference A+L11V+V89L+S101E+C-terminal alanine]), all having an N-terminal HIS6 tag, were tested for binding to pre-existing antibodies present in 96 serum samples (68 from human healthy subjects, 17 from healthy human volunteers whose sera contained pre-existing antibodies that were able to bind even in the presence of C-terminal alanine, and 11 from SLE patients). Immobilized human serum albumin was used to capture the compounds, and binding was measured using a ProteOn according to the protocol given earlier in this experimental section.

[0755] The results are shown in Figure 11 (for all samples) and 12 (for SLE samples only). Figure 13 Listed are the results for each sample that forms one of the data points in Figure 11 The results for each sample of one of the data points in

[0756] Also, for the tested serum albumin binders, a kinetic analysis (Langmuir, simultaneous ka / kd model) of the binding interaction with immobilized serum albumin was performed. The results are listed in Table E.

[0757] Table E:

[0758]

[0759] Example 4:

[0760] Binding of pre - existing antibodies present in 96 serum samples (68 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre - existing antibodies that can bind even in the presence of C - terminal alanine, and 11 from SLE patients) was tested with reference A (SEQ ID NO:119), reference A with C - terminal alanine, and three variants of the present invention (i.e., [reference A+L11V+R30T+V89L+C - terminal alanine], [reference A+L11V+S31D+V89L+C - terminal alanine], and [reference A+L11V+V89S+C - terminal alanine]), all of which have an N - terminal HIS6 tag. Immobilized human serum albumin was used to capture the compounds, and binding was measured using ProteOn according to the protocol given earlier in this experimental section.

[0761] Results are shown in Figure 14 (for all samples) and 15 (for SLE samples only). Figure 16 Results for each sample that forms one of the data points in Figure 14 are listed.

[0762] Also, for the serum albumin binders tested, kinetic analysis (Langmuir, simultaneous ka / kd model) of the binding interaction with immobilized serum albumin was performed. Results are listed in Table F in.

[0763] Table F:

[0764]

[0765] Example 5:

[0766] Binding of pre - existing antibodies present in 96 serum samples (69 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre - existing antibodies that can bind even in the presence of C - terminal alanine, and 10 from SLE patients) was tested with reference A (SEQ ID NO:119), reference A with C - terminal alanine, and three variants of the present invention (i.e., [reference A+L11V+V89N+C - terminal alanine], [reference A+L11V+V89N+T110K+C - terminal alanine], and [reference A+L11V+V89S+T110K+C - terminal alanine]), all of which have an N - terminal HIS6 tag. Immobilized human serum albumin was used to capture the compounds, and binding was measured using ProteOn according to the protocol given earlier in this experimental section.

[0767] Results are shown in Figure 17 (for all samples) and 18 (for SLE samples only). Figure 19 Results for each sample that formsFigure 17 The results for each sample of one of the data points in

[0768] And, for the tested serum albumin binders, a kinetic analysis (Langmuir, simultaneous ka / kd model) of the binding interaction with immobilized serum albumin was performed. The results are listed in Table G in

[0769] Table G:

[0770]

[0771] Example 6:

[0772] Reference A (SEQ ID NO:119), reference A with a C-terminal alanine, and three variants of the present invention (i.e., [reference A + L11V + V89L + S101H + C-terminal alanine], [reference A + L11V + V89L + R102D + C-terminal alanine], and [reference A + L11V + C-terminal alanine]) were tested for binding to pre-existing antibodies present in 96 serum samples (69 from human healthy subjects, 17 from healthy human volunteers whose sera contain pre-existing antibodies that can bind even in the presence of C-terminal alanine, and 10 from SLE patients), all of which have an N-terminal HIS6 tag. Immobilized human serum albumin was used to capture the compounds, and binding was measured using a ProteOn according to the protocol given earlier in this experimental section.

[0773] The results are shown in Figure 20 (for all samples) and 21 (for SLE samples only). Figure 22 Listed are the results for each sample that forms one of the data points in Figure 20 The results for each sample of one of the data points in

[0774] And, for the tested serum albumin binders, a kinetic analysis (Langmuir, simultaneous ka / kd model) of the binding interaction with immobilized serum albumin was performed. The results are listed in Table H in

[0775] Table H:

[0776]

[0777] Example 7: Influence of the amino acid residue pair at position 30 on the binding to serum albumin.

[0778] The kinetic binding data (association rate, dissociation rate, and affinity) for the binding of some representative serum albumin binders (all having V at position 5 and V at position 11) to guinea pig serum albumin, rat serum albumin, mouse serum albumin, cynomolgus monkey serum albumin, and human serum albumin were determined using ProteOn. The sequence of SEQ ID NO:245 was used as a reference. The results are shown in Figure 25 and indicate that the tested albumin binders (having S, T, and R at position 30, respectively) have comparable affinities (expressed as KD values) for the different mammalian serum albumins used (i.e., comparable for binding to guinea pig serum albumin, comparable for binding to rat serum albumin, etc.).

[0779] The entire contents of all references cited in this application (including literature citations, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated by reference, particularly with respect to the teachings mentioned above.

Claims

1. An immunoglobulin single variable domain capable of binding to human serum albumin, wherein: the immunoglobulin single variable domain has no more than 7 "amino acid differences" from the sequence of SEQ ID NO: 1, where any possible C-terminal extension is not considered when determining the "amino acid differences" and the L5V and L11V mutations are not considered; and the immunoglobulin single variable domain has reduced binding to interfering factors, wherein: the no more than 7 "amino acid differences" are: L5V + L11V + V89T + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); the no more than 7 "amino acid differences" are: L5V + L11V + V89T + T110K + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); the no more than 7 "amino acid differences" are: L5V + L11V + V89T + S104T + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); the no more than 7 "amino acid differences" are: L5V + L11V + V89A + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); the no more than 7 "amino acid differences" are: L5V + L11V + V89A + T110K + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); the no more than 7 "amino acid differences" are: L5V + L11V + V89L + S104G + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + V89L + S101G + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLGR (SEQ ID NO:58); The no more than 7 "amino acid differences" are: L5V + L11V + V89L + S104A + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + V89L + S101E + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLER (SEQ ID NO:57); The no more than 7 "amino acid differences" are: L5V + L11V + R30T + V89L + C-terminal alanine, and CDR1 is GFTFTSFGMS (SEQ ID NO:53), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + S31D + V89L + C-terminal alanine, and CDR1 is GFTFRDFGMS (SEQ ID NO:54), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + V89S + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + V89N + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + V89N + T110K + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + V89S + T110K + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7); The no more than 7 "amino acid differences" are: L5V + L11V + V89L + S101H + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLHR (SEQ ID NO:59); The no more than 7 "amino acid differences" are: L5V + L11V + V89L + R102D + C-terminal alanine, and CDR1 is GFTFRSFGMS (SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSD (SEQ ID NO:60); or The no more than 7 "amino acid differences" are: L5V + L11V + C-terminal alanine, and CDR1 is GFTFRSFGMS(SEQ ID NO:5), CDR2 is SISGSGSDTL (SEQ ID NO:6), and CDR3 is GGSLSR (SEQ ID NO:7).

2. The immunoglobulin single variable domain according to claim 1, wherein: - CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO:53); and - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7).

3. The immunoglobulin single variable domain according to claim 1, wherein: - CDR1 is the amino acid sequence GFTFRDFGMS (SEQ ID NO:54); and - CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO:6); and - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:7).

4. A construct comprising at least one immunoglobulin single variable domain according to any one of claims 1-3.

5. The construct according to claim 4, comprising at least one therapeutic moiety or entity.

6. A pharmaceutical composition comprising the construct according to any one of claims 4-5.

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