Improved serum albumin binder

Abstract

This provides an amino acid sequence that binds to serum albumin. [Solution] The present invention relates to improved immunoglobulin monovariate domains (also referred to herein as "ISV" or "ISVD") that bind to serum albumin, more specifically, improved heavy-chain immunoglobulin monovariate domains (also referred to herein as "ISV" or "ISVD"), and proteins, polypeptides, and other constructs, compounds, molecules, or chemical entities comprising such improved serum albumin binders.

Description

[Technical Field]

[0001] This invention relates to an amino acid sequence that binds to serum albumin.

[0002] In particular, the present invention relates to improved immunoglobulin monovariable domains (also referred to herein as "ISV" or "ISVD") that bind to serum albumin, more specifically, improved heavy-chain immunoglobulin monovariable domains (also referred to herein as "ISV" or "ISVD"), and proteins, polypeptides, and other constructs, compounds, molecules or chemical entities comprising such improved serum albumin binders.

[0003] In particular, the present invention relates to improved nanobodies that bind to serum albumin, and to proteins, polypeptides, and other constructs, compounds, molecules, or chemical entities that contain such improved serum albumin-binding nanobodies.

[0004] The improved serum albumin-binding ISVD provided by the present invention is also referred herein to as “amino acid sequence of the present invention,” “serum albumin binder of the present invention,” “albumin binder of the present invention,” or “serum albumin binder.” Furthermore, proteins, polypeptides, and other constructs, compounds, molecules, or chemical entities comprising at least one serum albumin binder of the present invention are also referred herein to as “compound of the present invention” or “polypeptide of the present invention.”

[0005] Preferably, the polypeptide of the present invention is a fusion protein.

[0006] Other aspects, embodiments, features, uses, and advantages of the present invention will be apparent to those skilled in the art based on the disclosure herein.

[0007] In this application, amino acid residues / positions within the variable domains of immunoglobulin heavy chains will be indicated using Kabat numbering. For convenience, Figure 1 shows a table listing some of the amino acid positions that will be specifically referred to herein and their numbering by some alternative numbering systems (e.g., Aho and IMGT). Note: Unless otherwise specified, Kabat numbering is used herein and in the claims; other numbering systems are shown for reference only.

[0008] With regard to CDRs, as is well known in the art, there are several conventions for defining and describing CDRs of VH or VHH fragments, including the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of the structural loop region). For example, this is mentioned on the website http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and the claims, Kabat CDRs may also be described, but CDRs are most preferably defined based on the AbM definition (which is based on Oxford Molecular's AbM antibody modeling software), because this is considered the best compromise between the Kabat and Chothia definitions. This is also mentioned on the website http: / / www.bioinf.org.uk / abs / .

[0009] Therefore, in this specification and in the claims, all CDRs are defined according to the conventions of Abm unless otherwise specified herein.

[0010] ISVDs (especially nanobodies) capable of binding to serum albumin and their uses are well known in the art from, for example, International Publication Nos. 2004 / 041865, 2006 / 122787, European Patent No. 2139918, 2011 / 006915, 2012 / 175400, and 2014 / 111550, which describe serum albumin-binding ISVDs and their uses for extending the serum half-lives (as defined in these applications) of therapeutic compounds, parts, and entities.

[0011] International Publication No. 2006 / 122787 discloses a humanized serum albumin-binding nanobody called Alb-8 as SEQ ID NO: 62 (see SEQ ID NO: 50 herein). International Publication No. 2012 / 175400 discloses a humanized serum albumin-binding nanobody called Alb-23D as SEQ ID NO: 6 (see SEQ ID NO: 1 herein), and also discloses that some amino acid differences between Alb-8 and Alb-23D (e.g., the presence of an SKN motif at positions 74-76) may have a favorable effect on the physical properties of albumin binders. The amino acid sequences of Alb-8 and Alb-23D and their CDRs (which are the same for Alb-8 and Alb-23D) are shown in Table A below as SEQ ID NO: 50, SEQ ID NO: 1, and SEQ ID NOs. 2-7, respectively. Table A also shows the sequence of "Reference A," a reference compound used in the laboratory, as SEQ ID NO: 119. Figures 3A-3C show various alignments of SEQ ID NOs: 1, 50, and 119. Figure 3D is a graph showing the binding of existing antibodies derived from six serum albumin-depleted serums to Alb-23D, Alb-23 (same as Alb-23D but without C-terminal alanine, SEQ ID NO: 1 in International Publication No. 2006 / 122787), Reference A with C-terminal alanine, Reference A, Alb-8 with C-terminal alanine, and Alb-8. Nanobodies were immobilized using HIS6-FLAG3 tags, and the binding of existing antibodies in the samples was determined using ProteOn, essentially following the protocol used in the laboratory. The results in Figures 3A-3D show that binding by existing antibodies was equivalent for three nanobodies without C-terminal extension (i.e., Alb23, reference A, and Alb-8) and for three nanobodies with C-terminal extension (i.e., Alb23D, reference A-Ala, and Alb-8-Ala). Furthermore, it can be observed that adding C-terminal alanine to each of the three nanobodies tested resulted in a similar reduction in binding by existing antibodies.

[0012] [Table 1]

[0013] The present invention aims to provide a serum albumin binder that is improved compared to the serum albumin binders disclosed in International Publication No. 2006 / 122787 and International Publication No. 2012 / 175400.

[0014] More specifically, the present invention aims to provide improved serum albumin-binding nanobodies, variants of serum albumin-binding nanobodies described in International Publication No. 2006 / 122787 and International Publication No. 2012 / 175400, which have reduced binding by interfering factors (generally referred to as “existing antibodies”) that may be present in the serum of several healthy human subjects and patients. This is referred to in International Publication No. 12 / 175741, International Publication No. 2013 / 024059 and, for example, in Holland et al. (J. Clin. Immunol. 2013, 33(7):1192-203), as well as in concurrently pending, unpublished PCT application PCT / EP2015 / 060643, filed on 13 May 2015 and titled “Improved immunoglobulin variable domains”.

[0015] The improved serum albumin binder provided by the present invention is also referred to herein as "the serum albumin binder of the present invention."

[0016] Among the serum albumin-binding agents listed in Table A, Alb-23D has a C-terminal alanine extension (sometimes referred to as "position 114") at the C-terminus of the ISVD sequence, i.e., an alanine residue, compared to the normal C-terminal sequence VTVSS (sequence number 116, as found in Alb-8). As described in International Publication No. 12 / 175741, this C-terminal alanine extension can prevent the binding of so-called "existing antibodies" (presumably IgG) to putative epitopes located in the C-terminal region of ISV. This epitope is expected to include, among other residues, the amino acid residue of the surface-exposed C-terminal sequence VTVSS, as well as the amino acid residue at position 14 (and adjacent / nearby amino acid residues in the amino acid sequence, such as positions 11, 13, and 15), and may also include the amino acid residue at position 83 (and adjacent / nearby amino acid residues in the amino acid sequence, such as positions 82, 82a, 82b, and 84) and / or the amino acid residue at position 108 (and adjacent / nearby amino acid residues in the amino acid sequence, such as position 107).

[0017] However, while the presence of such C-terminal alanine (or more commonly, C-terminal extension) can significantly reduce (and often even completely prevent) the binding of “pre-existing antibodies” that may be found in serum from a range of subjects (both healthy subjects and patients), it has been found that serum from some subjects (e.g., serum from patients with certain immune disorders such as SLE) can contain pre-existing antibodies that can bind to the C-terminal region of ISV (if such a region is exposed), even if the ISV contains such C-terminal alanine (or more commonly, such C-terminal extension). Here again, reference is made to the concurrently pending, unpublished PCT application PCT / EP2015 / 060643 filed on 13 May 2015 and titled “Improved immunoglobulin variable domains”.

[0018] Therefore, one particular object of the present invention is to provide a serum albumin-binding agent that is an improved variant of the serum albumin-binding nanobodies listed in Table A, and which exhibits reduced binding by so-called “existing antibodies,” particularly those of the type described in PCT / EP2015 / 060643 (i.e., existing antibodies capable of binding to the exposed C-terminal region of ISV even in the presence of C-terminal elongation), and also exhibits a low frequency and / or low degree of T cell response in dendritic cell-T cell proliferation assays (and / or a reduced frequency and / or low degree of T cell response in dendritic cell / T cell proliferation assays compared to SEQ ID NO: 1 and / or SEQ ID NO: 50). This assay, which measures the frequency and degree of T cell response, is designed to identify proteins that may induce helper T cell proliferation and thus lead to the development of a helper T cell immune response.

[0019] Generally, the present invention achieves this objective by providing an amino acid sequence that is a variant of the amino acid sequence of SEQ ID NO: 1 (as further described herein) and as further described herein (and as will be apparent to those skilled in the art based on the alignment shown in the figure and based on further disclosure herein, 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), so the serum albumin-binding nanobody disclosed herein is also a variant of the sequence of SEQ ID NO: 50).

[0020] These amino acid sequences are also referred to herein as “the amino acid sequences of the present invention” or “the serum albumin binding agent of the present invention.” Some preferred but non-limiting examples of the amino acid sequences of the present invention are shown in Figure 2 as SEQ ID NOs: 8-49 (sequences without C-terminal alanine elongation) and SEQ ID NOs: 61-102 (corresponding to the sequences of SEQ ID NOs: 8-49, but each representing an amino acid sequence with C-terminal alanine elongation); also see the alignments in Figures 4A and 4B and Figures 23 and 24, respectively. As can be found from these alignments and further disclosures herein, compared to the sequences of SEQ ID NO: 1 and / or SEQ ID NO: 50, the albumin binders of the present invention have (i) V at position 5 and V at position 11; and / or (ii) one or more of the following amino acid residues (i.e., in appropriate combinations) (29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T) (i.e., compared to the sequence of SEQ ID NO: 1, 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). They preferably have both V at position 5 and V at position 11, as well as one or more of the aforementioned amino acid residues / mutations. Also, as will be apparent from these alignments and further disclosures herein, the amino acid residue at position 89 is preferably selected from T, A, or L. In general, the albumin-binding amino acid sequences described herein preferably have at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongations and L5V and L11V mutations are not considered for the determination of sequence identity); and / or have 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" with respect to the sequence of SEQ ID NO: 1 (as defined herein, with no consideration of possible C-terminal elongations and no consideration of L5V and L11V mutations).Furthermore, in some aspects of the present invention, the amino acid sequences disclosed herein are defined with reference to the sequence of Sequence ID No. 50, in which case the albumin-binding amino acid sequences described herein preferably have at least 85%, preferably at least 90%, and more preferably at least 95% sequence identicality to the sequence of Sequence ID No. 50 (compared to Sequence ID No. 50, any possible C-terminal elongations and L11V mutations are not considered for the determination of sequence identicality); and / or have 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration of possible C-terminal elongations and no consideration of L11V mutations). Furthermore, if an aspect of the present invention is defined by reference to both sequence identity and / or the amount of amino acid difference compared to Sequence ID No. 1 (and / or possibly Sequence ID No. 50), and by reference to the presence of specific mutations (i.e., L5V, L11V, and any other specific mutations specified for that aspect of the present invention), and / or by reference to the presence of one or more specific CDRs, then these specified mutations and / or CDRs should also not be considered when determining the number of sequence identity and / or amino acid differences, respectively.

[0021] Some preferred amino acid residues / mutations that can be present in the amino acid sequence of the present invention are 29H (i.e., F29H compared to SEQ ID NO: 1), 101D (i.e., S101D compared to SEQ ID NO: 1), and 104T (i.e., S104T compared to SEQ ID NO: 1), or any two of these, or any suitable combination of all three of these. Furthermore, these preferred mutations are preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1), or alternatively with L at position 89 (i.e., V89L compared to SEQ ID NO: 1).

[0022] Several other preferred amino acid residues / mutations that may be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both of these. Also, these preferred mutations are preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively A at position 89 (i.e., V89A compared to SEQ ID NO: 1), or alternatively L at position 89 (i.e., V89L compared to SEQ ID NO: 1).

[0023] Another of several other preferred amino acid residues / mutations that may be present is 30S (i.e., R30S compared to SEQ ID NO: 1), which is combined with T, A or L (preferably L) at position 104, and with G or T (preferably further combined with some or all of the further preferred mutations described herein for this aspect of the invention, for example with L at position 89 and N at position 16). Accordingly, the amino acid sequence according to this aspect may have the same CDRs (according to Abm) as 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 compared to the sequence of SEQ ID NO: 50: (i) L11V; (ii) V89T, V89A or V89L (especially V89L), and (iii) S104G or S104T. Another preferred mutation is 31D: in particular, this may result in an amino acid sequence of the invention having CDR1 that is the sequence GFTFRDFGMS (SEQ ID NO: 54).

[0024] Some further preferred amino acid sequences of the present invention, comprising further combinations of some of the specific mutations described herein for the serum albumin binding agent of the present invention, are shown in Figure 2 as SEQ ID NOs: 121 to 144 [Note: the sequences of SEQ ID NO: 121 and 122, and the sequences of SEQ ID NO: 127 and 128 are identical]. Among these, SEQ ID NOs: 121 to 126 have E at position 1 and a C-terminal alanine, and SEQ ID NOs: 127 to 132 have D at position 1 and a C-terminal alanine. SEQ ID NOs: 133 to 138 and SEQ ID NOs: 139 to 143 correspond to SEQ ID NOs: 121 to 126 and SEQ ID NOs: 127 to 132, respectively, but do not have a C-terminal alanine. Figure 23 also shows an alignment of the sequences of SEQ ID NO: 1, 50 and 119 and the sequences of SEQ ID NOs: 121 to 132. Based on the disclosure herein, it will be apparent to those skilled in the art that, in practice, the albumin binding agents of SEQ ID NOs: 121 to 132, in particular SEQ ID NOs: 121 to 126, are often used as / are present at the C-terminus of the constructs in which they reside, and the albumin binding agents of SEQ ID NOs: 139 to 144 are often used as / are present at the N-terminus of the constructs in which they reside (similarly, the albumin binding agents of SEQ ID NOs: 133 to 138 are often present "in the middle of such constructs", and the serum albumin binding agents of SEQ ID NOs: 127 to 132 are particularly suitable for use in monovalent form, for example for research purposes). Each of the amino acid sequences of SEQ ID NOs: 121 to 144, as well as proteins, polypeptides and other compounds and constructs comprising the same (as further described herein) form further aspects of the present invention.

[0025] Several further preferred amino acid sequences of the present invention, including several further combinations of the specific mutations described herein for the serum albumin conjugates of the present invention, are shown in Figure 2 as SEQ ID NOs: 145-184, 185-208, and 209-244. Figures 24A-C also show the alignments of the sequences SEQ ID NOs: 145-184, 185-208, and 209-244, respectively, all aligned with the sequences SEQ ID NOs: 1, 50, and 119. As with the other albumin conjugates of the present invention described herein, the sequences SEQ ID NOs: 145-184, 185-208, and 209-244, having C-terminal elongation, will typically be present at the C-terminus of the (fusion) protein or construct of the present invention in which they exist. Similarly, those having D at position 1 (but without any C-terminal elongation) are preferably located at the N-terminus of the (fusion) protein or construct of the present invention in which they exist, and those having E at position 1 (but without any C-terminal elongation) may be located at the C-terminus or (in the case of trivalent constructs or constructs with even higher binding titers) "center" of the (fusion) protein or construct of the present invention in which they exist. The amino acid sequences of SEQ ID NOs. 145-184, SEQ ID NOs. 185-208, and SEQ ID NOs. 209-244, respectively, and the proteins, polypeptides, and other compounds and constructs (as further described herein) containing them, form further embodiments of the present invention.

[0026] Furthermore, some (mutations present in) the serum albumin conjugates disclosed herein may, in addition to exhibiting a (strong) reduced tendency to bind to existing antibodies as described herein, or essentially no tendency to bind at all, possess other desirable properties (i.e., compared to albumin conjugates disclosed in the prior art, such as, for example, SEQ ID NO: 1 and / or SEQ ID NO: 50, and / or compared to some of the other serum albumin conjugates of the present invention described herein). For example, but not limited to, some of the mutations (combinations thereof) present in the serum albumin conjugates disclosed herein may result in elevated expression levels in a desired host or expression system (e.g., E. coli, Pichia Pastoris, or mammalian cells), and / or a reduced tendency to form dimers (see, for example, International Publication No. 2010 / 100135), or reduced immunogenicity. For example, some of the mutations described herein may eliminate immunogenic epitopes (particularly T-cell epitopes) without essentially having a significant impact on other properties of the albumin conjugate, such as affinity. For example, and not limited to, serum albumin conjugates of the present invention having T at position 30 (as further described herein) (e.g., sequences of SEQ ID NOs. 25, 44, 45, 78, 97 and 98, in particular SEQ ID NOs. 121-144 and 145-184) are expected (based on standard computer predictions) to have reduced immunogenicity (i.e., because candidate T cell epitopes are removed) particularly compared to sequences of SEQ ID NOs. 1 and 50 of the prior art, but also (possibly) compared to other albumin conjugates of the present invention that do not contain T at position 30. Similarly, serum albumin conjugates of the present invention, such as SEQ ID NOs. 185-208, containing S at position 30 and G or T at position 104, are expected (again, based on standard computer predictions) to have reduced immunogenicity (i.e., because candidate T cell epitopes are removed) particularly compared to sequences of SEQ ID NOs. 1 and 50 of the prior art, but also (possibly) compared to other albumin conjugates of the present invention.Furthermore, some of the mutations described herein (e.g., 31D, see, e.g., amino acid sequences of SEQ ID NOs. 209-244) are expected to improve the expression and / or productivity of the amino acid sequences of the present invention (e.g., in Pichia pastrix or similar yeasts used as a host) compared, again, with the sequences of SEQ ID NOs. 1 and / or 50 of the prior art, and / or with other amino acid sequences of the present invention that do not contain the 31D mutation.

[0027] The serum albumin binder of the present invention has a CDR (by Abm) as described herein. Several preferred combinations of CDR, CDR2, and CDR3 that may be present in the serum albumin binder of the present invention are listed in Table B below. Particularly preferred combinations are indicated in bold / underlined.

[0028] [Table 2] TIFF2026143844000003.tif154165

[0029] One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0030] Other particularly preferred combinations of CDRs are GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLSR (SEQ ID NO: 7).

[0031] According to a non-limiting aspect of the present invention, if 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).

[0032] 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 GGSLER (SEQ ID NO: 57).

[0033] According to yet another aspect of the present invention, in the albumin binder 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, preferably N; (ii) the amino acid residue at position 45 is P or L, preferably L; (iii) the amino acid residues at positions 74-76 form an SKN or AKT motif, preferably an AKT motif; and (iv) the amino acid residue at position 89 is L, A, or T, preferably 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, preferably L.

[0034] As further described herein, if 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-terminus of said compound or polypeptide of the present invention, the serum albumin binder of the present invention (and, extended, the compound or polypeptide of the present invention) preferably has a C-terminal elongation (Xn) as further described herein. Sequence IDs 61-102 show some non-limiting examples of albumin binders of the present invention having a C-terminal elongation (in this case, a C-terminal alanine).

[0035] When the serum albumin binder of the present invention forms and / or is present at the N-terminus of the compound or polypeptide of the present invention (and, when extended, the compound or polypeptide of the present invention), the serum albumin binder of the present invention preferably has D at position 1 (i.e., an E1D mutation compared to the sequences of SEQ ID NOs: 8-49).

[0036] The amino acid sequence of the present invention preferably binds to (human) serum albumin with an affinity better than 100 nM, preferably better than 50 nM. For example, the albumin binder of the present invention may have an affinity for (human) serum albumin that is the same magnitude as the affinity of SEQ ID NO: 1 and / or SEQ ID NO: 50 for human serum albumin. See, for example, the kinetic data shown in Example 1.

[0037] Furthermore, the albumin binders provided by the present invention, and the compounds and polypeptides of the present invention containing them (as further described herein), preferably have a human half-life (defined as t1 / 2β) of 1 hour or more, preferably 2 hours or more, more preferably 6 hours or more, for example 12 hours or more, for example about 1 day, 2 days, 1 week, 2 weeks, and up to the time to the half-life of human serum albumin (estimated to be about 19 days), although the latter may not be very important. For example, the albumin binders of the present invention may have a human half-life equivalent to (preferably approximately the same as) that of SEQ ID NO: 1 and / or SEQ ID NO: 50. Also, the compounds or polypeptides of the present invention containing the albumin binders of the present invention may have a human half-life equivalent to (preferably approximately the same as) that of the same compounds or polypeptides containing SEQ ID NO: 1 and / or SEQ ID NO: 50 instead of the albumin binders of the present invention.

[0038] In a first aspect, the present invention relates to an amino acid sequence (the amino acid sequence is a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residues at positions -74 to -76 are the motif SKN; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; The amino acid sequence may, in some cases, -At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); and / or - Contains at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G and / or 104T (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T), The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0039] In one specific but non-limiting aspect, the serum albumin binder of the present invention is an amino acid sequence as described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting aspect, the serum albumin binder of the present invention is an amino acid sequence as described herein, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting aspect, the serum albumin binder of the present invention is an amino acid sequence as described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting aspect, the serum albumin binder of the present invention is an amino acid sequence as described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting aspect, the serum albumin binder of the present invention is an amino acid sequence as described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the serum albumin binder of the present invention is an amino acid sequence as described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is an amino acid sequence as described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In a still more specific but non-limiting aspect, the amino acid sequence of the present invention is an amino acid sequence as described in the previous 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.

[0040] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1) or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0041] With respect to this first aspect of the Invention and any other (specific and / or preferred) aspects, when it is said that the amino acid sequence of the Invention has (i) at least 85%, preferably at least 90%, more preferably at least 95% sequence identical to the sequence of Sequence ID No. 1 (any possible C-terminal elongations and L5V and L11V mutations are not considered for the determination of sequence identicalness); and / or (ii) 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no possible C-terminal elongations and no possible L5V and L11V mutations) to the sequence of Sequence ID No. 1, it should be noted that this also includes sequences that have no amino acid differences to the sequence of Sequence ID No. 1 other than (i) L5V and L11V mutations; (ii) any possible C-terminal elongations; and (iii) any specific amino acid residues / mutations and / or CDRs described for the foregoing aspects (i.e., those required to be present according to that aspect). It should also be noted that the amino acid sequences of the present invention in any embodiment described herein may have no amino acid differences from SEQ ID NO: 1 other than (i) L5V and L11V mutations; (ii) any possible C-terminal elongation; and (iii) any specific amino acid residue / mutation and / or CDR described for the embodiments (i.e., those required to be present according to that embodiment). As described herein, this is applied to the sequences defined herein with necessary modifications, with reference to the number of sequence identicals and / or amino acid differences to SEQ ID NO: 50 (instead of SEQ ID NO: 1).

[0042] Furthermore, if the amino acid sequence of the present invention according to any embodiment of the present invention has one or more amino acid differences with respect to the sequence of SEQ ID NO: 1 (other than the L5V and L11V mutations, and certain amino acid residues / mutations that may and / or may be required to be present according to the embodiments described herein), some specific but non-limiting examples of such mutations / amino acid differences (i.e., compared to the sequence of SEQ ID NO: 1) that may exist are, for example, E1D (i.e., when a serum albumin binder is at the N-terminus of the polypeptide of the present invention) and, for example, one or more appropriate "humanization" substitutions (or appropriate combinations thereof); for example, these are mentioned in International Publication No. 09 / 138519 (or in the prior art cited in International Publication No. 09 / 138519) and International Publication No. 08 / 020079 (or in the prior art cited in International Publication No. 08 / 020079), and in Tables A-3 to A-8 of International Publication No. 08 / 020079 (which is a list showing possible humanization substitutions). As described herein, this applies to the sequences defined herein with necessary modifications, taking into account the number of sequence identicals and / or amino acid differences for SEQ ID NO: 50 (instead of SEQ ID NO: 1).

[0043] In another embodiment, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, particularly a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residues at positions -74 to -76 are the motif SKN; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; In the amino acid sequence: -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); The amino acid sequence is sequence identical to the sequence of Sequence ID No. 1 by at least 85%, preferably at least 90%, and more preferably at least 95% (any possible C-terminal elongations and L5V and L11V mutations are not considered for determining sequence identicalness); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0044] In one specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In yet another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In a still more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous 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.

[0045] As described, one particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLSR (SEQ ID NO: 7).

[0046] In a further embodiment, the present invention relates to one of the amino acid sequences of SEQ ID NOs. 8, SEQ ID NOs. 9, SEQ ID NOs. 10, SEQ ID NOs. 11, SEQ ID NOs. 12, SEQ ID NOs. 13, SEQ ID NOs. 14, SEQ ID NOs. 15, SEQ ID NOs. 16, SEQ ID NOs. 17, SEQ ID NOs. 18, SEQ ID NOs. 19, SEQ ID NOs. 20, SEQ ID NOs. 21, SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 24, SEQ ID NOs. 25, SEQ ID NOs. 26, SEQ ID NOs. 27, SEQ ID NOs. 28, SEQ ID NOs. 29, SEQ ID NOs. 30, SEQ ID NOs. 31, SEQ ID NOs. 32, SEQ ID NOs. 33, SEQ ID NOs. 34, SEQ ID NOs. 35, SEQ ID NOs. 36, SEQ ID NOs. 37, SEQ ID NOs. 38, SEQ ID NOs. 39, SEQ ID NOs. 40, SEQ ID NOs. 41, SEQ ID NOs. 42, SEQ ID NOs. 43, SEQ ID NOs. 44, SEQ ID NOs. 45, SEQ ID NOs. 46, SEQ ID NOs. 47, SEQ ID NOs. 48, or SEQ ID NOs. 49, or a combination of these. The present invention relates to an amino acid sequence that is one of the amino acid sequences of sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65, sequence number 66, sequence number 67, sequence number 68, sequence number 69, sequence number 70, sequence number 71, sequence number 72, sequence number 73, sequence number 74, sequence number 75, sequence number 76, sequence number 77, sequence number 78, sequence number 79, sequence number 80, sequence number 81, sequence number 82, sequence number 83, sequence number 84, sequence number 85, sequence number 86, sequence number 87, sequence number 88, sequence number 89, sequence number 90, sequence number 91, sequence number 92, sequence number 93, sequence number 94, sequence number 95, sequence number 96, sequence number 97, sequence number 98, sequence number 99, sequence number 100, sequence number 101, or sequence number 102. Each of these amino acid sequences of the present invention forms a further embodiment of the present invention.

[0047] In a further embodiment, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NOs: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144. Each of these amino acid sequences of the present invention forms a further embodiment of the present invention.

[0048] In a further embodiment, 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 a further embodiment of the present invention.

[0049] In a further embodiment, 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 a further embodiment of the present invention.

[0050] In a further aspect, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NOs: 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, and 208. Each of these amino acid sequences of the present invention forms a further aspect of the present invention.

[0051] In a further embodiment, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NOs: 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, and 244. Each of these amino acid sequences of the present invention forms a further embodiment of the present invention.

[0052] In a specific but non-limiting embodiment, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, and more particularly a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residues at positions -74 to -76 are the motif SKN; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; In the amino acid sequence, -CDR1 has the amino acid sequence GFTFRSFGMS (SEQ ID NO: 5); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 7); The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0053] In one specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In yet another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T.

[0054] In a further embodiment, 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 a further embodiment of the present invention.

[0055] In a further embodiment, 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.

[0056] In a further embodiment, 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.

[0057] In a further embodiment, 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.

[0058] In a further embodiment, 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.

[0059] In a further embodiment, 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.

[0060] In a further embodiment, 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.

[0061] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residues at positions -74 to -76 are the motif SKN; The amino acid residue at position -89 (according to Kabat) is selected from L, T, or V; The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; In the amino acid sequence: -CDR1 has the amino acid sequence GFTFRSFGMS (SEQ ID NO: 5); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 7); The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0062] In one specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In yet another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T.

[0063] In a further embodiment, 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 a further embodiment of the present invention.

[0064] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residues at positions -74 to -76 are the motif SKN; The amino acid residue at position -89 (according to Kabat) is selected from A, N, or S; The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; At the relevant amino acid residue: -CDR1 has the amino acid sequence GFTFRSFGMS (SEQ ID NO: 5); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 7); The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0065] In one specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In yet another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T.

[0066] In a further embodiment, 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 a further embodiment of the present invention.

[0067] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residues at positions -74 to -76 are the motif SKN; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, or T; The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; In the amino acid sequence: -CDR1 has the amino acid sequence GFTFRSFGMS (SEQ ID NO: 5); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 7); The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0068] In one specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In yet another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is A.

[0069] In a further embodiment, 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 a further embodiment of the present invention.

[0070] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; The amino acid sequence is, -At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); and / or - Containing at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, or 102D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, or R102D), The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0071] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residue at positions 74-76 is preferably the motif SKN (however, it may also be AKT, as exemplified by the sequences of SEQ ID NOs. 124-126 and 130-132, for example). In one specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In a still more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous 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.

[0072] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One particularly preferred combination of CDRs (the CDR containing the 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 shown in SEQ ID NOs: 169-184.

[0073] Other particularly preferred combinations of CDRs (which contain a 30T residue but not a 101E residue) are 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 also contain G or N at position 16, P or L at position 45, an SKN or AKT motif at positions 74-76, L, T, or A at position 89, and preferably G at position 104 (for the remainder, these amino acid sequences of the present invention may be as further defined herein). Some specific but non-limiting examples of such amino acid sequences of the present invention are shown as SEQ ID NOs: 121-144 and SEQ ID NOs: 145-168.

[0074] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; The amino acid sequence is, - Containing at least one amino acid residue selected from -29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); The amino acid sequence is, - Containing at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, or 102D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, or R102D), The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0075] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN. In one specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In a still more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous 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.

[0076] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0077] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; In the amino acid sequence, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); If CDR1 is GFTFRSFGMS (sequence number 5), then CDR2 is not GGSLSR (sequence number 7) (and vice versa). The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0078] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residue at positions 74-76 is preferably the motif SKN (however, it may also be AKT, as exemplified by the sequences of SEQ ID NOs. 124-126 and 130-132, for example). In one specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In a still more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous 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 described, one particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0080] Other particularly preferred combinations of CDRs are 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 also contain G or N at position 16, P or L at position 45, an SKN or AKT motif at positions 74-76, L, T or A at position 89, and preferably G at position 104 (for the remainder, these amino acid sequences of the present invention may be as further defined herein). Some specific but non-limiting examples of such amino acid sequences of the present invention are shown as SEQ ID NOs: 121-132.

[0081] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; In the amino acid sequence, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0082] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN. In one specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In an even more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous 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.

[0083] As described, one particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0084] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; The amino acid residue is, -At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); and / or - Containing at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, or 102D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, or R102D), In the amino acid sequence, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0085] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residue at positions 74-76 is preferably the motif SKN (however, it may also be AKT, as exemplified by the sequences of SEQ ID NOs. 124-126 and 130-132, for example). In one specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In a still more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous 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.Furthermore, according to this embodiment, the amino acid sequence of the present invention should contain at least one mutation as described herein (compared to the sequence of SEQ ID NO: 1) at any of the positions 29, 30, 31, 99, 101, or 102. Therefore, it should be noted that in the amino acid sequence of the present invention according to this embodiment, if CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 cannot be GGSLSR (SEQ ID NO: 7), and vice versa.

[0086] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., L89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One 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). If the amino acid sequences of the present invention contain these CDRs, the amino acid sequences may also contain G or N at position 16, P or L at position 45, an SKN or AKT motif at positions 74-76, L, T or A at position 89, and preferably G at position 104 (for the remainder, these amino acid sequences of the present invention may be as further defined herein). Some specific but non-limiting examples of such amino acid sequences of the present invention are shown as SEQ ID NOs: 121-132.

[0087] In a further embodiment, the present invention relates to one of the amino acid sequences of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49, or This invention relates to an amino acid sequence that is one of the amino acid sequences of SEQ ID NOs. 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, or 102. Each of these amino acid sequences of the present invention forms a further embodiment of the present invention.

[0088] In yet another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be A in particular); The amino acid residue at position -104 (according to Kabat) is selected from A, G, S, or T, and is particularly likely to be S, or may be selected from A, G, or T (and is particularly likely to be T if selected from A, G, and T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; The amino acid sequence is, - Containing at least one amino acid residue selected from -29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); The amino acid sequence is, - Containing at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, or 102D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, or R102D), In the amino acid sequence, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 is an amino acid sequence selected from the following amino acid sequences: GGLSR (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); The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there must be 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). It holds.

[0089] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residues at positions 74-76 are preferably the motif SKN. In one specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from L, T, or V. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is T. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is selected from A, S, or N. In a more specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 89 (according to Kabat) is A. In another specific but non-limiting embodiment, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is S. In another specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. In a more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous paragraph, wherein the amino acid residue at position 104 (according to Kabat) is T. In a still more specific but non-limiting aspect, the amino acid sequence of the present invention is the amino acid sequence described in the previous 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.

[0090] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0091] In a further embodiment, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 45, 46, 47, or 48, or one of the amino acid sequences of SEQ ID NOs: 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 91, 93, 95, 97, 98, 99, 100, or 101. Each of these amino acid sequences of the present invention forms a further embodiment of the present invention.

[0092] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -16 (according to Kabat) is either G or N; The amino acid residue at position -45 (according to Kabat) is either P or L; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and is particularly likely to be A, L, or T; The amino acid residue at position -104 (according to Kabat) is G; The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; The amino acid sequence is, -CDR1 is GFTFTSFGMS (SEQ ID NO: 53), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7). Contains; The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 1 (any possible C-terminal elongations and mutations L5V, L11V, R30T, and S104G are not considered for determining sequence identity); and / or - No more than 7, preferably 5 or fewer, "amino acid differences" relative to the sequence of Sequence ID No. 1, for example, just 3, 2, or 1 (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V, L11V, R30T, and S104G mutations). It holds.

[0093] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residues at positions 74 to 76 are preferably motif SKN or motif AKT.

[0094] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), The amino acid residue at position -5 (according to Kabat) is V; The amino acid residue at position -11 (according to Kabat) is V; The amino acid residue at position -16 (according to Kabat) is either G or N; The amino acid residue at position -45 (according to Kabat) is either P or L; The amino acid residue at position -89 (according to Kabat) is selected from A, L, N, S, T, or V, and is particularly likely to be A, L, or T; The amino acid residue at position -104 (according to Kabat) is G; The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; The amino acid residue at position -112 (according to Kabat) is selected from S, K, or Q; The amino acid sequence is, -CDR1 is GFTFTSFGMS (SEQ ID NO: 53), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7). Contains; The amino acid sequence is, - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of SEQ ID NO: 50 (any possible C-terminal elongations and mutations L5V, L11V, R30T, and S104G are not considered for determining sequence identity); and / or - No more than 7, preferably 5 or fewer, "amino acid differences" (as defined herein, with no consideration of possible C-terminal elongation, and no consideration of L5V, L11V, R30T, and S104G mutations) relative to the sequence of SEQ ID NO: 50. It holds.

[0095] In the amino acid sequence of the present invention as described in the previous paragraph, the amino acid residues at positions 74 to 76 are preferably motif SKN or motif AKT.

[0096] In a further embodiment, the present invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NOs: 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, or 144. Each of these amino acid sequences of the present invention forms a further embodiment of the present invention.

[0097] In another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), wherein the amino acid sequence is - At least 85%, preferably at least 90%, more preferably at least 95% of the sequence of sequence number 1; and / or - A difference of 7 or less, preferably 5 or less, relative to the sequence of SEQ ID NO: 1, for example, just 3, 2, or 1 amino acid. The amino acid sequence is such that the amino acid residue at position 89 is selected from A, N, and S (according to Kabat). Preferably, in the amino acid sequence according to this embodiment, (i) the amino acid residue at position 5 (according to Kabat) is L or V (more preferably V); (ii) the amino acid residue at position 11 (according to Kabat) is L or V (more preferably V); (iii) the amino acid residue at position 104 (according to Kabat) is selected from A, G, S, or T, and may be particularly S, or may be selected from A, G, or T (if selected from A, G, and T, it may be particularly 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. Furthermore, the amino acid sequence according to this embodiment may optionally contain (i) at least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain 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 (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T).Most preferably, in the amino acid sequence according to this embodiment, (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 embodiment, CDR1 is GFTFRSFGMS (SEQ ID NO: 5), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7). Furthermore, an amino acid sequence in this embodiment in which the amino acid residue at position 89 is A (or alternatively T) is particularly preferred.

[0098] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0099] In a further embodiment, the present invention relates to one of the amino acid sequences of SEQ ID NOs: 14, 15, 16, 17, 18, 19, 36, 37, 38, 39, 43, 44, 45, 46, 47, 48, or 49, or SEQ ID NOs: 67, 68, 69, 70, 71, 72, 89, 90, 91, 92, 96, 97, 98, 99, 100, sequence The present invention relates to one of the amino acid sequences of number 101 or sequence number 102 (in particular, one of the amino acid sequences of sequence number 14, sequence number 15, sequence number 36, sequence number 37, sequence number 38, sequence number 39, sequence number 43, sequence number 44, sequence number 45, sequence number 46, sequence number 47, sequence number 48 or sequence number 49, or one of the amino acid sequences of sequence number 67, sequence number 68, sequence number 89, sequence number 90, sequence number 91, sequence number 92, sequence number 96, sequence number 97, sequence number 98, sequence number 99, sequence number 100, sequence number 101 or sequence number 102). Each of these amino acid sequences of the present invention forms a further embodiment of the present invention.

[0100] In yet another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), wherein the amino acid sequence is - At least 85%, preferably at least 90%, more preferably at least 95% of the sequence of sequence number 1; and / or - A difference of 7 or less, preferably 5 or less, relative to the sequence of SEQ ID NO: 1, for example, just 3, 2, or 1 amino acid. The amino acid sequence is such that the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A. Preferably, in the amino acid sequence according to this embodiment, (i) the amino acid residue at position 5 (according to Kabat) is L or V (more preferably V); (ii) the amino acid residue at position 11 (according to Kabat) is L or V (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 may be selected particularly from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be particularly 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. Furthermore, in the amino acid sequence according to this embodiment, the amino acid sequence may optionally contain (i) at least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain 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 (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T). In particular, they may contain the S101D mutation compared to SEQ ID NO: 1.Most preferably, in the amino acid sequence according to this embodiment, (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 embodiment, CDR1 is GFTFRSFGMS (SEQ ID NO: 5), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7). Furthermore, an amino acid sequence in this embodiment in which the amino acid residue at position 89 is T (or alternatively A) is particularly preferred.

[0101] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0102] In a further embodiment, the present invention relates to one of the amino acid sequences of SEQ ID NOs: 20, 21, 22, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49, or SEQ ID NOs: 73, 74, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 The present invention relates to an amino acid sequence that is one of the amino acid sequences of 01 or SEQ ID NO: 102 (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 a further embodiment of the present invention.

[0103] In yet another specific but non-limiting aspect, the present invention relates to an amino acid sequence (the amino acid sequence being a serum albumin-binding ISVD, in particular a serum albumin-binding nanobody), wherein the amino acid sequence is - At least 85%, preferably at least 90%, more preferably at least 95% of the sequence of sequence number 1; and / or - A difference of 7 or less, preferably 5 or less, relative to the sequence of SEQ ID NO: 1, for example, just 3, 2, or 1 amino acid. Having; In the amino acid sequence, the amino acid residue at position 89 (according to Kabat) is selected from A, N, and S, preferably A, and the amino acid residue at position 104 (according to Kabat) is selected from G, T, or A, preferably T. Preferably, in the amino acid sequence according to this embodiment, (i) the amino acid residue at position 5 (according to Kabat) is L or V (more preferably V); (ii) the amino acid residue at position 11 (according to Kabat) is L or V (more preferably V); (iii) the amino acid residue at position 110 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. Furthermore, in the amino acid sequence according to this embodiment, the amino acid sequence may optionally contain (i) at least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain 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 (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T). In particular, they may contain the S101D mutation compared to SEQ ID NO: 1.Most preferably, in the amino acid sequence according to this embodiment, (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 embodiment, CDR1 is GFTFRSFGMS (SEQ ID NO: 5), CDR2 is SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLSR (SEQ ID NO: 7). Furthermore, an amino acid sequence according to this embodiment is particularly preferred in which the amino acid residue at position 89 is A or the amino acid residue at position 89 is T.

[0104] As described, some preferred amino acid residues / mutations that can be present are 30T (i.e., R30T compared to SEQ ID NO: 1) and 101E (i.e., S101E compared to SEQ ID NO: 1), preferably both. These preferred mutations are also preferably combined with T at position 89 (i.e., V89T compared to SEQ ID NO: 1), or alternatively with A at position 89 (i.e., V89A compared to SEQ ID NO: 1). One particularly preferred combination of CDRs is GFTFTSFGMS (SEQ ID NO: 53), SISGSGSDTL (SEQ ID NO: 6), and GGSLER (SEQ ID NO: 57).

[0105] In a further embodiment, 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 (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 a further embodiment of the present invention.

[0106] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); If CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa); the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0107] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in whole or as part thereof) as further described herein.

[0108] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Most preferably, the 89th position is T and the 104th position is T (again, especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0109] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); If CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa); the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 50, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0110] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0111] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Most preferably, the 89th position is T and the 104th position is T (again, especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0112] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56); If CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa); the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0113] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0114] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Most preferably, the 89th position is T and the 104th position is T (again, especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0115] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56); If CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa); the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 50, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0116] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0117] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Most preferably, the 89th position is T and the 104th position is T (again, especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0118] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -CDR1 is the amino acid sequence GFTHRSFGMS (SEQ ID NO: 52); and -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 has the amino acid sequence GGSLDR (SEQ ID NO: 56); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0119] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0120] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T. Most preferably, the 89th position is T and the 104th position is T. Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0121] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0122] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -CDR1 is the amino acid sequence GFTHRSFGMS (SEQ ID NO: 52); and -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 has the amino acid sequence GGSLDR (SEQ ID NO: 56); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0123] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0124] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T. Most preferably, the 89th position is T and the 104th position is T. Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0125] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0126] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0127] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Most preferably, the 89th position is T and the 104th position is T (again, especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0128] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0129] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0130] In the serum albumin binder according to this embodiment, the 89th position is preferably T. Also, the 104th position is preferably T (especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Most preferably, the 89th position is T and the 104th position is T (again, especially when CDR3 is GGSLDR (SEQ ID NO: 56)). Also preferably, the 5th position is V and / or the 11th position is V, preferably the 5th position is V and the 11th position is V.

[0131] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -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 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), preferably GGSLSR (SEQ ID NO: 7); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences at positions 5 and 11 are not considered, and amino acid differences in the CDR are not considered).

[0132] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0133] In this embodiment of the serum albumin binder, position 89 is preferably L, A, or T, and position 104 is preferably G. Also, position 16 is preferably G or N; position 45 is preferably P or L; and positions 74-76 are preferably SKN or AKT motifs.

[0134] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -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 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), preferably GGSLSR (SEQ ID NO: 7); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences at positions 5 and 11 are not considered, and amino acid differences in the CDR are not considered).

[0135] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0136] In this embodiment of the serum albumin binder, position 89 is preferably L, A, or T, and position 104 is preferably G. Also, position 16 is preferably G or N; position 45 is preferably P or L; and positions 74-76 are preferably SKN or AKT motifs.

[0137] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -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 has the amino acid sequence GGSLSR (SEQ ID NO: 7); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences at positions 5 and 11 are not considered, and amino acid differences in the CDR are not considered).

[0138] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0139] In this embodiment of the serum albumin binder, position 89 is preferably L, A, or T, and position 104 is preferably G. Also, position 16 is preferably G or N; position 45 is preferably P or L; and positions 74-76 are preferably SKN or AKT motifs.

[0140] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -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 has the amino acid sequence GGSLSR (SEQ ID NO: 7); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences at positions 5 and 11 are not considered, and amino acid differences in the CDR are not considered).

[0141] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0142] In this embodiment of the serum albumin binder, position 89 is preferably L, A, or T, and position 104 is preferably G. Also, position 16 is preferably G or N; position 45 is preferably P or L; and positions 74-76 are preferably SKN or AKT motifs.

[0143] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -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 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), preferably GGSLSR (SEQ ID NO: 7) or GGSLER (SEQ ID NO: 57); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences at positions 5 and 11 are not considered, and amino acid differences in the CDR are not considered).

[0144] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0145] In this embodiment of the serum albumin binder, position 89 is preferably L, A, or T, and position 104 is preferably G. Also, position 16 is preferably G or N; position 45 is preferably P or L; and positions 74-76 are preferably SKN or AKT motifs.

[0146] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -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 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), preferably GGSLSR (SEQ ID NO: 7) or GGSLER (SEQ ID NO: 57); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences at positions 5 and 11 are not considered, and amino acid differences in the CDR are not considered).

[0147] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0148] In this embodiment of the serum albumin binder, position 89 is preferably L, A, or T, and position 104 is preferably G. Also, position 16 is preferably G or N; position 45 is preferably P or L; and positions 74-76 are preferably SKN or AKT motifs.

[0149] Some preferred but non-limiting examples of the amino acid sequences of the present invention having CDR1, which is sequence number 54, are the amino acid sequences of sequence numbers 26, 46, 47, 79, 99, 100, and 209-244, in particular the amino acid sequences of sequence numbers 209-244.

[0150] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, wherein the amino acid residue at position 5 is V, the amino acid residue at position 11 is V, and the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0. The amino acid sequence according to this embodiment 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), 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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56). According to one specific but non-limiting aspect of these amino acid sequences of the present invention having V at position 11 or L at position 11, if CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa).

[0151] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0152] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, wherein the amino acid residue at position 5 is V, the amino acid residue at position 11 is V, and the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0. The amino acid sequence according to this embodiment 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), 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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56). According to one specific but non-limiting embodiment of these amino acid sequences of the present invention having V or L at position 11, if 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 albumin binders according to this embodiment may contain S at position 30, as is present in CDR1 of SEQ ID NO: 50 (according to Abm).

[0153] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0154] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable immunoglobulin domain capable of binding to (human) serum albumin, wherein it contains at least one or more suitable combinations (i.e., in a suitable combination) of the following amino acid residues (29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T) (i.e., compared to the sequence of SEQ ID NO: 1, 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 (i) has 7 or fewer, preferably 5 or fewer, amino acid differences with respect to the sequence of SEQ ID NO: 1, for example, 5, 4, 3, 2, 1, or 0.

[0155] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0156] In the amino acid sequence according to this embodiment, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Furthermore, the amino acid sequence according to this embodiment preferably has the following CDR: (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), preferably selected from GFTFRSFGMS (SEQ ID NO: 5) and GFTHRSFGMS (SEQ ID NO: 52); (ii) CDR2 is an amino acid sequence SISGSGSDTL (SEQ ID NO: 6) (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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56), and if CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa).

[0157] In a further embodiment, the present invention relates to an amino acid sequence that is a monovariable immunoglobulin domain capable of binding to (human) serum albumin, wherein it contains (i.e., in an appropriate combination) at least one or more suitable combinations of the following amino acid residues (29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T) (i.e., SEQ ID NO: Compared to sequence 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 (i) has 7 or fewer, preferably 5 or fewer, amino acid differences with respect to sequence number 50, for example 5, 4, 3, 2, 1, or 0.

[0158] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0159] In the amino acid sequence according to this embodiment, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Furthermore, the amino acid sequence according to this embodiment preferably has the following CDR: (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), preferably selected from GFTFRSFGMS (SEQ ID NO: 5) and GFTHRSFGMS (SEQ ID NO: 52); (ii) CDR2 is an amino acid sequence SISGSGSDTL (SEQ ID NO: 6) (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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56), and if 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 albumin binders according to this embodiment may contain S at position 30, as is present in CDR1 of SEQ ID NO: 50 (according to Abm).

[0160] In a further embodiment, the present invention relates to an amino acid sequence which is a monovariate immunoglobulin domain capable of binding to (human) serum albumin, wherein it contains at least one of the following amino acid residues (29H, 101D, and 104T) (i.e., in an appropriate combination) (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least F29H, S101D, and S104T amino acid mutations); the amino acid sequence (i) has 7 or fewer, preferably 5 or fewer, amino acid differences, for example, 5, 4, 3, 2, 1, or 0, compared to the sequence of SEQ ID NO: 1.

[0161] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0162] In the amino acid sequence according to this embodiment, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Furthermore, the amino acid sequence according to this embodiment preferably has the following CDR: (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), preferably selected from GFTFRSFGMS (SEQ ID NO: 5) and GFTHRSFGMS (SEQ ID NO: 52); (ii) CDR2 is an amino acid sequence SISGSGSDTL (SEQ ID NO: 6) (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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56), and if CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa).

[0163] In a further embodiment, the present invention relates to an amino acid sequence which is a monovariate immunoglobulin domain capable of binding to (human) serum albumin, wherein it contains at least one of the following amino acid residues (29H, 101D, and 104T) (i.e., in an appropriate combination) (i.e., compared to the sequence of SEQ ID NO: 50, they contain at least F29H, S101D, and S104T amino acid mutations); the amino acid sequence (i) has 7 or fewer, preferably 5 or fewer, amino acid differences, for example, 5, 4, 3, 2, 1, or 0, compared to the sequence of SEQ ID NO: 50.

[0164] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0165] In the amino acid sequence according to this embodiment, the amino acid residue at position 5 is V, and the amino acid residue at position 11 is V. Furthermore, the amino acid sequence according to this embodiment preferably has the following CDR: (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), preferably selected from GFTFRSFGMS (SEQ ID NO: 5) and GFTHRSFGMS (SEQ ID NO: 52); (ii) CDR2 is an amino acid sequence SISGSGSDTL (SEQ ID NO: 6) (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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLDR (SEQ ID NO: 56), and if 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 albumin binders according to this embodiment may contain S at position 30, as is present in CDR1 of SEQ ID NO: 50 (according to Abm).

[0166] More specific but non-limiting aspects of the present invention relate to serum albumin binders as defined herein, wherein (i) CDR1 is GFTFRSFGMS (SEQ ID NO: 5), CDR2 is SISGSGSDTL (SEQ ID NO: 6), CDR3 is GGSLSR (SEQ ID NO: 7), and (ii) the amino acid residue at position 5 is preferably V, and / or the amino acid at position 11 is preferably V (preferably V at both positions). Albumin binders according to this embodiment preferably have 7 or fewer, preferably 5 or fewer, amino acid differences relative to the sequence of SEQ ID NO: 1, e.g., 5, 4, 3, 2, 1, or 0, and / or 7 or fewer, preferably 5 or fewer, amino acid differences relative to the sequence of SEQ ID NO: 50, e.g., 5, 4, 3, 2, 1, or 0. Albumin binders according to this embodiment are preferably as further described herein. For example, they preferably have affinity for serum albumin as described herein, and / or they have serum half-lives (as the polypeptide of the present invention or as part thereof) as further described herein.

[0167] More specific but non-limiting aspects of the present invention relate to serum albumin binders as defined herein, wherein (i) CDR1 is GFTFSSFGMS (SEQ ID NO: 120), CDR2 is SISGSGSDTL (SEQ ID NO: 6), CDR3 is GGSLSR (SEQ ID NO: 7), and (ii) the amino acid residue at position 5 is preferably V, and / or the amino acid at position 11 is preferably V (preferably V at both positions). Albumin binders according to this embodiment preferably have 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 1, for example, 5, 4, 3, 2, 1, or 0, and / or 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 50, for example, 5, 4, 3, 2, 1, or 0. Albumin binders according to this embodiment 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 (in the polypeptide of the present invention as is or as part thereof) as further described herein. Some preferred but non-limiting examples of serum albumin binders of this group of the present invention (i.e., based on CDR1 having V at positions 5 and 11, which is SEQ ID NO: 120) are shown by SEQ ID NOs: 185-208.

[0168] In particular, in the serum albumin binder according to this embodiment, position 16 may be G or N, preferably N; position 45 may be P or L, preferably L; positions 74-76 may be an SKN or AKT motif, preferably an AKT motif; position 89 may be L, A, or T, preferably L; and position 104 may be G or T.

[0169] Herein, some preferred but non-limiting examples of serum albumin binders of this group of the present invention (i.e., based on CDR1 having V at positions 5 and 11, which is SEQ ID NO: 120) are shown by SEQ ID NOs: 185-208.

[0170] Therefore, a more specific but non-limiting aspect of the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (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), The amino acid residue at position -5 is V; The amino acid residue at position -11 is V; The amino acid residue at position -16 is G or N, preferably N; The amino acid residue at position -45 is P or L, preferably L; The amino acid residues at positions -74 to -76 form an SKN or AKT motif, preferably an AKT motif; The amino acid residue at position -89 is L, A, or T, preferably L; and The amino acid residue at position -104 is either G or T; The amino acid sequence preferably has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (mutations at CDR and the positions specified above are not considered).

[0171] More specific but non-limiting aspects of the present invention relate to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (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), The amino acid residue at position -5 is V; The amino acid residue at position -11 is V; The amino acid residue at position -16 is N; The amino acid residue at position -45 is L; The amino acid residues at positions -74 to -76 form an AKT motif; The amino acid residue at position -89 is L, A, or T, preferably L; and The amino acid residue at position -104 is either G or T; The amino acid sequence preferably has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (mutations at CDR and the positions specified above are not considered).

[0172] Another specific but non-limiting aspect of the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (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), The amino acid residue at position -5 is V; The amino acid residue at position -11 is V; The amino acid residue at position -16 is G or N, preferably N; The amino acid residue at position -45 is P or L, preferably L; The amino acid residues at positions -74 to -76 form an SKN or AKT motif, preferably an AKT motif; The amino acid residue at position -89 is L, A, or T, preferably L; and The amino acid residue at position -104 is either G or T; The amino acid sequence preferably has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (mutations at CDR and the positions specified above are not considered).

[0173] More specific but non-limiting aspects of the present invention relate to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (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), The amino acid residue at position -5 is V; The amino acid residue at position -11 is V; The amino acid residue at position -16 is N; The amino acid residue at position -45 is L; The amino acid residues at positions -74 to -76 form an AKT motif; The amino acid residue at position -89 is L, A, or T, preferably L; and The amino acid residue at position -104 is either G or T; The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (mutations at CDR and the positions specified above are not considered).

[0174] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO: 7) and GGSLER (SEQ ID NO: 57); If CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa); the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0175] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0176] In the serum albumin binder according to this embodiment, the 89th position is preferably T (or alternatively A).

[0177] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 has the amino acid sequence GGSLER (SEQ ID NO: 57); The amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

[0178] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0179] In the serum albumin binder according to this embodiment, the 89th position is preferably T (or alternatively A).

[0180] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -CDR1 is an amino acid sequence selected from the following amino acid sequences: GFTFRSFGMS (SEQ ID NO: 5) and GFTFTSFGMS (SEQ ID NO: 53); -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 is an amino acid sequence selected from the following amino acid sequences: GGSLSR (SEQ ID NO: 7) and GGSLER (SEQ ID NO: 57); When CDR1 is GFTFRSFGMS (SEQ ID NO: 5), CDR3 is not GGSLSR (SEQ ID NO: 7), and vice versa; said amino acid sequence has 7 or fewer, preferably 5 or fewer, for example 5, 4, 3, 2, 1, or 0 amino acid differences relative to the sequence of SEQ ID NO: 50 (amino acid differences in CDRs are not taken into account).

[0181] The albumin binding agents according to this embodiment are preferably as further described herein. For example, they preferably have affinity for serum albumin as described herein, and / or they have a serum half-life (as the polypeptide of the present invention per se or as a part thereof) as further described herein.

[0182] In the serum albumin binding agent according to this embodiment, position 89 is preferably T (or alternatively A).

[0183] In a further aspect, the present invention relates to an amino acid sequence that is an immunoglobulin single variable domain capable of binding to (human) serum albumin, -CDR1 is the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); -CDR2 is the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57); said amino acid sequence has 7 or fewer, preferably 5 or fewer, for example 5, 4, 3, 2, 1, or 0 amino acid differences relative to the sequence of SEQ ID NO: 50 (amino acid differences in CDRs are not taken into account).

[0184] The albumin binding agents according to this embodiment are preferably as further described herein. For example, they preferably have affinity for serum albumin as described herein, and / or they have a serum half-life (as the polypeptide of the present invention per se or as a part thereof) as further described herein.

[0185] In the serum albumin binder according to this embodiment, the 89th position is preferably T (or alternatively A).

[0186] In a further embodiment, the present invention relates to an amino acid sequence that is a monovariate immunoglobulin domain capable of binding to (human) serum albumin, wherein it contains at least one of the following amino acid residues (30T and / or 101E) (i.e., in an appropriate combination) (i.e., compared to the sequence of Sequence ID No. 1, they contain at least the amino acid mutations R30T and / or S101E, preferably both); The amino acid sequence (i) has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0. In this embodiment of the amino acid sequence, 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.

[0187] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0188] Furthermore, the amino acid sequence according to this embodiment preferably has the following CDR: (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), preferably selected from GFTFRSFGMS (SEQ ID NO: 5) and GFTFTSFGMS (SEQ ID NO: 53); (ii) CDR2 is an amino acid sequence SISGSGSDTL (SEQ ID NO: 6) (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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLER (SEQ ID NO: 57), and if 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 SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLER (SEQ ID NO: 57).

[0189] In a further embodiment, the present invention relates to an amino acid sequence that is a monovariate immunoglobulin domain capable of binding to (human) serum albumin, wherein it contains at least one of the following amino acid residues (30T and / or 101E) (i.e., in an appropriate combination) (i.e., compared to the sequence of SEQ ID NO: 50, they contain at least the amino acid mutations S30T and / or S101E, preferably both); The amino acid sequence (i) has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0. In the amino acid sequence according to this embodiment, 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.

[0190] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0191] Furthermore, the amino acid sequence according to this embodiment preferably has the following CDR: (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), preferably selected from GFTFRSFGMS (SEQ ID NO: 5) and GFTFTSFGMS (SEQ ID NO: 53); (ii) CDR2 is an amino acid sequence SISGSGSDTL (SEQ ID NO: 6) (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), preferably selected from GGSLSR (SEQ ID NO: 7) and GGSLER (SEQ ID NO: 57), and if 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 SISGSGSDTL (SEQ ID NO: 6), and CDR3 is GGSLER (SEQ ID NO: 57).

[0192] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; -11th place is V; and -30th place is T; The amino acid sequence (i) has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (mutations at positions 5, 11, and 30 are not considered).

[0193] The serum albumin binder according to this embodiment preferably also has N or P at position 16; P or L at position 45; an SKN or AKT motif at positions 74-76; A, L, or T at position 89; and G at position 104.

[0194] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0195] Furthermore, the amino acid sequence according to this embodiment preferably has the following CDR: (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 preferably GGSLSR (SEQ ID NO: 7).

[0196] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -30th place is T; and Said amino acid sequence has (i) 7 or fewer, preferably 5 or fewer, for example 5, 4, 3, 2, 1, or 0 amino acid differences compared to the sequence of SEQ ID NO: 50 (mutations at positions 5, 11 and 30 are not considered).

[0197] The serum albumin binding agent according to this embodiment preferably also has N or P at position 16; P or L at position 45; a SKN or AKT motif at positions 74 to 76; A, L, or T at position 89; and G at position 104.

[0198] The albumin binding agents according to this embodiment are preferably as further described herein. For example, they preferably have affinity for serum albumin as described herein and / or they have a serum half-life (as or as part of a polypeptide of the invention) as further described herein.

[0199] Also, the amino acid sequence according to this embodiment preferably has CDRs as follows: (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).

[0200] In a further aspect, the invention relates to an amino acid sequence that is an immunoglobulin single variable domain capable of binding to (human) serum albumin, - position 5 is V; and - position 11 is V; and - position 30 is T; and - position 89 is A, L, or T; and - position 104 is G; and The amino acid sequence (i) has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (mutations at positions 5, 11, 30, 89, and 104 are not considered).

[0201] The serum albumin binder according to this embodiment preferably also has an N or P at position 16; a P or L at position 45; and an SKN or AKT motif at positions 74-76.

[0202] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0203] Furthermore, the amino acid sequence according to this embodiment 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), preferably GGSLSR (SEQ ID NO: 7).

[0204] In a further embodiment, the present invention relates to an amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -5th place is V; and -11th place is V; and -30th place is T; and -89th place is A, L, or T; and -104th place is G; and The amino acid sequence (i) has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 50, for example, 5, 4, 3, 2, 1, or 0 (mutations at positions 5, 11, 30, 89, and 104 are not considered).

[0205] The serum albumin binder according to this embodiment preferably also has an N or P at position 16; a P or L at position 45; and an SKN or AKT motif at positions 74-76.

[0206] Albumin binders according to this embodiment 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 (of the polypeptide of the present invention, either in its entirety or as part thereof) as further described herein.

[0207] Furthermore, the amino acid sequence according to this embodiment 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), preferably GGSLSR (SEQ ID NO: 7).

[0208] The serum albumin binders of the present invention are preferably also of formula (X) if they are present at and / or form the C-terminus of the compound or polypeptide of the present invention (or otherwise have an "exposed" C-terminus in the protein, polypeptide, or other compound or construct, thereby generally meaning that the C-terminus of the ISV is not associated with or linked to a constant domain (e.g., the CH1 domain); here again, referencing International Publication No. 12 / 175741 and PCT / EP2015 / 06043). n It also has a C-terminal elongation as shown by n, where n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4 or 5 (and preferably 1 or 2, for example 1); each X is an (preferably natural) amino acid residue independently selected from a natural amino acid residue (according to one preferred embodiment, it does not contain any cysteine ​​residues), preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (V) or isoleucine (I).

[0209] Such C-terminal elongation (X) n According to some preferred but non-limiting embodiments, X and n may be as follows: (a) n=1 and X=Ala; (b) n=2 and each X=Ala; (c) n=3 and each X=Ala; (d) n=2 and at least one X=Ala (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (e) n=3 and at least one X=Ala (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (f) n=3 and at least two X=Ala (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (g)n=1 and X=Gly; (h)n=2 and each X=Gly; (i) n=3 and each X=Gly; (j)n=2 and at least one X=Gly (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (k)n=3 and at least one X=Gly (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (l)n=3 and at least two X=Gly (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (m)n=2 and each X=Ala or Gly; (n)n=3 and each X=Ala or Gly; (o)n=3 and at least one X=Ala or Gly (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); or (p)n=3 and at least two X=Ala or Gly (the remaining amino acid residue(s) X are independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); Embodiments (a), (b), (c), (g), (h), (i), (m), and (n) are particularly preferred, the embodiment where n=1 or 2 is preferred, and the embodiment where n=1 is particularly preferred.

[0210] Furthermore, it should be noted that, preferably, any C-terminal extensions present in the serum albumin binder of the present invention do not contain (free) cysteine ​​residues (where such cysteine ​​residues are not used or intended for further functionalization, for example, for PEGylation).

[0211] Some specific but non-limiting examples of useful C-terminal elongation are the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA, or GAG.

[0212] If the serum albumin binder of the present invention contains a mutation at position 110 or 112 (in part, in combination with a mutation at position 11 and / or 89 as described herein), the C-terminal amino acid residue of framework 4 (starting at position 109) may be as follows: (i) if there is no C-terminal elongation at all: VTVKS (SEQ ID NO: 104), VTVQS (SEQ ID NO: 105), VKVSS (SEQ ID NO: 106), or VQVSS (SEQ ID NO: 107); or (ii) if there is C-terminal elongation: VTVKSX (n) (Sequence ID 108), VTVQSX(n) (Sequence ID 109), VKVSSX(n) (Sequence ID 110), or VQVSSX (n) (SEQ ID NO: 111), for example, VTVKSA (SEQ ID NO: 112), VTVQSA (SEQ ID NO: 113), VKVSSA (SEQ ID NO: 114), or VQVSSA (SEQ ID NO: 115). If the serum albumin binder of the present invention does not contain a mutation at position 110 or 112 (but only mutations at position 11 and / or 89 as described herein), the C-terminal amino acid residue of framework 4 (starting at position 109) would typically be: (i) if there is no C-terminal elongation at all: VTVSS (SEQ ID NO: 116), as in the sequences of SEQ ID NOs. 8-49; or (ii) if there is C-terminal elongation: VTVSSX (n) (Sequence ID 117), for example, VTVSSA (Sequence ID 118) (as in sequences 61-102). In these C-terminal sequences, X and n are as defined herein with respect to C-terminal extension.

[0213] Furthermore, if the serum albumin binder of the present invention is present at and / or forms the N-terminus of the compound or polypeptide of the present invention, the serum albumin binder preferably has D at position 1 (i.e., an E1D mutation compared to the sequences shown in SEQ ID NOs: 1, 8-50, and 61-102).

[0214] Furthermore, generally speaking, if the compound or polypeptide of the present invention has a heavy chain ISVD at its C-terminus (which may be a serum albumin binder of the present invention, but may also be, for example, an ISVD that binds to a therapeutic target), the C-terminal ISVD (extended to the compound or polypeptide of the present invention) preferably has a C-terminal extension X(n) as described herein. Similarly, if the compound or polypeptide of the present invention has a heavy chain ISVD at its N-terminus (which may be a serum albumin binder of the present invention, but may also be, for example, an ISVD that binds to a therapeutic target), the N-terminal ISVD (extended to the compound or polypeptide of the present invention) preferably has a D at position 1.

[0215] Preferably, if the compound or polypeptide of the present invention contains one or more other ISVDs in addition to the albumin-binding agents(group) of the present invention (the other ISVD(group) may be, for example, one or more ISVDs against a therapeutic target), then preferably all ISVDs present in the compound or polypeptide contain one or more framework mutations in their sequences that reduce binding by existing antibodies. In particular, if these other ISVDs are VH domains, consist essentially of VH domains, and / or nanobody or (single) domain antibodies derived from VH domains, they may essentially contain amino residues / mutations (or appropriate combinations thereof) at positions 11, 89, 110, and / or 112, as described in PCT / EP2015 / 060643.

[0216] As described herein, the amino acid sequences provided by the present invention are proteins capable of binding to human serum albumin, and in particular, specifically (as described herein). Therefore, they can be used, for example, as binding units or binding domains for binding to (human) serum albumin to confer an extension of the half-life (as defined herein) to a therapeutic compound, part, or entity. The use of serum albumin-binding domains for extending the half-life of a therapeutic compound, part, or entity is generally referred to in International Publication No. 2004 / 041865, International Publication No. 2006 / 122787, European Patent No. 2139918, International Publication No. 2011 / 006915, International Publication No. 2012 / 175400 and / or International Publication No. 2014 / 111550. The albumin-binding agents of the present invention can generally be used in the same manner and for the same purposes as the serum albumin-binding agents described in these documents.

[0217] The present invention also relates to proteins, polypeptides, and other constructs, molecules, or chemical entities comprising (one or more) serum albumin-binding agents of the present invention as described herein; methods for expressing / producing the improved heavy immunoglobulin variable domain of the present invention, and / or expressing / producing proteins, polypeptides, and other constructs, molecules, or chemical entities comprising the same; compositions and products (e.g., pharmaceutical compositions and pharmaceuticals) comprising the improved heavy immunoglobulin variable domain of the present invention, and / or proteins, polypeptides, and other constructs, molecules, or chemical entities comprising the same; nucleotide sequences and nucleic acids encoding and / or proteins or polypeptides comprising the improved heavy immunoglobulin variable domain of the present invention; and the use (particularly therapeutic, prophylactic, and diagnostic) of the improved heavy immunoglobulin variable domain of the present invention, and proteins, polypeptides, and other constructs, molecules, or chemical entities comprising the same.

[0218] Further aspects, embodiments, advantages, applications, and uses of the present invention will become apparent from further description herein.

[0219] In this specification: - The term “immunoglobulin monovariate domain” (also referred to as “ISV” or “ISVD”) is generally used to refer to an immunoglobulin monovariate domain (which may be a heavy or light chain domain containing a VH, VHH, or VL domain) that can form a functional antigen-binding site without interaction with another variable domain (for example, without the VH / VL interaction required between the VH and VL domains of a conventional quadruple-chain monoclonal antibody). Examples of ISVDs will be obvious to those skilled in the art and include, for example, nanobodies (including VHH, humanized VHH, and / or camelid VH, e.g., camelid human VH), IgNARs, domains, (monodomain) antibodies that are or are derived from a VH domain (e.g., dAb's®), and (monodomain) antibodies that are or are derived from a VL domain (e.g., dAb's®). Unless otherwise specified herein, ISVDs based on and / or derived from heavy chain variable domains (e.g., VH domains or VHH domains) are generally preferred. Most preferably, unless otherwise specified herein, the ISVD will be a nanobody.

[0220] The term “nanobody” is generally defined as in International Publication No. 2008 / 020079 or International Publication No. 2009 / 138519, and therefore, in specific embodiments, generally refers to VHH, humanized VHH, or camelized VH (e.g., camelized human VH), or generally sequence-optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). It should be noted that the terms nanobody or nanobodies are registered trademarks of Ablynx NV and therefore may also be referred to as nanobody® and / or nanobodies®.

[0221] Generally, unless otherwise specified herein, the ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs referred to herein will be intended for use in the prevention or treatment of diseases or disorders in humans (and / or optionally also in homeothermic animals, particularly mammals). Therefore, generally, the ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs described herein are preferably capable of being used as (biological) drugs or other pharmaceutically or therapeutically active compounds and / or pharmaceuticals or pharmaceutical compositions, and / or appropriately as part thereof. Such drugs, compounds, or products are preferably suitable for administration to humans, for example, for the prevention or treatment of subjects in need of such prevention or treatment, or, for example, as part of a clinical trial. As further described herein, for this purpose, such drugs or compounds may contain other parts, entities, or binding units in addition to the ISVD provided by the present invention (these may be one or more other further therapeutic parts and / or one or more other parts that affect, for example, the pharmacokinetic or pharmacodynamic properties of the ISVD-based or nanobody-based biologic, such as its half-life, as also described herein). Suitable examples of such further therapeutic parts or other parts will be obvious to those skilled in the art, and may generally include any therapeutically effective protein, polypeptide, or other binding domain or binding unit, as well as modifications such as those described on pages 149-152 of International Publication No. 2009 / 138159. ISVD-based biologics or nanobody-based biologics are preferably therapeutic agents or intended for use as therapeutic agents (including prophylactic and diagnostic), and for this purpose preferably contain at least one ISVD against a therapeutically appropriate target (e.g., RANK-L, vWF, IgE, RSV, CXCR4, IL-23, or other interleukins).Some specific but non-limiting examples of such ISVD-based biologics or nanobody-based biologics are referred to in Examples 8–18, and also in various applications by Ablynx NV, for example (e.g., International Publication Nos. 2004 / 062551, 2006 / 122825, 2008 / 020079 and 2009 / 068627), as well as applications such as (but not limited to) International Publication Nos. 2006 / 038027, 2006 / 059108, 2007 / 063308, 2007 / 063311, 2007 / 066016 and 2007 / 085814. Furthermore, as further described herein, additional portions may be ISVDs or nanobodies directed toward (human) serum proteins such as (human) serum albumin, and such ISVDs or nanobodies may also find therapeutic use in and / or for the extension of the half-life of TNF binders as described herein. For example, see International Publication Nos. 2004 / 041865, International Publication Nos. 2006 / 122787 and International Publication Nos. 2012 / 175400, which generally describe the use of serum albumin-binding nanobodies for half-life extension. Furthermore, in this specification, unless otherwise specifically stated herein, all terms used herein have the meanings set forth in International Publication No. 2009 / 138519 (or in the prior art cited in International Publication No. 2009 / 138519) or International Publication No. 2008 / 020079 (or in the prior art cited in International Publication No. 2008 / 020079). Also, if a method or technique is not specifically described herein, it may be implemented as described in International Publication No. 2009 / 138519 (or in the prior art cited in International Publication No. 2009 / 138519) or International Publication No. 2008 / 020079 (or in the prior art cited in International Publication No. 2008 / 020079).Furthermore, as described herein, any pharmaceutical or pharmaceutical composition comprising any ISVD or compound of the present invention may also contain one or more further components known by itself for use in the pharmaceutical or pharmaceutical composition (i.e., depending on the intended dosage form), and / or one or more other compounds or active ingredients intended, for example, for therapeutic use (i.e., to provide a combination product).

[0222] Furthermore, as used herein or in the claims, the following terms have the same meanings as set forth in International Publication No. 2009 / 138519, pages 62-75, and / or, where applicable, may be determined in the manner set forth in International Publication No. 2009 / 138519, pages 62-75: “agonist,” “antagonist,” “reverse agonist,” “nonpolar uncharged amino acid residue,” “polar uncharged amino acid residue,” “polar charged amino acid residue,” “sequence identity,” “exactly the same,” and “amino acid difference” (when referring to a sequence comparison of two amino acid sequences), “essentially isolated (in form),” “domain,” “binding domain,” “antigenic determinant,” “epitope,” “(antigen)” or “directed to,” “specificity,” and “half-life.” Furthermore, the terms “regulating” and “regulating,” “interaction site,” “specific to,” “cross-blocking,” “cross-blocked,” and “cross-blocked,” as well as “essentially pH-independent,” are defined (and / or may be determined as described therein) on pages 74-79 of International Publication No. 2010 / 130832 of Ablynx NV. Also, when referring to the constructs, compounds, proteins, or polypeptides of the present invention, terms such as “monovalent,” “divalent” (or “polyvalent”), “dual-specific” (or “multi-specific”) and “dual-paratope” (or “multi-paratope”) may have the meanings set forth in International Publication No. 2009 / 138519, International Publication No. 2010 / 130832, or International Publication No. 2008 / 020079.

[0223] The term “half-life” as used herein in relation to ISVDs, nanobodies, ISVD-based biologics, nanobodies-based biologics, or any other amino acid sequences, compounds, or polypeptides may generally be defined as set forth in paragraph o) on page 57 of International Publication No. 2008 / 020079, as set forth herein, and refers to the time required for the serum concentration of an amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, for example, due to degradation of the sequence or compound and / or exclusion or capture of the sequence or compound by natural mechanisms. The in vivo half-life of the amino acid sequences, compounds, or polypeptides of the present invention may be determined by any known method, such as by pharmacokinetic analysis. Appropriate techniques will be apparent to those skilled in the art, and this may generally be as set forth in paragraph o) on page 57 of International Publication No. 2008 / 020079. As also stated in paragraph o) on page 57 of International Publication No. 2008 / 020079, the half-life can be expressed using parameters such as t1 / 2α, t1 / 2β, and area under the curve (AUC). In this regard, it should be noted that the term “half-life” as used herein specifically refers to t1 / 2β or the terminal phase half-life (where t1 / 2α and / or AUC, or both, may not be considered). For example, the following experimental sections, as well as standard handbooks, such as Kenneth, A et al.: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic analysis: A Practical Approach (1996), are referenced. Also referenced is “Pharmacokinetics” by Marcel Dekker, M Gibaldi & D Perron, revised edition (1982). Similarly, the terms “extended half-life” or “extended half-life” are defined in paragraph o) on page 57 of International Publication No. 2008 / 020079, and refer in particular to an increase in t1 / 2β, which may or may not be accompanied by an increase in t1 / 2α and / or AUC or both.

[0224] Unless a term is specifically defined herein, it has its ordinary meaning in the art, which will be obvious to those skilled in the art. For example, standard handbooks such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd edition), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, "Genes II", John Wiley & Sons, New York, NY, (1985); Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., "Immunology" (6th edition), Mosby / Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 10th edition, Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th edition), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background technology cited herein are referenced.

[0225] Furthermore, as already shown herein, the amino acid residues of the nanobodies are numbered according to the general numbering for VH shown by Kabat et al. ("Sequence of proteins of immunological interest," U.S. Public Health Service, National Institutes of Health, Benesda, MD, publication number 91), as applied to VHH domains derived from camelids in the paper Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195, or as referred herein. According to this numbering, nanobody FR1 contains amino acid residues at positions 1-30, nanobody CDR1 contains amino acid residues at positions 31-35, nanobody FR2 contains amino acids at positions 36-49, nanobody CDR2 contains amino acid residues at positions 50-65, nanobody FR3 contains amino acid residues at positions 66-94, nanobody CDR3 contains amino acid residues at positions 95-102, and nanobody FR4 contains amino acid residues at positions 103-113. [In this regard, it should be noted that, as is well known in the art with respect to VH domains and VHH domains, the total number of amino acid residues within each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions indicated by Kabat numbering may not be present in the actual sequence, or the actual sequence may contain more amino acid residues than is possible by Kabat numbering). This generally means that the numbering by Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.However, generally speaking, according to Kabat numbering, regardless of the amino acid residue number within the CDR, position 1 according to Kabat numbering corresponds to the start site of FR1 and vice versa; position 36 according to Kabat numbering corresponds to the start site of FR2 and vice versa; position 66 according to Kabat numbering corresponds to the start site of FR3 and vice versa; and position 103 according to Kabat numbering corresponds to the start site of FR4 and vice versa.

[0226] An alternative method for numbering amino acid residues in VH domains (this method can also be similarly applied to VHH domains and nanobodies derived 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, unless otherwise noted, this specification, embodiments, and drawings will follow the numbering by Kabat, as applied to VHH domains by Riechmann and Muyldermans.

[0227] Furthermore, it should be noted that the drawings, any sequence listings, and experimental sections / examples are provided solely for the purpose of further illustrating the present invention and should not be construed or understood to limit the scope of the present invention and / or the appended claims unless otherwise specifically stated herein.

[0228] As further described herein, the serum albumin binders of the present invention may be advantageously used as a portion, binding unit, or fusion pair for extending the half-life of therapeutic portions such as polypeptides, proteins, compounds (including but not limited to low molecular weights) or other therapeutic entities.

[0229] Accordingly, in another embodiment, the present invention provides polypeptides, proteins, constructs, compounds, or other chemical entities comprising, or essentially comprising, the serum albumin binder of the present invention and one or more other amino acid sequences, (binding) domains, binding units, or other parts or chemical entities.

[0230] In particular, the present invention provides polypeptides, proteins, constructs, compounds, or other chemical entities comprising the serum albumin binder of the present invention and one or more (e.g., one or two) therapeutic moieties (which may be the same or different, and may be directed to the same target or to different targets, and if they are directed to the same target, they may be directed to the same or different epitopes, moieties, domains, or subunits of said target) that are appropriately linked to one another, either directly or via one or more suitable linkers or spacers. Such polypeptides, proteins, or constructs may be, but are not limited to, fusion proteins, as further described herein.

[0231] The present invention further relates to the therapeutic use of such polypeptides, proteins, constructs, or compounds, and to pharmaceutical compositions comprising such polypeptides, proteins, constructs, or compounds.

[0232] In one embodiment, at least one therapeutic moiety comprises or essentially comprises a therapeutic protein, polypeptide, compound, factor, or other entity. In a preferred embodiment, the therapeutic moiety is directed toward a desired antigen or target, can bind to a desired antigen (in particular, can bind specifically to a desired antigen), and / or can interact with a desired target. In another embodiment, at least one therapeutic moiety comprises or essentially comprises a therapeutic protein or polypeptide. In further embodiments, at least one therapeutic moiety comprises or essentially comprises a binding domain or binding unit, such as an immunoglobulin or immunoglobulin sequence (including but not limited to immunoglobulin fragments), such as an antibody or antibody fragment (including but not limited to ScFv fragments), or another suitable protein backbone, such as a protein A domain (e.g., Affibodies®), tendamistat, fibronectin, lipocalin, CTLA-4, T cell receptor, designed ankyrin repeat sequence, avimer, and PDZ domain (Binz et al., Nat. Biotech 2005, Vol 23:1257), and a DNA or RNA-based binding moiety, including but not limited to a DNA or RNA aptamer (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

[0233] In yet another embodiment, at least one therapeutic portion comprises or essentially comprises an antibody-variable domain, such as a heavy-chain variable domain or a light-chain variable domain.

[0234] In a preferred embodiment, at least one therapeutic portion comprises or essentially comprises at least one immunoglobulin monovariable domain, e.g., a domain antibody, a single-domain antibody, "dAb", or a nanobody (e.g., VHH, humanized VHH, or camelid VHH) or an IgNAR domain.

[0235] In specific embodiments, at least one therapeutic portion comprises or essentially consists of at least one monovalent nanobody, or a divalent, polyvalent, bispecific, or multiplespecific nanobody construct.

[0236] Polypeptides, (fusion) proteins, constructs, or compounds comprising the serum albumin binder of the present invention and one or more therapeutic moieties may generally be prepared and used as described in the prior art referenced above (for example, International Publication Nos. 04 / 041865 and International Publication Nos. 06 / 122787), but the serum albumin binder of the present invention is used instead of the half-life-extending moieties described in the prior art.

[0237] The polypeptide, (fusion) protein, construct, or compound comprising the serum albumin binder and one or more therapeutic moieties of the present invention generally and preferably has an extended half-life compared to the therapeutic moiety or group of therapeutic moieties themselves.

[0238] Generally, the compounds, polypeptides, constructs, or fusion proteins described herein preferably have a half-life (as measured in humans or suitable animals, e.g., mice or cynomolgus monkeys) of at least 1.5 times, preferably at least 2 times, e.g., at least 5 times, e.g., at least 10 times, or more than 20 times, of the half-life of the corresponding therapeutic portion itself.

[0239] Furthermore, preferably, any such compound, polypeptide, fusion protein, or construct has a half-life in humans that is extended by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example more than 12 hours, compared to the half-life of the corresponding therapeutic portion itself.

[0240] Furthermore, preferably, the compound or polypeptide of the present invention has a half-life (preferably defined as t1 / 2β) in humans of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example more than 12 hours, 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).

[0241] Half-life can generally be defined as the time required for the serum concentration of a polypeptide to decrease by 50% in vivo, for example, due to ligand degradation and / or ligand elimination or capture by natural mechanisms. In particular, half-life may be as defined in International Publication No. 2009 / 068627.

[0242] 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). It is also mentioned in "Pharmacokinetics" by Marcel Dekker, M Gibaldi & D Perron, revised edition (1982).

[0243] As described, in one embodiment, the serum albumin conjugate of the present invention may be used to extend the half-life of (one or more) immunoglobulin monovariate domains, e.g., domain antibodies, single-domain antibodies, "dAb", VHH, or nanobodies (e.g., VHH, humanized VHH, or camelid VH, e.g., camelid human VH).

[0244] Accordingly, one embodiment of the present invention relates to a polypeptide, construct, or fusion protein comprising the serum albumin conjugate of the present invention and one or more (e.g., one or two) immunoglobulin monovariable domain sequences, which are appropriately linked to one another, either directly or optionally via one or more suitable linkers or spacers. As described herein, each such immunoglobulin monovariable domain present in such polypeptide, construct, or fusion protein may independently be a domain antibody, a single-domain antibody, a "dAb", or a nanobody (e.g., VHH, humanized VHH, or camelid VH, e.g., camelid human VH); according to one specific but non-limiting embodiment, at least one (and up to all) of these immunoglobulin monovariable domains include two or three disulfide bridges.

[0245] As described, if the polypeptide, construct, or fusion protein has a heavy chain ISVD at its C-terminus (the ISVD may be an ISVD for the serum albumin-binding agent or therapeutic target of the present invention, for example, a nanobody for the therapeutic target), the polypeptide, construct, or fusion protein (the ISVD present at its C-terminus) preferably has a C-terminal extension at its C-terminus. Here again, the C-terminal extension is given by formula (X) n The expression is shown as follows, where n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (preferably 1 or 2, for example 1); each X is an (preferably natural) amino acid residue independently selected from natural amino acid residues (according to one preferred embodiment, it does not contain any cysteine ​​residues), preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (V), or isoleucine (I).

[0246] Furthermore, as described above, if the polypeptide, construct, or fusion protein has a heavy chain ISVD at its N-terminus (the ISVD may be an ISVD for the serum albumin-binding agent or therapeutic target of the present invention, for example, a nanobody for the therapeutic target), the polypeptide, construct, or fusion protein (the ISVD present at its C-terminus) preferably has a D or E1D mutation at position 1.

[0247] Therefore, in another embodiment, the present invention is - comprising or essentially consisting of at least one (preferably just one) serum albumin binder of the present invention and at least one (e.g., one, two, or three) therapeutic portion or entity (the serum albumin binder and one or more therapeutic portions or entities are appropriately linked, optionally via one or more suitable linkers); -Its C-terminus has a heavy chain ISVD (where ISVD at the C-terminus is C-terminal elongation (X) n (Having as further described herein) With respect to proteins, polypeptides, or other compounds; - The protein, polypeptide, or other compound may also have a heavy chain ISVD at its N-terminus, in which case the N-terminus ISVD terminus preferably has D or E1D at position 1.

[0248] In addition, in a preferred embodiment, if one or more other ISVDs are present in addition to the serum albumin-binding agent of the present invention (i.e., if one or more therapeutic portions present are ISVDs), then (one or all) of the “therapeutic” ISVDs preferably also have amino acid residues / mutations (or combinations thereof) that reduce binding by existing antibodies. If the ISVDs are heavy chain ISVDs, these mutations may be one or more mutations (or appropriate combinations thereof) at positions 11, 89, 110, and 112, in particular, as described herein for the serum albumin-binding agent of the present invention, as described in PCT / EP2015 / 060643. Preferably, if such other ISVDs are present at the C-terminus, at least the therapeutic ISVDs include such mutations at positions 11, 89, 110, and / or 112 (i.e., in addition to the C-terminal elongation as described herein).

[0249] In one specific embodiment, all therapeutic portions present in a construct, fusion protein, or polypeptide are ISVDs (i.e., ISVDs for therapeutic targets), particularly heavy chain ISVDs, and more specifically, nanobodies (i.e., nanobodies for therapeutic targets).

[0250] For example, and not limited to, constructs, fusion proteins, or polypeptides containing the serum albumin binder of the present invention are: -1 copy of the serum albumin binder of the present invention and one ISVD (preferably a nanobody) for a therapeutic target; or -1 copy of the serum albumin binder of the present invention and two ISVDs (preferably two nanobodies) directed to a therapeutic target (the ISVDs may be the same or different, and if different, they may be directed to the same target, to different epitopes on the same target, or to different therapeutic targets); or -1 copy of the serum albumin binding agent of the present invention and three ISVDs (preferably three Nanobodies) against a therapeutic target (said ISVDs may be the same or different, and when they are different, they may be directed against the same target, against different epitopes on the same target, or against different therapeutic targets) may be included.

[0251] Some non-limiting examples of the constructs, fusion proteins or polypeptides of the present invention may be illustrated in the figures as follows, where "[Alb]" represents the serum albumin binding agent of the present invention, "[Therapeutic Moiety 1]" and "[Therapeutic Moiety 2]" represent therapeutic moieties (which, as described, may each independently be an immunoglobulin single variable domain), "-" represents a suitable linker (which is optional; suitable examples are 9GS and 35GS linkers), the N-terminus is on the left hand side and the C-terminus is on the right hand side: [Alb]-[Therapeutic Moiety 1] [Therapeutic Moiety 1]-[Alb]-X (n) [Alb]-[Therapeutic Moiety 1]-[Therapeutic Moiety 1] [Therapeutic Moiety 1]-[Therapeutic Moiety 1]-[Alb]-X (n) [Therapeutic Moiety 1]-[Alb]-[Therapeutic Moiety 1] [Alb]-[Therapeutic Moiety 1]-[Therapeutic Moiety 2] [Therapeutic Moiety 1]-[Therapeutic Moiety 2]-[Alb]-X (n) [Therapeutic Moiety 1]-[Alb]-[Therapeutic Moiety 2].

[0252] When the therapeutic portion is an ISVD (preferably a nanobody) directed at a therapeutic target, preferred but non-limiting constructs, fusion proteins, or polypeptides of the present invention may be shown in the figure as follows: "[Alb]" indicates the serum albumin binder of the present invention, "[ISVD1 for Therapy]" and "[ISVD2 for Therapy]" indicate ISVDs directed at a therapeutic target (the ISVDs may be the same or different, and if different, they may be directed to the same target, to different epitopes on the same target, or to different therapeutic targets), "[-]" indicates a suitable linker (which is optional), and X(n) indicates a C-terminal extension as described herein, with the N-terminus being left-handed and the C-terminus being right-handed: [Alb]-[Therapeutic ISVD1]-X (n) [Therapeutic ISVD1]-[Alb]-X (n) [Alb]-[Therapeutic ISVD1]-[Therapeutic ISVD1]-X (n) [Therapeutic ISVD1]-[Therapeutic ISVD1]-[Alb]-X (n) [Therapeutic ISVD1]-[Alb]-[Therapeutic ISVD1]-X (n) [Alb]-[Therapeutic ISVD1]-[Therapeutic ISVD2]-X (n) [Therapeutic ISVD1]-[Therapeutic ISVD2]-[Alb]-X (n) [Therapeutic ISVD1]-[Alb]-[Therapeutic ISVD2]-X (n) .

[0253] Accordingly, in another embodiment, the present invention relates to a multispecific (particularly bispecific) nanobody construct comprising the serum albumin binder of the present invention and at least one other nanobody (e.g., one or two other nanobodyes, which may be the same or different) (the said at least one other nanobody is preferably directed toward a desired target (which is preferably a therapeutic target)), and / or another nanobody that is useful or suitable for therapeutic, preventive, and / or diagnostic purposes. Here again, the serum albumin binder of the present invention and the other nanobody may be appropriately linked to one another directly or optionally via one or more suitable linkers or spacers.

[0254] For a general description of polyvalent and multispecific polypeptides containing one or more nanobodies and their preparations, see Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; and also see, for example, International Publication Nos. 96 / 34103, 99 / 23221, 04 / 041862, 2006 / 122786, 2008 / 020079, 2008 / 142164, or 2009 / 068627.

[0255] Some specific examples of multiple specific and / or polyvalent polypeptides of the present invention may be found in the application by Ablynx NV described herein. In particular, a general description of polyvalent and multispecific constructs comprising at least one nanobody for serum proteins to extend half-life, nucleic acids encoding them, compositions comprising them, the preparation of them, and their use is referred to in International Publication Nos. 04 / 041865 and 06 / 122787 of the aforementioned international applications (the serum albumin binders of the present invention described herein may generally be used in the same way as the half-life-extending nanobodies described therein, such as Alb-8), as well as in the general descriptions and specific examples of such constructs shown in, for example, International Publication Nos. 04 / 041862, 2006 / 122786, 2008 / 020079, 2008 / 142164, or 2009 / 068627.

[0256] In one embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs (e.g., nanobodies or (single) domain antibodies containing or derived from a VH domain), wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: The amino acid residue at position -11 is preferably selected from L or V; and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L; and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q; and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q; Therefore, (i) the 89th position is T; or (ii) the 89th position is L and the 11th position is V; or (iii) the 89th position is L and the 110th position is K or Q; or (iv) the 89th position is L and the 112th position is K or Q; or (v) the 89th position is L, the 11th position is V and the 110th position is K or Q; or (vi) the 89th position is L, the 11th position is V and the 112th position is K or Q; or (vii) the 11th position is V and the 110th position is K or Q; or (vii) the 11th position is V and the 112th position is K or Q.

[0257] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: -89T; or 89L combined with -11V; or -89L in combination with 110K or 110Q; or -89L in combination with 112K or 112Q; or -89L in combination with -11V and 110K or 110Q; or -89L in combination with 11V and 112K or 112Q; or 11V combined with -110K or 110Q; or -11V when combined with 112K or 112Q.

[0258] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: The amino acid residue at position -11 is preferably selected from L or V; and The amino acid residue at position -89 is T; and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q (preferably T); and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q (preferably S).

[0259] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: The amino acid residue at position -11 is V; and The amino acid residue at position -89 is L; and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q; and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q.

[0260] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: -11V in combination with 89L; or 11V in combination with -110K or 110Q; -11V in combination with 112K or 112Q; -11V in combination with 89L and 110K or 110Q; or -11V when combined with 89L and 112K or 112Q.

[0261] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: 89L combined with -11V; or -89L in combination with 110K or 110Q; or -89L in combination with 112K or 112Q; or -89L in combination with -11V and 110K or 110Q; or -89L in combination with -11V and 112K or 112Q.

[0262] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: 110K or 110Q combined with -11V; or -110K or 110Q in combination with 89L; or 110K or 110Q when combined with -11V and 89L.

[0263] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain the following amino acid residues: 112K or 112Q combined with -11V; or -112K or 112Q in combination with 89L; or 112K or 112Q in combination with -11V and 89L.

[0264] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain T at position 89.

[0265] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein all of the one or more further heavy chain ISVDs contain V at position 11 and L at position 89.

[0266] Herein too, if such a protein, polypeptide, or other compound or construct has a (heavy chain)ISVD at its C-terminus, it preferably contains a C-terminal extension X(n) (as described herein), and if such a protein, polypeptide, or other compound or construct has a (heavy chain)ISVD at its N-terminus, it preferably has D at position 1. Such a protein, polypeptide, or other compound or construct also preferably has a half-life as further described herein.

[0267] 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 further includes a gene construct comprising the aforementioned nucleotide sequence or nucleic acid and one or more elements for a gene construct known by itself. The gene construct may be in the form of a plasmid or a vector. Here again, such constructs may generally be as described in the published patent applications of Ablynx NV, such as, for example, International Publication No. 04 / 041862, International Publication No. 2006 / 122786, International Publication No. 2008 / 020079, International Publication No. 2008 / 142164, or International Publication No. 2009 / 068627.

[0268] The present invention also relates to a host or host cell that contains such nucleotide sequences or nucleic acids and / or expresses (or can express) the albumin-binding agents, compounds, or polypeptides of the present invention. Herein, such host cells may generally be as described in the published patent applications of Ablynx NV, such as International Publication No. 04 / 041862, International Publication No. 2006 / 122786, International Publication No. 2008 / 020079, International Publication No. 2008 / 142164, or International Publication No. 2009 / 068627.

[0269] The present invention also relates to a method for preparing the albumin-binding agent, compound, or polypeptide of the present invention, the method comprising the steps of culturing or maintaining host cells as described herein under conditions such that the host cells produce or express the albumin-binding agent, compound, or polypeptide of the present invention, and optionally further comprising the steps of isolating the albumin-binding agent, compound, or polypeptide of the present invention thus produced. Herein too, such a method may be carried out as generally described in Ablynx NV's published patent applications, such as International Publication No. 04 / 041862, International Publication No. 2006 / 122786, International Publication No. 2008 / 020079, International Publication No. 2008 / 142164, or International Publication No. 2009 / 068627.

[0270] 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 preparations, carriers, excipients, and diluents are generally as described in Ablynx NV's published patent applications, such as International Publication No. 04 / 041862, International Publication No. 2006 / 122786, International Publication No. 2008 / 020079, International Publication No. 2008 / 142164, or International Publication No. 2009 / 068627.

[0271] However, since the compounds or polypeptides of the present invention have an extended half-life, they are preferably administered in circulation. Therefore, they can be administered in any suitable manner that allows the compounds or polypeptides of the present invention to enter circulation, for example, intravenously, by injection or infusion, or in any other suitable manner (including oral administration, subcutaneous administration, intramuscular administration, transcutaneous administration, intranasal administration, transpulmonary administration, etc.). Suitable methods and routes of administration will be apparent to those skilled in the art from the doctrines of Ablynx NV's published patent applications, such as, for example, International Publication No. 04 / 041862, International Publication No. 2006 / 122786, International Publication No. 2008 / 020079, International Publication No. 2008 / 142164, or International Publication No. 2009 / 068627.

[0272] Accordingly, in another embodiment, 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 the use of the compound or polypeptide of the present invention, the method comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of the compound or polypeptide of the present invention and / or a pharmaceutical composition containing the same. Diseases and disorders that can be prevented or treated by the use of the compound or polypeptide of the present invention as described herein will generally be the same as diseases and disorders that can be prevented or treated by the use of the compound or polypeptide of the present invention / a therapeutic portion or therapeutic subgroup present in the compound or polypeptide of the present invention.

[0273] In the context of the present invention, the term “prevention and / or treatment” includes not only the prevention and / or treatment of a disease, but also generally the prevention of the onset of a disease, the slowing or reversal of the progression of a disease, the prevention or slowing of the onset of one or more symptoms associated with a disease, the reduction and / or remission of one or more symptoms associated with a disease, the reduction of the severity and / or duration of any symptoms associated with a disease, and / or the prevention of further increases in the severity of any symptoms associated with a disease, the prevention, reduction or reversal of any physiological impairment caused by a disease, and generally any pharmacological effects that are beneficial to the patient being treated.

[0274] The subjects treated may be any warm-blooded animal, but more specifically mammals, and more specifically humans. As will be apparent to those skilled in the art, the subjects treated will particularly be humans suffering from or at risk of the diseases and disorders described herein.

[0275] In another embodiment, the present invention relates to a method for immunotherapy, particularly for passive immunotherapy, the method comprising administering to a subject who has or is at risk of having one of the diseases and disorders described herein, a pharmaceutically effective amount of the compound or polypeptide of the present invention and / or a pharmaceutical composition containing the same.

[0276] The compounds or polypeptides of the present invention and / or compositions containing them are administered in accordance with a treatment plan suitable for preventing and / or treating the disease or disorder to be prevented or treated. A clinician may generally determine an appropriate treatment plan depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease and / or its symptoms to be treated, 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 patient's age, sex, weight, diet, overall condition, and similar factors well known to clinicians.

[0277] Generally, the treatment plan involves administering one or more pharmaceutically effective amounts or doses of one or more compounds or polypeptides of the present invention, or one or more compositions containing them. The specific amount(s) or dose(s) to be administered may be determined by the clinician based on the factors cited herein.

[0278] Generally, for the prevention and / or treatment of the diseases and disorders described herein, the compounds or polypeptides of the present invention will generally be administered in amounts of 1 g to 0.01 μg / kg(body weight) / day, preferably 0.1 g to 0.1 μg / kg(body weight) / day, for example, about 1, 10, 100, or 1000 μg / kg(body weight) / day, either as a single dose once daily or in multiple divided doses throughout the day, depending on the specific disease or disorder to be treated, the potency and / or half-life of the compound or polypeptide to be used, the specific route of administration, and the specific pharmaceutical formulation or composition used. Generally, some guidelines regarding the dosage can be derived from the usual dosages of equivalent conventional antibodies or antibody fragments targeting the same target and administered via essentially the same route, but taking into account differences in affinity / binding capacity, efficacy, biodistribution, half-life, and similar factors well known to those skilled in the art.

[0279] Furthermore, since the compounds of the present invention contain serum albumin binders of the present invention with extended half-lives, they do not inherently need to be administered continuously (e.g., by infusion), but they can be administered at appropriate intervals (determined by those skilled in the art). For example, they can be administered (in appropriate doses) once every two days, once every four days, once a week, once every two weeks, and possibly once every four weeks, or even less frequently, for example, by injection or infusion.

[0280] One aspect of the present invention relates to a pharmaceutical composition comprising at least one compound or polypeptide of the present invention, which is intended to be administered at intervals of once a week to once every four weeks, particularly once every seven days to once every 21 days, for example, once every seven days or once every 14 days.

[0281] Typically, a single polypeptide of the present invention would be used in the methods described above. However, using two or more polypeptides of the present invention in combination is within the scope of the invention.

[0282] The polypeptides of the present invention may also be used in combination with one or more further pharmaceutically active compounds or components, i.e., as a combination treatment plan, which may or may not result in synergistic effects. Here again, clinicians will be able to select such further compounds or components, as well as an appropriate combination treatment plan, based on the factors cited above and their professional judgment.

[0283] In particular, the polypeptides of the present invention may be used in combination with other pharmaceutically active compounds or components used or may be used for the prevention and / or treatment of diseases and disorders that can be prevented or treated using the fusion proteins or constructs of the present invention, and as a result, a synergistic effect may or may not be obtained.

[0284] The effectiveness of treatment plans used in accordance with the present invention can be determined and / or tracked in any manner known in itself for the disease or disorder involved, as is evident to the clinician. The clinician may also modify or alter specific treatment plans as appropriate and / or as necessary, thereby achieving the desired therapeutic effect and avoiding, limiting, or reducing undesirable side effects, and / or striking an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting, or reducing undesirable side effects on the other hand.

[0285] Generally, the treatment plan will be followed until the desired therapeutic effect is achieved and / or maintained. Again, this can be determined by the clinician.

[0286] Other aspects, embodiments, advantages, and applications of the present invention will become apparent from further description herein.

[0287] The present invention will be further described with reference to the following non-limiting preferred embodiments, examples, and drawings. [Brief explanation of the drawing]

[0288] [Figure 1] Figure 1 is a table listing some of the amino acid positions specifically mentioned herein and their numbering according to some alternative numbering systems (e.g., Aho and IMGT). [Figure 2-1] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-2] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-3] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-4] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-5] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-6] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-7] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-8] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-9] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-10] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-11]Figure 2 lists the amino acid sequences referred to herein; [Figure 2-12] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-13] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-14] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-15] Figure 2 lists the amino acid sequences referred to herein; [Figure 2-16] Figure 2 lists the amino acid sequences referred to herein; [Figure 3A] Figures 3A-C show the alignments of SEQ ID NOs: 1, 50, and 119, and Figure 3D shows the binding of existing antibodies derived from six serum albumin-depleted serums. [Figure 3B] Figures 3A-C show the alignments of SEQ ID NOs: 1, 50, and 119, and Figure 3D shows the binding of existing antibodies derived from six serum albumin-depleted serums. [Figure 3C] Figures 3A-C show the alignments of SEQ ID NOs: 1, 50, and 119, and Figure 3D shows the binding of existing antibodies derived from six serum albumin-depleted serums. [Figure 3D] Figures 3A-C show the alignments of SEQ ID NOs: 1, 50, and 119, and Figure 3D shows the binding of existing antibodies derived from six serum albumin-depleted serums. [Figure 4A] Figure 4A shows the alignment of sequence numbers 1 and 8-50, and Figure 4B shows the alignment of sequence numbers 1, 50 and 61-102. [Figure 4B] Figure 4A shows the alignment of sequence numbers 1 and 8-50, and Figure 4B shows the alignment of sequence numbers 1, 50 and 61-102. [Figure 5]Figure 5 shows plots of two corresponding data points obtained in Example 1 when 96 serum samples (68 from healthy human subjects, 17 from healthy human test volunteers whose serum contained pre-existing antibodies capable of binding 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 + V89T + C-terminal alanine], [Reference A + L11V + V89T + T110K + C-terminal alanine], and [Reference A + L11V + V89T + S104T + C-terminal alanine], respectively). Compared to the sequences of Prior Art Sequence IDs 1 and 50, it should be noted that Reference A already contains the L5V mutation, and therefore all three variants contain V at positions 5 and 11, in addition to the specific mutations shown. Each point indicates the binding level for one of the 96 samples tested. The data points shown in the right-hand and left-hand panels are the same; in the right-hand panel, the data points measured using each individual sample for each compound tested are connected by lines (consequently, a downward slope of the line indicates the degree to which binding by existing antibodies decreases when the mutation and C-terminal alanine of the present invention are introduced). [Figure 6] Figure 6 shows in detail the data from Figure 5 obtained in Example 1 for 11 samples derived from SLE patients. [Figure 7-1] Figure 7 is a table listing the combined data of the data points edited in Figure 5. [Figure 7-2] Figure 7 is a table listing the combined data of the data points edited in Figure 5. [Figure 7-3] Figure 7 is a table listing the combined data of the data points edited in Figure 5. [Figure 8]Figure 8 shows plots of two corresponding data points obtained in Example 2 when 96 serum samples (68 from healthy human subjects, 17 from healthy human test volunteers whose serum contained pre-existing antibodies capable of binding 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 + V89T + C-terminal alanine], [Reference A + L11V + V89L + T110K + C-terminal alanine], and [Reference A + L11V + V89L + S104G + C-terminal alanine], respectively). Compared to the sequences of Prior Art Sequence IDs 1 and 50, it should be noted that Reference A already contains the L5V mutation, and therefore all three variants contain V at positions 5 and 11, in addition to the specific mutations shown. Each point indicates the binding level for one of the 96 samples tested. The data points shown in the right-hand and left-hand panels are the same; in the right-hand panel, the data points measured using each individual sample for each compound tested are connected by lines (consequently, a downward slope of the line indicates the degree to which binding by the existing antibody decreases when the mutation and C-terminal alanine of the present invention are introduced). [Figure 9] Figure 9 shows in detail the data from Figure 8 obtained in Example 2 for 11 samples derived from SLE patients. [Figure 10-1] Figure 10 is a table listing the combined data of the data points edited in Figure 8. [Figure 10-2] Figure 10 is a table listing the combined data of the data points edited in Figure 8. [Figure 10-3] Figure 10 is a table listing the combined data of the data points edited in Figure 8. [Figure 11]Figure 11 shows plots of two corresponding data points obtained in Example 3 when 96 serum samples (68 from healthy human subjects, 17 from healthy human test volunteers whose serum contained pre-existing antibodies capable of binding 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 + V89L + S101G + C-terminal alanine], [Reference A + L11V + V89L + S104A + C-terminal alanine] and [Reference A + L11V + V89L + S101E + C-terminal alanine], respectively). Compared to the sequences of Prior Art SEQ ID NOs. 1 and 50, it should be noted that Reference A already contains the L5V mutation, and therefore all three variants contain V at positions 5 and 11, in addition to the specific mutations shown. Each point indicates the binding level for one of the 96 samples tested. The data points shown in the right-hand and left-hand panels are the same; in the right-hand panel, the data points measured using each individual sample for each compound tested are connected by lines (consequently, a downward slope of the line indicates the degree to which binding by existing antibodies decreases when the mutation and C-terminal alanine of the present invention are introduced). [Figure 12] Figure 12 shows in detail the data from Figure 11 obtained in Example 3 for 11 samples derived from SLE patients. [Figure 13-1] Figure 13 is a table listing the combined data of the data points edited in Figure 11. [Figure 13-2] Figure 13 is a table listing the combined data of the data points edited in Figure 11. [Figure 13-3] Figure 13 is a table listing the combined data of the data points edited in Figure 11. [Figure 14]Figure 14 shows plots of two corresponding data points obtained in Example 4 when 96 serum samples (68 from healthy human subjects, 17 from healthy human test volunteers whose serum contained pre-existing antibodies capable of binding 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 + R30T + V89L + C-terminal alanine], [Reference A + L11V + S31D + V89L + C-terminal alanine], and [Reference A + L11V + V89S + C-terminal alanine], respectively). Compared to the sequences of Prior Art SEQ ID NOs. 1 and 50, it should be noted that Reference A already contains the L5V mutation, and therefore all three variants contain V at positions 5 and 11, in addition to the specific mutations shown. Each point indicates the binding level for one of the 96 samples tested. The data points shown in the right-hand and left-hand panels are the same; in the right-hand panel, the data points measured using each individual sample for each compound tested are connected by lines (consequently, a downward slope of the line indicates the degree to which binding by existing antibodies decreases when the mutation and C-terminal alanine of the present invention are introduced). [Figure 15] Figure 15 shows in detail the data from Figure 14 obtained in Example 4 for 11 samples derived from SLE patients. [Figure 16-1] Figure 16 is a table listing the combined data of the data points edited in Figure 14. [Figure 16-2] Figure 16 is a table listing the combined data of the data points edited in Figure 14. [Figure 16-3] Figure 16 is a table listing the combined data of the data points edited in Figure 14. [Figure 17]Figure 17 shows plots of two corresponding data points obtained in Example 5 when 96 serum samples (69 from healthy human subjects, 17 from healthy human test volunteers whose serum contained pre-existing antibodies capable of binding 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 + V89N + C-terminal alanine], [Reference A + L11V + V89N + T110K + C-terminal alanine], and [Reference A + L11V + V89S + T110K + C-terminal alanine], respectively). Compared to the sequences of Prior Art Sequence IDs 1 and 50, it should be noted that Reference A already contains the L5V mutation, and therefore all three variants contain V at positions 5 and 11, in addition to the specific mutations shown. Each point indicates the binding level for one of the 96 samples tested. The data points shown in the right-hand and left-hand panels are the same; in the right-hand panel, the data points measured using each individual sample for each compound tested are connected by lines (consequently, a downward slope of the line indicates the degree to which binding by existing antibodies decreases when the mutation and C-terminal alanine of the present invention are introduced). [Figure 18] Figure 18 shows in detail the data from Figure 17 obtained in Example 5 for 10 samples derived from SLE patients. [Figure 19-1] Figure 19 is a table listing the combined data of the data points edited in Figure 17. [Figure 19-2] Figure 19 is a table listing the combined data of the data points edited in Figure 17. [Figure 19-3] Figure 19 is a table listing the combined data of the data points edited in Figure 17. [Figure 20]Figure 20 shows plots of two corresponding data points obtained in Example 6 when 96 serum samples (69 from healthy human subjects, 17 from healthy human test volunteers whose serum contained pre-existing antibodies capable of binding 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). Compared to the sequences of Prior Art SEQ ID NOs. 1 and 50, it should be noted that Reference A already contains the L5V mutation, and therefore all three variants contain V at positions 5 and 11, in addition to the specific mutations shown. Each point indicates the binding level for one of the 96 samples tested. The data points shown in the right-hand and left-hand panels are the same; in the right-hand panel, the data points measured using each individual sample for each compound being tested are connected by lines (consequently, a downward slope of the line indicates the degree to which binding by existing antibodies decreases when the mutation and C-terminal alanine of the present invention are introduced). [Figure 21] Figure 21 shows in detail the data from Figure 20 obtained in Example 6 for 10 samples derived from SLE patients. [Figure 22-1] Figure 22 is a table listing the combined data of the data points edited in Figure 20. [Figure 22-2] Figure 22 is a table listing the combined data of the data points edited in Figure 20. [Figure 22-3] Figure 22 is a table listing the combined data of the data points edited in Figure 20. [Figure 23] Figure 23 shows the alignment of sequences 1, 50, 119, and 121-132. [Figure 24A]Figures 24A to C show the alignment of sequences 145 to 184, 185 to 208, and 209 to 244, respectively, aligned with sequences 1, 50, and 119 in each case. [Figure 24B] Figures 24A to C show the alignment of sequences 145 to 184, 185 to 208, and 209 to 244, respectively, aligned with sequences 1, 50, and 119 in each case. [Figure 24C] Figures 24A to C show the alignment of sequences 145 to 184, 185 to 208, and 209 to 244, respectively, aligned with sequences 1, 50, and 119 in each case. [Figure 25] Figure 25 shows binding data for several representative albumin binders that have S, R, or T at position 30.

[0289] Experiment Department The human samples used in the following experiments were obtained from either the supplier or human trial volunteers (after all necessary consent and approvals had been obtained) and were used in accordance with applicable legal and regulatory requirements, including but not limited to medical confidentiality and patient privacy requirements.

[0290] In the following examples, unless otherwise specified, the binding of pre-existing antibodies present in the samples used (i.e., derived from healthy test volunteers, rheumatoid arthritis (RA) patients, and SLE patients) to the nanobodies under test was determined using ProteOn as follows.

[0291] Nanobodies were captured either on serum albumin or via a FLAG3 tag using a monoclonal anti-FLAG M2 antibody.

[0292] The binding of existing antibodies to nanobodies captured on human serum albumin (HSA) was evaluated using ProteOn XPR36 (Bio-Rad Laboratories). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) was used as the running buffer, and the experiment was conducted at 25°C. The ligand lane of the ProteOn GLC sensor chip was activated using EDC / NHS (flow rate 30 μl / min), and 10 μg / ml of HSA in ProteOn acetate buffer (pH 4.5) was injected (flow rate 100 μl / min) to an immobilization level of approximately 3200 RU. After immobilization, the surface was deactivated using ethanolamine HCl (flow rate 30 μl / min). Nanobodies were injected onto the HSA surface at a rate of 45 μl / min over 2 minutes to achieve a nanobody capture level of approximately 200 RU. Samples containing the existing antibody were centrifuged at 14,000 rpm for 2 minutes. The supernatant was diluted 1:10 with PBS-Tween20 (0.005%), then injected at 45 μl / min for 2 minutes, followed by a 400-second dissociation step. After each cycle (i.e., before the capture of new nanobodies and the injection of blood samples), the HSA surface was regenerated by injecting HCl (100 mM) at 45 μl / min for 2 minutes. Sensorgram processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories). Sensorgrams showing the binding of the existing antibody were obtained after double normalization by subtracting 1) nanobody-HSA dissociation and 2) nonspecific binding to the reference ligand lane. The binding level of the existing antibody was determined by setting the reporting time at 125 seconds (5 seconds after the end of association). The reduction in binding of existing antibodies was calculated by comparing it to the binding level of a reference nanobody at 125 seconds.

[0293] The binding of an existing antibody to FLAG-tagged nanobodies captured on monoclonal anti-FLAG M2 antibody (Sigma-A) was evaluated using ProteOn XPR36 (Bio-Rad Laboratories). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) was used as the running buffer, and the experiment was conducted at 25°C. The ligand lane of the ProteOn GLC sensor chip was activated using EDC / NHS (flow rate 30 μl / min), and 10 μg / ml of anti-FLAG M2 mAb in ProteOn acetate buffer (pH 4.5) was injected (flow rate 100 μl / min) to an immobilization level of approximately 4000 RU. After immobilization, the surface was deactivated using ethanolamine HCl (flow rate 30 μl / min). Nanobodies were injected onto the anti-FLAG M2 antibody surface at a rate of 45 μl / min over 2 minutes to achieve a nanobody capture level of approximately 100 RU. To reduce nonspecific binding of blood samples to the anti-FLAG M2 antibody surface, 100 nM 3× FLAG peptide (Sigma-A) was added to the blood sample. Samples containing the existing antibody were centrifuged at 14,000 rpm for 2 minutes, the supernatant was diluted 1:10 with PBS-Tween20 (0.005%), and then injected at 45 μl / min for 2 minutes, followed by a 600-second dissociation step. After each cycle (i.e., before the capture of new nanobodies and the injection of new blood samples), the anti-FLAG M2 antibody surface was regenerated by injecting glycine (pH 1.5) (10 mM) at 150 μl / min for 10 seconds. Sensorgram processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories). Sensorgrams showing the binding of existing antibodies were obtained after double normalization by subtracting 1) dissociation of nanobody-anti-FLAG M2 antibody and 2) nonspecific binding of reference ligand to the lane. The binding level of existing antibodies was determined by setting the reporting time at 125 seconds (5 seconds after the end of association). The rate of decrease in binding of existing antibodies was calculated by comparing it with the binding level of the reference nanobody at 125 seconds.

[0294] Example 1: Reference compound A (SEQ ID NO: 119), Reference compound A with C-terminal alanine, and three variants of the present invention (i.e., [Reference compound A + L11V + V89T + C-terminal alanine], [Reference compound A + L11V + V89T + T110K + C-terminal alanine], and [Reference compound A + L11V + V89T + S104T + C-terminal alanine], respectively) (all possessing an N-terminal HIS6 tag) were tested for binding by pre-existing antibodies present in 96 serum samples (68 from healthy human subjects, 17 from healthy human trial volunteers whose serum contained pre-existing antibodies capable of binding even in the presence of C-terminal alanine, and 11 from SLE patients). The compounds were captured using immobilized human serum albumin, and binding was measured using ProteOn according to the protocol set forth in the preface to this experimental section.

[0295] The results are shown in Figure 5 (all samples) and Figure 6 (SLE samples only). Figure 7 lists the results for each sample that forms one of the data points in Figure 5.

[0296] Furthermore, the dynamics of the binding interactions between the tested serum albumin binders and immobilized serum albumin were analyzed (Langmuir model, simultaneous ka / kD model). The results are listed in Table C.

[0297] [Table 3]

[0298] Example 2: Reference compound A (SEQ ID NO: 119), Reference compound A with C-terminal alanine, and three variants of the present invention (i.e., [Reference compound A + L11V + V89A + C-terminal alanine], [Reference compound A + L11V + V89A + T110K + C-terminal alanine], and [Reference compound A + L11V + V89L + S104G + C-terminal alanine] (all possessing an N-terminal HIS6 tag), were tested for binding by pre-existing antibodies present in 96 serum samples (68 from healthy human subjects, 17 from healthy human trial volunteers whose serum contained pre-existing antibodies capable of binding even in the presence of C-terminal alanine, and 11 from SLE patients). The compounds were captured using immobilized human serum albumin, and binding was measured using ProteOn according to the protocol set forth in the preface of this experimental section.

[0299] The results are shown in Figure 8 (all samples) and Figure 9 (SLE samples only). Figure 10 lists the results for each sample that forms one of the data points in Figure 8.

[0300] Furthermore, the dynamics of the binding interactions between the tested serum albumin binders and immobilized serum albumin were analyzed (Langmuir model, simultaneous ka / kD model). The results are listed in Table D.

[0301] [Table 4]

[0302] Example 3: Reference compound A (SEQ ID NO: 119), Reference compound A with C-terminal alanine, and three variants of the present invention (i.e., [Reference compound A + L11V + V89A + S101G + C-terminal alanine], [Reference compound A + L11V + V89A + S104A + C-terminal alanine], and [Reference compound A + L11V + V89L + S101E + C-terminal alanine] (all possessing an N-terminal HIS6 tag), were tested for binding by pre-existing antibodies present in 96 serum samples (68 from healthy human subjects, 17 from healthy human trial volunteers whose serum contained pre-existing antibodies capable of binding even in the presence of C-terminal alanine, and 11 from SLE patients). The compounds were captured using immobilized human serum albumin, and binding was measured using ProteOn according to the protocol set forth in the preface of this experimental section.

[0303] The results are shown in Figure 11 (all samples) and Figure 12 (SLE samples only). Figure 13 lists the results for each sample that forms one of the data points in Figure 11.

[0304] Furthermore, the dynamics of the binding interactions between the tested serum albumin binders and immobilized serum albumin were analyzed (Langmuir model, simultaneous ka / kD model). The results are listed in Table E.

[0305] [Table 5]

[0306] Example 4: Reference compound A (SEQ ID NO: 119), Reference compound A with C-terminal alanine, and three variants of the present invention (i.e., [Reference compound A + L11V + R30T + V89L + C-terminal alanine], [Reference compound A + L11V + S31D + V89L + C-terminal alanine], and [Reference compound A + L11V + V89S + C-terminal alanine] (all possessing an N-terminal HIS6 tag), were tested for binding by pre-existing antibodies present in 96 serum samples (68 from healthy human subjects, 17 from healthy human trial volunteers whose serum contained pre-existing antibodies capable of binding even in the presence of C-terminal alanine, and 11 from SLE patients). The compounds were captured using immobilized human serum albumin, and binding was measured using ProteOn according to the protocol set forth in the preface of this experimental section.

[0307] The results are shown in Figure 14 (all samples) and Figure 15 (SLE samples only). Figure 16 lists the results for each sample that forms one of the data points in Figure 14.

[0308] Furthermore, the dynamics of the binding interactions between the tested serum albumin binders and immobilized serum albumin were analyzed (Langmuir model, simultaneous ka / kD model). The results are listed in Table F.

[0309] [Table 6]

[0310] Example 5: Reference compound A (SEQ ID NO: 119), Reference compound A with C-terminal alanine, and three variants of the present invention (i.e., [Reference compound A + L11V + V89N + C-terminal alanine], [Reference compound A + L11V + V89N + T110K + C-terminal alanine], and [Reference compound A + L11V + V89S + T110K + C-terminal alanine] (all possessing an N-terminal HIS6 tag) were tested for binding by pre-existing antibodies present in 96 serum samples (69 from healthy human subjects, 17 from healthy human trial volunteers whose serum contained pre-existing antibodies capable of binding even in the presence of C-terminal alanine, and 10 from SLE patients). The compounds were captured using immobilized human serum albumin, and binding was measured using ProteOn according to the protocol set forth in the preface of this experimental section.

[0311] The results are shown in Figure 17 (all samples) and Figure 18 (SLE samples only). Figure 19 lists the results for each sample that forms one of the data points in Figure 17.

[0312] Furthermore, the dynamics of the binding interactions between the tested serum albumin binders and immobilized serum albumin were analyzed (Langmuir model, simultaneous ka / kd model). The results are listed in Table G.

[0313] [Table 7]

[0314] Example 6: Reference compound A (SEQ ID NO: 119), Reference compound A with C-terminal alanine, and three variants of the present invention (i.e., [Reference compound A + L11V + V89L + S101H + C-terminal alanine], [Reference compound A + L11V + V89L + R102D + C-terminal alanine], and [Reference compound A + L11V + C-terminal alanine] (all possessing an N-terminal HIS6 tag), were tested for binding by pre-existing antibodies present in 96 serum samples (69 from healthy human subjects, 17 from healthy human trial volunteers whose serum contained pre-existing antibodies capable of binding even in the presence of C-terminal alanine, and 10 from SLE patients). The compounds were captured using immobilized human serum albumin, and binding was measured using ProteOn according to the protocol set forth in the preface of this experimental section.

[0315] The results are shown in Figure 20 (all samples) and Figure 21 (SLE samples only). Figure 22 lists the results for each sample that forms one of the data points in Figure 20.

[0316] Furthermore, the dynamics of the binding interactions between the tested serum albumin binders and immobilized serum albumin were analyzed (Langmuir model, simultaneous ka / kd model). The results are listed in Table H.

[0317] [Table 8]

[0318] Example 7: Effect of the amino acid residue at position 30 bound to serum albumin The kinetic binding data (association rate, dissociation rate, and affinity) obtained for several representative serum albumin binders (all with V at position 5 and V at position 11) for binding to guinea pig serum albumin, rat serum albumin, mouse serum albumin, cynomolgus monkey serum albumin, and human serum albumin was determined using ProteOn. Sequence ID No. 245 was used as the reference. The results are shown in Figure 25, which shows that the tested albumin binders with S, T, and R at position 30, respectively, had equivalent affinity (expressed as KD value) to the various mammalian serum albumins used (i.e., equivalent for binding to guinea pig serum albumin, equivalent for binding to rat serum albumin, etc.).

[0319] The entire contents of all documents cited throughout this application (including references, issued patents, published patent applications, and concurrently pending patent applications) are incorporated herein by reference, in particular for the purposes of the doctrines referenced herein above.

Claims

1. An amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, wherein the amino acid residue at position 5 is V, the amino acid residue at position 11 is V, and the sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (possible C-terminal elongation is not considered at all, and L5V and L11V mutations are not considered).

2. The amino acid sequence according to claim 1, comprising (i.e., in appropriate combinations) at least one or more suitable combinations of the following amino acid residues: 29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T (i.e., compared to the sequence of SEQ ID NO: 1, 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).

3. The amino acid sequence according to claim 1 or claim 2, wherein the amino acid residue at position 89 is selected from T, A, or L.

4. -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 -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); The amino acid sequence according to any one of claims 1, 2, or 3, wherein if CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa).

5. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to any one of claims 1, 2, 3, or 4, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

6. The amino acid sequence according to any one of claims 1, 2, 3, 4, or 5, wherein CDR3 is GGSLSR (SEQ ID NO: 7) and position 104 is G or T.

7. CDR3 is GGSLER (sequence number 57), and in particular, -CDR1 has the amino acid sequence GFTFRSFGMS (SEQ ID NO: 5); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - Is CDR3 the amino acid sequence GGSLER (SEQ ID NO: 57)? Or, -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to any one of claims 1, 2, 3, or 4, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

8. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to any one of claims 1, 2, 3, 4, or 6, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

9. -CDR1 has the amino acid sequence GFTFSSFGMS (SEQ ID NO: 120); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to any one of claims 1, 2, or 3, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

10. The amino acid residue at position -16 is G or N, preferably N; - The amino acid residue at position 45 is P or L, preferably L; The amino acid residues at positions -74 to -76 form an SKN or AKT motif, preferably an AKT motif; - The amino acid residue at position 89 is L, A, or T, preferably L; and The amino acid sequence according to claim 9, wherein the amino acid residue at position -104 is G or T.

11. The amino acid residue at position -16 is G or N, preferably N; - The amino acid residue at position 45 is P or L, preferably L; The amino acid residues at positions -74 to -76 form an SKN or AKT motif, preferably an AKT motif; - The amino acid residue at position 89 is L, A, or T, preferably L; and The amino acid sequence according to claim 10, wherein the amino acid residue at position -104 is G or T.

12. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to any one of claims 1, 2, or 3, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

13. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to any one of claims 1, 2, or 3, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

14. The amino acid residue at position -16 is G or N, preferably N; - The amino acid residue at position 45 is P or L, preferably L; The amino acid residues at positions -74 to -76 form an SKN or AKT motif, preferably an AKT motif; - The amino acid residue at position 89 is L, A, or T, preferably L; and The amino acid sequence according to claim 12 or 13, wherein the amino acid residue at position -104 is G or T.

15. An amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, comprising (i.e., in appropriate combinations) at least one or more suitable combinations of the following amino acid residues (29A, 29H, 30T, 31D, 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G, and / or 104T) (i.e., compared to the sequence of SEQ ID NO: 1, 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); (i) the amino acid sequence having 7 or fewer, preferably 5 or fewer, amino acid differences with respect to the sequence of SEQ ID NO: 1, for example, 5, 4, 3, 2, 1, or 0.

16. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 15, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

17. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 15, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

18. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 15, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

19. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); - The amino acid sequence according to claim 15, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

20. An amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, -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 -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); If CDR1 is GFTFRSFGMS (SEQ ID NO: 5), then CDR3 is not GGSLSR (SEQ ID NO: 7) (and vice versa); the amino acid sequence has 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of SEQ ID NO: 1, for example, 5, 4, 3, 2, 1, or 0 (amino acid differences in CDR are not considered).

21. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 20, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

22. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 20, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

23. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 20, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

24. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 20, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

25. An amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (human) serum albumin, - The amino acid residue at position 5 (according to Kabat) is V; - The amino acid residue at position 11 (according to Kabat) is V; - The amino acid residues at positions 74 to 76 are the motif SKN; - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected particularly from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be particularly from A); - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S, or T, and may be particularly S, or may be selected from A, G, or T (if selected from A, G, and T, it may be particularly T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; - The amino acid residue at position 112 (according to Kabat) is selected from S, K, or Q; Depending on the circumstances, - At least one amino acid residue selected from 29A, 29H, 30T and / or 31D (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from F29A, F29H, R30T, or S31D); and / or - Containing at least one amino acid residue selected from 99G, 101D, 101E, 101G, 101H, 102D, 104A, 104G and / or 104T (i.e., compared to the sequence of SEQ ID NO: 1, they contain at least one amino acid mutation selected from S99G, S101D, S101E, S101G, S101H, R102D, S104A, S104G and / or S104T), - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of Sequence ID No. 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there are 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). The amino acid sequence having the above characteristics.

26. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 25, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

27. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 25, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

28. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 25, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

29. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); - The amino acid sequence according to claim 25, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

30. - The amino acid residue at position 5 (according to Kabat) is V; - The amino acid residue at position 11 (according to Kabat) is V; - The amino acid residues at positions 74 to 76 are the motif SKN; - The amino acid residue at position 89 (according to Kabat) is selected from A, L, N, S, T, or V, and may be selected particularly from L, V, or T, or may be selected from A, S, or N (if selected from A, S, or N, it may be particularly from A); - The amino acid residue at position 104 (according to Kabat) is selected from A, G, S, or T, and may be particularly S, or may be selected from A, G, or T (if selected from A, G, and T, it may be particularly T); The amino acid residue at position -110 (according to Kabat) is selected from T, K, or Q; - The amino acid residue at position 112 (according to Kabat) is selected from S, K, or Q; In the amino acid sequence: -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 -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and -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); - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to the sequence of Sequence ID No. 1 (any possible C-terminal elongation and L5V and L11V mutations are not considered for determining sequence identity); and / or - With respect to the sequence of Sequence ID No. 1, there are 7 or fewer, preferably 5 or fewer, for example, just 3, 2, or 1 "amino acid differences" (as defined herein, with no consideration given to any possible C-terminal elongation, and no consideration given to L5V and L11V mutations). The amino acid sequence having the above characteristics.

31. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 30, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

32. -CDR1 has the amino acid sequence GFTFTSFGMS (SEQ ID NO: 53); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 30, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

33. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); and - The amino acid sequence according to claim 30, wherein CDR3 is the amino acid sequence GGSLSR (SEQ ID NO: 7).

34. -CDR1 has the amino acid sequence GFTFRDFGMS (SEQ ID NO: 54); and -CDR2 has the amino acid sequence SISGSGSDTL (SEQ ID NO: 6); - The amino acid sequence according to claim 30, wherein CDR3 is the amino acid sequence GGSLER (SEQ ID NO: 57).

35. An amino acid sequence that is a single variable domain of immunoglobulin capable of binding to (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), - The amino acid residue at position 5 is V; - The amino acid residue at position 11 is V; The amino acid residue at position -16 is G or N, preferably N; - The amino acid residue at position 45 is P or L, preferably L; The amino acid residues at positions -74 to -76 form an SKN or AKT motif, preferably an AKT motif; - The amino acid residue at position 89 is L, A, or T, preferably L; and - The amino acid residue at position 104 is G or T; The amino acid sequence having 7 or fewer, preferably 5 or fewer, amino acid differences from the sequence of Sequence ID No. 1, for example, 5, 4, 3, 2, 1, or 0 (CDR, and mutations at positions 5, 11, 16, 45, 74-76, 89, and 104 are not considered).

36. - The amino acid residue at position 5 is V; - The amino acid residue at position 11 is V; - The amino acid residue at position 16 is N; - The amino acid residue at position 45 is L; - The amino acid residues at positions 74 to 76 form an AKT motif; - The amino acid residue at position 89 is L, A, or T, preferably L; and The amino acid sequence according to claim 35, wherein the amino acid residue at position -104 is G or T.

37. A protein, polypeptide, or other construct, compound, molecule, or chemical entity comprising at least one amino acid sequence as described in any of claims 1 to 36.

38. A protein, polypeptide, or other construct, compound, molecule, or chemical entity according to claim 37, comprising at least one therapeutic portion or entity.

39. A fusion protein, the protein, polypeptide, or other construct, compound, molecule, or chemical entity according to claim 37 or 38.

40. A pharmaceutical composition comprising a protein, polypeptide, or other construct, compound, molecule, or chemical entity according to any one of claims 37 to 39.

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