Improved serum albumin-binding immunoglobulin variable domains

Abstract

Improved immunoglobulin single variable domains that bind to serum albumin are provided. [Solution] An immunoglobulin single variable domain has specific CDR1, CDR2, and CDR3 (by Abm) and has seven or fewer, for example, five or fewer "amino acid differences" from a specific amino acid sequence, whereby (i) position 89 is T; or (ii) position 89 is L and position 11 is V; or (iii) position 89 is L and position 110 is K or Q; or (iv) position 89 is L and position 112 is K or Q; or (v) position 89 is L, position 11 is V, and position 110 is K or Q; or (vi) position 89 is L, position 11 is V, and position 112 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (vii) position 11 is V and position 112 is K or Q.

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 single variable domains (hereinafter also referred to as "ISV" or "ISVD") that bind to serum albumin, and especially to improved heavy-chain immunoglobulin single variable domains and proteins, polypeptides and other constructs, compounds, molecules or chemical components comprising such improved serum albumin binders.

[0003] 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.

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

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

[0006] In this application, amino acid residues / positions in the immunoglobulin heavy chain variable domain will be indicated by numbering according to Kabat. For convenience, Figure 1 provides a table listing some of the amino acid positions that will be specifically referred to herein, along with their numbering according to several alternative numbering systems (e.g., Aho and IMGT; Note: Unless otherwise specified, Kabat numbering is definitive for this description and the claims, and other numbering systems are provided for reference only).

[0007] With respect to CDRs, as is well known in the art, there are several conventions for defining and describing CDRs of VH or VHH fragments, e.g., the Kabat definition (based on sequence variability and most commonly used) and the Chothia definition (based on the location of structural loop regions). See, for example, the websites http: / / www.bioinf. or g.uk / abs / . For the purposes of this specification and the claims, even when CDRs are referred to according to Kabat, CDRs are most preferably defined based on the AbM definition (which is based on Oxf or d Molecular's AbM antibody modeling software). At the same time, this is considered the best compromise between the Kabat definition and the Chothia definition. Again, see, the websites http: / / www.bioinf. or g.uk / abs / .

[0008] ISVDs capable of binding to serum albumin and their uses are well known in the art, for example, from WO 2004 / 041865, 2006 / 122787, EP 2139918, WO 2011 / 006915, 2012 / 175400, and 2014 / 111550. These documents describe serum albumin-binding ISVDs and their use to extend the serum half-life (as defined in these uses) of therapeutic compounds, parts, and components.

[0009] The present invention aims to provide an improved serum albumin binder, particularly compared to the serum albumin binders disclosed in WO 2011 / 006915 and 2014 / 111550. Representative examples of serum albumin binders known from these two PCT applications are provided below in Table A as "Reference A" to "Reference D," respectively. Alignments of these reference sequences are provided in Figure 2. The CDRs (combinations) of these reference compounds (according to Kabat and Abm conventions, respectively) are listed in Table B.

[0010] In particular, the present invention aims to provide improved serum albumin-binding ISVDs, which are variants of the serum albumin-binding ISVDs mentioned in Table A, and which have reduced binding affinity due to interfering factors (generally referred to as “existing antibodies”) that may be present in the serum of some healthy human subjects and patients. See WO Nos. 12 / 175741 and 2013 / 024059, and for example, Holland et al. (J. Clin. Immunol. 2013, 33(7):1192-203), and concurrently pending unpublished PCT application PCT / EP No. 2015 / 060643 (published on November 19, 2015 as WO No. 2015 / 173325), filed by the assignee on 13 May 2015, with the title of the invention “Improved Immunoglobulin Variable Domains”.

[0011] [Table 1]

[0012] [Table 2]

[0013] As further described herein, the serum albumin binder of the present invention preferably has the same combination of CDRs (i.e., CDR1, CDR2, and CDR3) as present in one of references A, B, C, or D, and most preferably has the same combination of CDRs as present in reference C or reference D (which have the same CDRs).

[0014] Among the serum albumin binders listed in Table A, the binder with Sequence ID No. 4 exhibits C-terminal alanine elongation, i.e., an alanine residue at the C-terminus of the ISVD sequence (sometimes referred to as "position 114") compared to the normal C-terminal sequence VTVSS (the same as that present in binders with Sequence ID No. 113, Sequence IDs 1-3). As described in WO No. 12 / 175741 (and also, for example, in WO No. 2013 / 024059), this C-terminal alanine elongation can prevent so-called "existing antibodies" (presumably IgG) from binding to a hypothetical epitope located in the C-terminal region of ISV. This epitope is presumed to include, among other residues, the surface-exposed amino acid residue of the C-terminal sequence VTVSS and the amino acid residue at position 14 (and the amino acid residues immediately following or near that residue in the amino acid sequence, for example, at positions 11, 13, and 15), and may also include the amino acid residue at position 83 (and the amino acid residues immediately following or near that residue in the amino acid sequence, for example, at positions 82, 82a, 82b, and 84) and / or the amino acid residue at position 108 (and the amino acid residue immediately following or near that residue in the amino acid sequence, for example, at position 107).

[0015] On the other hand, the presence of such C-terminal alanine (or more generally, C-terminal extension) can significantly reduce (and in many cases even completely prevent) the binding affinity of “existing antibodies” that can be found in serum from a certain range of subjects (both healthy subjects and patients), however, it has been found that serum from some subjects (e.g., serum from patients with certain immune diseases, e.g., SLE) may contain existing antibodies that can bind to the C-terminal region of ISV (if such region is exposed), even if ISV contains such C-terminal alanine (or more generally, such C-terminal extension). Again, refer to concurrently pending unpublished PCT application PCT / EP 2015 / 060643, filed by the Assignee on 13 May 2015, with the title of the invention “Improved Immunoglobulin Variable Domains”.

[0016] Thus, one specific object of the present invention is to provide a serum albumin binder which is a variant of the serum albumin-binding ISVD listed in Table A and has a reduced binding ability by so-called "existing antibodies" and in particular "existing antibodies" of the type described in PCT / EP 2015 / 060643 (i.e., existing antibodies capable of binding to the exposed C-terminal region of ISV even in the presence of a C-terminal extension).

[0017] Generally, the present invention relates to the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NOs: 1 to 4) -89T or 89L in combination 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 in combination with -110K or 110Q or 11V in combination with -112K or 112Q By providing a variant of the sequence of SEQ ID NOs: 1 to 4 (and in particular a variant of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4) of serum albumin-binding ISV containing the above, this object is achieved.

[0018] In a specific embodiment, in the serum albumin binder of the present invention, The amino acid residue at position -11 is preferably selected from L, V or K, 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, Thereby, (i) the 89th position is T; or (ii) the 110th position is K or Q; or (iii) the 112th position is K or Q; or (iv) the 89th position is L and the 11th position is V; or (v) the 89th position is L and the 110th position is K or Q; or (vi) the 89th position is L and the 112th position is K or Q; or (vi) the 89th position is L and the 11th position is V and the 110th position is K or Q; or (vii) the 89th position is L and the 11th position is V and the 112th position is K or Q; or (viii) the 11th position is V and the 110th position is K or Q; or (ix) the 11th position is V and the 112th position is K or Q.

[0019] Among the amino acid sequences provided by the present invention, amino acid sequences in which position 89 is T or position 11 is V and position 89 is L are particularly preferred (in cases, in appropriate combinations with mutations of 110K or 110Q and / or mutations of 112K or 112Q, and especially in combination with mutations of 110K or 110Q). In cases, amino acid sequences in which position 11 is V and position 89 is L are even more preferred, along with mutations of 110K or 110Q.

[0020] The amino acid sequences of the present invention preferably bind to (human) serum albumin with an affinity better than 100 nM, and preferably better than 50 nM. For example, albumin binders of the present invention that are variants of reference A or reference B may have substantially the same affinity to (human) serum albumin as described for reference A or reference B, respectively, in WO 2011 / 006915, and similarly, albumin binders of the present invention that are variants of reference C and / or reference D may have substantially the same affinity to (human) serum albumin as described for reference C and / or reference D, respectively, in WO 2014 / 111550 (where the affinity is measured as described in WO 2011 / 006915 or WO 2014 / 111550, respectively).

[0021] Furthermore, the albumin binders and compounds and polypeptides provided by the present invention preferably have a half-life (defined as t1 / 2 beta) in humans, which is, for example, more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example more than 12 hours and, for example, about 1 day, 2 days, 1 week, 2 weeks and at most, the half-life of serum albumin in humans (estimated to be about 19 days). However, the latter may be less important.

[0022] For example, the albumin binders of the present invention that are variants of reference A or reference B may have a human half-life comparable to (and preferably substantially the same as) that of reference A or reference B, respectively (see again WO 2011 / 006915), and similarly, the albumin binders of the present invention that are variants of reference C and / or reference D may have a human half-life comparable to (and preferably substantially the same as) that of reference C and / or reference D, respectively (see again WO 2014 / 111550).

[0023] Furthermore, the compounds or polypeptides of the present invention containing albumin binders that are variants of reference A or reference B, respectively, may have a human half-life comparable to (and preferably substantially the same as) that of the same compounds or polypeptides having reference A or reference B, respectively, instead of the albumin binder of the present invention. Similarly, the compounds or polypeptides of the present invention containing albumin binders that are variants of reference C or reference D, respectively, may have a human half-life comparable to (and preferably substantially the same as) that of the same compounds or polypeptides having reference C or reference D, respectively.

[0024] Table C lists several non-limiting possible combinations of amino acid residues that may be present at positions 11, 89, 110, and 112 in the serum albumin binder of the present invention. Particularly preferred combinations are shown in bold, and the most preferred combinations are shown in bold / underlined.

[0025] [Table 3]

[0026] The serum albumin binders of the present invention are as further described in the description, examples and drawings herein. That is, they have the CDR described herein, have the overall degree of sequence identity described herein to one of the sequences of SEQ ID NOs: 1 to 4 disclosed herein, and / or may have a limited number of "amino acid differences" described herein to one of these reference sequences.

[0027] The serum albumin binder of the present invention preferably has the following CDR (according to Kabat convention): -The following sequences are selected from TGEMA (sequence number: 5) and TSSML (sequence number: 10), preferably TSSML (sequence number: 10), and CDR1 (by Kabat) and -The following sequences are selected from SISSSGATTYYADSVKG (sequence number: 6) and VIHQSGTPTYYADSVKG (sequence number: 11), preferably VIHQSGTPTYYADSVKG (sequence number: 11) for CDR2 (by Kabat) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (by Kabat) Includes.

[0028] More preferably, the CDRs are as follows (again, given according to Kabat convention): CDR1 is TSSML (SEQ ID NO: 10); CDR2 is VIHQSGTPTYYADSVKG (SEQ ID NO: 11); and CDR3 is FPSTHGKFDY (SEQ ID NO: 12) or FPSSRMKFDY (SEQ ID NO: 15). Most preferably, CDR1 is TSSML (SEQ ID NO: 10); CDR2 is VIHQSGTPTYYADSVKG (SEQ ID NO: 11); and CDR3 is FPSSRMKFDY (SEQ ID NO: 15).

[0029] Alternatively, if the CDR is given according to Abm convention, the serum albumin binder of the present invention is preferably the following CDR: -The following sequences are selected from GFTFSTGEMA (sequence number: 8) and GFTFDTSSML (sequence number: 13), preferably GFTFDTSSML (sequence number: 13) CDR1 (by Abm) and -The following sequences are selected from SISSSGATTY (sequence number: 9) and VIHQSGTPTY (sequence number: 14), preferably VIHQSGTPTY (sequence number: 14) CDR2 (by Abm) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (according to Abm) Includes.

[0030] When given according to Abm convention, preferably the CDRs are as follows: CDR1 is GFTFDTSSML (SEQ ID NO: 13), CDR2 is VIHQSGTPTY (SEQ ID NO: 14), and CDR3 is FPSTHGKFDY (SEQ ID NO: 12) or FPSSRMKFDY (SEQ ID NO: 15). Most preferably, CDR1 is GFTFDTSSML (SEQ ID NO: 13), CDR2 is VIHQSGTPTY (SEQ ID NO: 14), and CDR3 is FPSSRMKFDY (SEQ ID NO: 15).

[0031] Furthermore, the serum albumin binder of the present invention is preferably, -Sequence identity to one of the sequences of sequence numbers 1 to 4, of at least 85%, preferably at least 90%, and more preferably at least 95%, (any C-terminal extensions and CDRs that may be present in the sequence are not considered in determining the degree of sequence identity), and in particular, sequence identity to sequence number 3 or 4, of at least 85%, preferably at least 90%, and more preferably at least 95%, (again, any C-terminal extensions and CDRs that may be present in the sequence are not considered in determining the degree of sequence identity), and / or -Five or fewer, preferably three or fewer, "amino acid differences" (as defined herein, without regard to any of the listed mutations at positions 11, 89, 110 or 112, which may exist, and without regard to any C-terminal elongations that may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) and in particular, five or fewer, preferably three or fewer, such amino acid differences (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) for any of the sequences of sequence number 3 or sequence number 4 (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) It also possesses.

[0032] With respect to various and preferred embodiments of the albumin binder of the present invention, if the degree of sequence identity with respect to one of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3 and / or SEQ ID NOs: 4 is such that, and if applicable, the number and types of "amino acid differences" that may be present in such binder of the present invention (i.e., compared to one of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3 and / or SEQ ID NOs: 4): (i) the amino acid sequence of the present invention has, if applicable, at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity with respect to SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3 and / or SEQ ID NOs: 4 (in the same sequence, CDR, any C-terminal extensions that may be present, and positions 11, 89, 110 and / or 112 as required in the specific embodiments): If it is said that the sequence has (mutations in which are not considered in determining the degree of sequence identity), and / or (ii) if the amino acid sequence of the present invention is said to have, where applicable, seven or fewer, preferably five or fewer, for example, only three, two or one "amino acid difference" to each of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4 (again, without considering any C-terminal extensions that may be present, and without considering mutations at positions 11, 89, 110 and / or 112 as required for the specific embodiment in question), then it should be noted that the albumin binder of the present invention also includes sequences that do not have amino acid differences to each of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4, other than the mutations at positions 11, 89, 110 and / or 112 as required for the specific embodiment in question, and any C-terminal extensions that may be present.

[0033] Therefore, in one specific embodiment of the present invention, the albumin binder of the present invention may, where applicable, have 100% sequence identity to each of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4 (including CDR, but without considering the mutations or combinations of mutations at positions 11, 89, 110 and / or 112 as disclosed herein, and / or any C-terminal elongations that may exist), and / or, where applicable, have no amino acid differences to each of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4 (amino acid differences other than the mutations or combinations of mutations at positions 11, 89, 110 and / or 112 as disclosed herein, and any C-terminal elongations that may exist).

[0034] If any amino acid differences exist (i.e., any C-terminal elongation and mutations at positions 11, 89, 110 and / or 112 required for specific embodiments of the present invention), these amino acid differences may be present in the CDR and / or framework region, but preferably only in the framework region (as defined by Abm convention, i.e., not in the CDR as defined according to Abm convention). Thus, the albumin binder of the present invention has, where applicable, the same combination of CDRs (as defined according to Abm convention) present in one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0035] The serum albumin binder of the present invention is preferably of formula (X) if the serum albumin binder of the present invention is present at and / or forms the C-terminus of the compound or polypeptide of the present invention (or if the serum albumin binder of the present invention has in some way an "exposed C-terminus" in the protein, polypeptide or other compound or construct in which the serum albumin binder of the present invention is present (this generally means that the C-terminus of the ISV does not associate with or bind to a constant domain (e.g., the CH1 domain); again, see WO 12 / 175741 and PCT / EP 2015 / 06043). n It also has a C-terminal elongation represented by [wherein 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), and each X is a (preferably natural) amino acid residue, independently selected from natural amino acid residues (however, according to one preferred embodiment, none of which are cysteine ​​residues), and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)].

[0036] Such C-terminal elongation X (n) According to some preferred but non-limiting examples, X and n are as follows: (a) n=1 and X=Ala, (b) n=2 and each X=Ala, (c) n=3 and each X=Ala, (d) n=2 and at least one X=Ala (where the remaining amino acid residue X is 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 (where the remaining amino acid residue X is 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 (where the remaining amino acid residue X is independently selected from any native amino acid, 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 (where the remaining amino acid residue X is 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 (where the remaining amino acid residue X is 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 (where the remaining amino acid residue X is independently selected from any native amino acid, 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 (where the remaining amino acid residue X is independently selected from any native amino acid, preferably independently selected from Val, Leu and / or Ile) or (p)n=3 and at least two X=Ala or Gly (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu and / or Ile) It can be, Here, embodiments (a), (b), (c), (g), (h), (i), (m), and (n) are particularly preferred, embodiments where n=1 or n=2 are preferred, and embodiments where n=1 are particularly preferred.

[0037] 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 (unless such cysteine ​​residues are used for further functionality, such as pegylation, or are intended to provide such functionality).

[0038] Some specific and 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.

[0039] If the serum albumin binder of the present invention contains a mutation at position 110 or 112 (in the eventual combination with the mutations at positions 11 and / or 89 as described herein), the C-terminal amino acid residue of Framework 4 (starting at position 109) is as follows: (i) if C-terminal elongation is absent: VTVKS (SEQ ID NO: 101), VTVQS (SEQ ID NO: 102), VKVSS (SEQ ID NO: 103), or VQVSS (SEQ ID NO: 104); or (ii) if C-terminal elongation is present: VTVKSX (n) (Sequence ID: 105), VTVQSX( n )(Sequence number: 106), VKVSSX( n )(Sequence ID: 107) or VQVSSX (n) (SEQ ID NO: 108), for example, VTVKSA (SEQ ID NO: 109), VTVQSA (SEQ ID NO: 110), VKVSSA (SEQ ID NO: 111), or VQVSSA (SEQ ID NO: 112). If the serum albumin binder of the present invention does not contain mutations at positions 110 or 112 (but contains only the mutations at positions 11 and / or 89 as described herein), the C-terminal amino acid residue of framework 4 (starting at position 109) is usually either: (i) if there is no C-terminal elongation: VTVSS (SEQ ID NO: 113) (the same as in the sequence of SEQ ID NO: 3); or (ii) if there is a C-terminal elongation: VTVSSX (n)(Sequence ID: 114), for example, VTVSSA (Sequence ID: 115) (the same as in the sequence of Sequence ID: 4). In these C-terminal sequences, X and n are as defined herein for C-terminal extension.

[0040] 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 a D at position 1 (i.e., an E1D mutation compared to the sequences given in SEQ ID NOs: 1-4 and 16-99).

[0041] Furthermore, generally speaking, if the compound or polypeptide of the present invention has a heavy chain ISVD at its C-terminus (the heavy chain ISVD may be the 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 (and, by extension, the compound or polypeptide of the present invention) preferably has the C-terminal extension X(n) described herein. Similarly, if the compound or polypeptide of the present invention has a heavy chain ISVD at its N-terminus (the heavy chain ISVD may be the 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 (and, by extension, the compound or polypeptide of the present invention) preferably has D at position 1.

[0042] Preferably, if the compound or polypeptide of the present invention contains one or more other ISVDs other than the albumin binder of the present invention (these other ISVDs 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 sequence that reduce binding by existing antibodies. In particular, if these other ISVDs are (single) domain antibodies consisting essentially of and / or derived from nanobodies or VH domains, these other ISVDs may contain (appropriate combinations) of amino acid residues / mutations at positions 11, 89, 110 and / or 112 as described in PCT / EP 2015 / 060643 and / or as described herein for the albumin binder of the present invention.

[0043] As mentioned, the amino acid sequences provided by the present invention are proteins that can bind to human serum albumin, and in particular, specifically (as described herein). Therefore, the amino acid sequences provided by the present invention can be used, for example, as binding units or binding domains for binding to (human) serum albumin to confer an extension of the (as defined herein) half-life of a therapeutic compound, partial, or component. For the use of serum albumin-binding domains to extend the half-life of therapeutic compounds, partials, or components, referentially see WO 2004 / 041865, 2006 / 122787, EP 2139918, WO 2011 / 006915, 2012 / 175400 and / or 2014 / 111550. The albumin binders of the present invention can generally be used in the same ways and for the same purposes as the serum albumin binders described in these references.

[0044] Some preferred and non-limiting examples of the ISVs of the present invention are given in SEQ ID NOs: 16-99, each of which constitutes a further aspect of the present invention (similarly, a protein, polypeptide, or other compound or construct comprising one of these sequences). Among these, Sequence numbers 16-29 are examples of variants of sequence number 1. These sequences have CDR1 (defined according to Kabat), which is sequence number 5; CDR2 (defined according to Kabat), which is sequence number 6; and CDR3 (defined according to Kabat), which is sequence number 7. Sequence IDs 30-43 are examples of variants of sequence ID 1 having a C-terminal alanine (preferably non-limiting examples of C-terminal elongation are as described herein). These sequences have CDR1 (defined according to Kabat), which is sequence ID 5; CDR2 (defined according to Kabat), which is sequence ID 6; and CDR3 (defined according to Kabat), which is sequence ID 7. Sequence numbers 44-57 are examples of variants of sequence number 2. These sequences have CDR1 (defined according to Kabat), which is sequence number 10; CDR2 (defined according to Kabat), which is sequence number 11; and CDR3 (defined according to Kabat), which is sequence number 12. Sequence IDs 58-71 are examples of variants of sequence ID 2 having a C-terminal alanine (preferably non-limiting examples of C-terminal elongation are as described herein). These sequences have CDR1 (defined according to Kabat), which is sequence ID 10; CDR2 (defined according to Kabat), which is sequence ID 11; and CDR3 (defined according to Kabat), which is sequence ID 12. Sequence numbers 72-85 are examples of variants of sequence number 3. These sequences have CDR1 (defined according to Kabat), which is sequence number 10; CDR2 (defined according to Kabat), which is sequence number 11; and CDR3 (defined according to Kabat), which is sequence number 15. Sequence IDs 86-99-54 are examples of variants of sequence ID 4 (which is sequence ID 3 with C-terminal alanine extension). These sequences have CDR1 (defined according to Kabat), which is sequence ID 10; CDR2 (defined according to Kabat), which is sequence ID 11; and CDR3 (defined according to Kabat), which is sequence ID 15.

[0045] Among these variants, sequences of sequence numbers 72-85 (when C-terminal elongation is not required) and sequences of sequence numbers 86-99 (when C-terminal elongation is required) are the most preferred.

[0046] Therefore, in the first aspect, the present invention is -The following sequences are selected from TGEMA (sequence number: 5) and TSSML (sequence number: 10), preferably TSSML (sequence number: 10), and CDR1 (by Kabat) and -The following sequences are selected from SISSSGATTYYADSVKG (sequence number: 6) and VIHQSGTPTYYADSVKG (sequence number: 11), preferably VIHQSGTPTYYADSVKG (sequence number: 11) for CDR2 (by Kabat) and - The following sequences: CDR3 selected from PRHPQGGVTFDY (sequence number: 7) and FPSTHGKFDY (sequence number: 12) (by Kabat) It has, and - At least 85%, preferably at least 90%, and more preferably at least 95% sequence identity to the amino acid sequence of Sequence ID No. 1 (any C-terminal elongation and CDR that may be present at the same amino acid are not considered in determining the degree of sequence identity). and / or - An amino acid difference of seven or fewer, for example, five or fewer, preferably three or fewer, for example, three, two or one, relative to the amino acid sequence of SEQ ID NO: 1 (as defined herein, without regard to any of the above-listed mutations at positions 11, 89, 110 or 112, which may exist, and without regard to any C-terminal elongations which may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR). It has, And, as may be the case (especially if ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention): -C-terminal extension (X) n [In the same extension, 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), and each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)] Having And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, Here, 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. This means that (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 and the 11th position is V and the 110th position is K or Q; or (vi) the 89th position is L and 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; Regarding immunoglobulin single variable domains.

[0047] In a further embodiment, the present invention relates to an immunoglobulin single variable domain, -The following sequences are selected from TGEMA (sequence number: 5) and TSSML (sequence number: 10), preferably TSSML (sequence number: 10), and CDR1 (by Kabat) and -The following sequences are selected from SISSSGATTYYADSVKG (sequence number: 6) and VIHQSGTPTYYADSVKG (sequence number: 11), preferably VIHQSGTPTYYADSVKG (sequence number: 11) for CDR2 (by Kabat) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (by Kabat) It has, and, -At least 85%, preferably at least 90%, more preferably at least about 95% sequence identity to one of the sequences of SEQ ID NOs: 1 to 4 (in the same sequence, any C-terminal extension and CDR that may be present are not considered in determining the degree of sequence identity), and in particular, at least 85%, preferably at least 90%, more preferably at least about 95% sequence identity to the sequence of SEQ ID NO: 3 or 4 (in the same sequence, again, any C-terminal extension and CDR that may be present are not considered in determining the degree of sequence identity) and / or -Five or fewer, preferably three or fewer, for example, only 3, 2 or 1 "amino acid differences" (as defined herein, without considering the mutations listed above at positions 11, 89, 110 or 112 that may be present and without considering any C-terminal extension that may be present) to one of the sequences of SEQ ID NOs: 1 to 4 (where, when such amino acid differences are present, they may be present in the framework and / or CDR, but preferably are present only in the framework and not in the CDR), and in particular, five or fewer, preferably three or fewer, for example, only 3, 2 or 1 such amino acid differences to the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 (where, when such amino acid differences are present, they may be present in the framework and / or CDR, but preferably are present only in the framework and not in the CDR) having and optionally, (especially when ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention): -C-terminal extension (X) n [During the extension, 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), and each X is an independently selected (preferably natural) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I)] having And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, The immunoglobulin single variable domain has the following amino acid residues at the indicated location (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): -89T or 89L or in combination with -11V 89L in combination with -110K or 110Q or -89L in combination with 112K or 112Q or -89L or in combination with 11V and 110K or 110Q -89L or in combination with -11V and 112K or 112Q -11V in combination with 110K or 110Q or 11V in combination with -112K or 112Q This relates to immunoglobulin single variable domains, including those mentioned above.

[0048] In particular, the serum albumin binder of the present invention preferably has five or fewer, preferably three or fewer, "amino acid differences" (as defined herein, without regard to any of the listed mutations at positions 11, 89, 110, or 112, which may be present, and without regard to any C-terminal elongations that may be present) for one of the sequences of SEQ ID NOs: 1 to 4 (wherein the case of such amino acid differences, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) and in particular, five or fewer, preferably three or fewer, such amino acid differences (wherein the case of such amino acid differences, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR).

[0049] Some specific and non-limiting examples of such mutations / amino acid differences that may exist (i.e., compared to the sequences of SEQ ID NOs: 1-4) are E1D, P14A, P41A, P41L, P41S or P41T (and especially P41A), P42E or T87A. Other examples of mutations are one or more appropriate “camelization” substitutions (in appropriate combinations). See, for example, Tables A-3 to A-8 from Davies and Riechmann, Protein Engineering, vol.9, no.6, 531-537, 1996 and Davies and Riechmann, FEBS Letters 399 (1004), 285-290 and WO No. 08 / 020079.

[0050] As mentioned, in the present invention, amino acid sequences in which position 89 is T or position 11 is V and position 89 is L are particularly preferred (in cases, in appropriate combinations with mutations of 110K or 110Q and / or mutations of 112K or 112Q, and in particular in combination with mutations of 110K or 110Q). In cases, amino acid sequences in which position 11 is V and position 89 is L are more preferred together with mutations of 110K or 110Q.

[0051] Therefore, in one preferred embodiment, the present invention is -The following sequences are selected from TGEMA (sequence number: 5) and TSSML (sequence number: 10), preferably TSSML (sequence number: 10), and CDR1 (by Kabat) and -The following sequences are selected from SISSSGATTYYADSVKG (sequence number: 6) and VIHQSGTPTYYADSVKG (sequence number: 11), preferably VIHQSGTPTYYADSVKG (sequence number: 11) for CDR2 (by Kabat) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (by Kabat) It has, and - Sequence identity to the amino acid sequence of Sequence ID No. 1 of at least 85%, preferably at least 90%, and more preferably at least 95% (any C-terminal elongation and CDR that may be present in the same amino acid sequence are not considered in determining the degree of sequence identity). and / or - An amino acid difference of seven or fewer, for example, five or fewer, preferably three or fewer, for example, three, two or one, relative to the amino acid sequence of SEQ ID NO: 1 (as defined herein, without regard to any of the above-listed mutations at positions 11, 89, 110 or 112, which may exist, and without regard to any C-terminal elongations which may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR). It has, And, as may be the case (especially if ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention): -C-terminal extension (X) n [In the same extension, 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), and each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)] It has, And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, Here, 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 (and preferably T), and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q (and preferably S). Regarding immunoglobulin single variable domains.

[0052] In another preferred embodiment, the present invention is -The following sequences are selected from TGEMA (sequence number: 5) and TSSML (sequence number: 10), preferably TSSML (sequence number: 10), and CDR1 (by Kabat) and -The following sequences are selected from SISSSGATTYYADSVKG (sequence number: 6) and VIHQSGTPTYYADSVKG (sequence number: 11), preferably VIHQSGTPTYYADSVKG (sequence number: 11) for CDR2 (by Kabat) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (by Kabat) It has, and - Sequence identity to the amino acid sequence of Sequence ID No. 1 of at least 85%, preferably at least 90%, and more preferably at least 95% (any C-terminal elongation and CDR that may be present in the same amino acid sequence are not considered in determining the degree of sequence identity). and / or - An amino acid difference of seven or fewer, for example, five or fewer, preferably three or fewer, for example, three, two or one, relative to the amino acid sequence of SEQ ID NO: 1 (as defined herein, without regard to any of the above-listed mutations at positions 11, 89, 110 or 112, which may exist, and without regard to any C-terminal elongations which may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR). Having And, as may be the case (especially if ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention): -C-terminal extension (X) n [In the same extension, 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), and each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)] It has, And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, Here, 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. Regarding immunoglobulin single variable domains.

[0053] In specific and non-limiting embodiments, the serum albumin binder of the present invention is: -TSSML (sequence number: 10) is CDR1 (by Kabat) and -VIHQSGTPTYYADSVKG (Sequence ID: 11) is CDR2 (by Kabat) and -The following sequence: Selected from FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), preferably FPSSRMKFDY (sequence number: 15), CDR3 (by Kabat), In addition, -TSSML (sequence number: 10) is CDR1 (by Kabat) and -VIHQSGTPTYYADSVKG (Sequence ID: 11) is CDR2 (by Kabat) and -FPSSRMKFDY (Sequence ID: 15) is CDR3 (by Kabat). It holds.

[0054] In one specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the indicated positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): -11V or when combined with 89L 11V in combination with -110K or 110Q 11V in combination with -112K or 112Q -11V or in combination with 89L and 110K or 110Q - 11V in combination with 89L and 112K or 112Q Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0055] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the mentioned positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): 89L or in combination with -11V 89L in combination with -110K or 110Q or -89L in combination with 112K or 112Q or -89L or in combination with 11V and 110K or 110Q -89L in combination with -11V and 112K or 112Q Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0056] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the mentioned positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): 110K or 110Q in combination with -11V or -110K or 110Q in combination with 89L or -11K or 110Q when combined with 11V and 89L Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0057] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the mentioned positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): 112K or 112Q in combination with -11V or -112K or 112Q in combination with 89L or 112K or 112Q when combined with -11V and 89L. Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0058] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises a T at position 89 and has a CDR, and has a degree of overall sequence identity to the reference sequences described herein.

[0059] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises V at position 11 and L at position 89, and has a CDR, and has a degree of overall sequence identity to the reference sequence described herein.

[0060] In another embodiment, the present invention is -The following sequences are selected from GFTFSTGEMA (sequence number: 8) and GFTFDTSSML (sequence number: 13), preferably GFTFDTSSML (sequence number: 13) CDR1 (by Abm) and -The following sequences are selected from SISSSGATTY (sequence number: 9) and VIHQSGTPTY (sequence number: 14), preferably VIHQSGTPTY (sequence number: 14) CDR2 (by Abm) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (according to Abm) It has, and - Sequence identity to the amino acid sequence of Sequence ID No. 1 of at least 85%, preferably at least 90%, and more preferably at least 95% (any C-terminal elongation and CDR that may be present in the same amino acid sequence are not considered in determining the degree of sequence identity). and / or - An amino acid difference of seven or fewer, for example, five or fewer, preferably three or fewer, for example, three, two or one, relative to the amino acid sequence of SEQ ID NO: 1 (as defined herein, without regard to any of the above-listed mutations at positions 11, 89, 110 or 112, which may exist, and without regard to any C-terminal elongations which may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR). It has, And, depending on the circumstances (especially if ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention), -C-terminal extension (X) n [In the same extension, 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), and each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)] It has, And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, Here, 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. This means that (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 and the 11th position is V and the 110th position is K or Q; or (vi) the 89th position is L and 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; Regarding immunoglobulin single variable domains.

[0061] In another embodiment, the present invention relates to an immunoglobulin single variable domain, -The following sequences are selected from GFTFSTGEMA (sequence number: 8) and GFTFDTSSML (sequence number: 13), preferably GFTFDTSSML (sequence number: 13) CDR1 (by Abm) and -The following sequences are selected from SISSSGATTY (sequence number: 9) and VIHQSGTPTY (sequence number: 14), preferably VIHQSGTPTY (sequence number: 14) CDR2 (by Abm) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (according to Abm) It has, and -Sequence identity to one of the sequences of sequence numbers 1 to 4, of at least 85%, preferably at least 90%, and more preferably at least 95% (any C-terminal extensions and CDRs that may be present in the sequence are not considered in determining the degree of sequence identity); and, in particular, sequence identity to sequence number 3 or 4, of at least 85%, preferably at least 90%, and more preferably at least 95% (again, any C-terminal extensions and CDRs that may be present in the sequence are not considered in determining the degree of sequence identity). and / or -Five or fewer, preferably three or fewer, "amino acid differences" (as defined herein, without regard to any of the listed mutations at positions 11, 89, 110 or 112, which may exist, and without regard to any C-terminal elongations that may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) and in particular, five or fewer, preferably three or fewer, such amino acid differences (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) for any of the sequences of sequence number 3 or sequence number 4 (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) It has, And, as may be the case (especially if ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention): -C-terminal extension (X) n [In the same extension, 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), and each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)] It has, And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, The immunoglobulin single variable domain has the following amino acid residues at the indicated location (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): -89T or 89L or in combination with -11V 89L in combination with -110K or 110Q or -89L in combination with 112K or 112Q or -89L or in combination with 11V and 110K or 110Q -89L or in combination with -11V and 112K or 112Q -11V in combination with 110K or 110Q or 11V in combination with -112K or 112Q This relates to immunoglobulin single variable domains, including those mentioned above.

[0062] In particular, the serum albumin binder of the present invention preferably has five or fewer, preferably three or fewer, "amino acid differences" (as defined herein, without regard to any of the listed mutations at positions 11, 89, 110, or 112, which may be present, and without regard to any C-terminal elongations that may be present) for one of the sequences of SEQ ID NOs: 1 to 4 (wherein the case of such amino acid differences, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR) and in particular, five or fewer, preferably three or fewer, such amino acid differences (wherein the case of such amino acid differences, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR).

[0063] As mentioned, in the present invention, amino acid sequences in which position 89 is T or position 11 is V and position 89 is L are particularly preferred (in part, in appropriate combinations with the 110K or 110Q mutation and / or the 112K or 112Q mutation, and especially in combination with the 110K or 110Q mutation), and in part, amino acid sequences in which position 11 is V and position 89 is L are more preferred, in part, with the 110K or 110Q mutation.

[0064] Therefore, in one preferred embodiment, the present invention is -The following sequences are selected from GFTFSTGEMA (sequence number: 8) and GFTFDTSSML (sequence number: 13), preferably GFTFDTSSML (sequence number: 13) CDR1 (by Abm) and -The following sequences are selected from SISSSGATTY (sequence number: 9) and VIHQSGTPTY (sequence number: 14), preferably VIHQSGTPTY (sequence number: 14) CDR2 (by Abm) and -The CDR3 (according to Abm) has the following sequence: selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), most preferably FPSSRMKFDY (sequence number: 15). and, - Sequence identity to the amino acid sequence of Sequence ID No. 1 of at least 85%, preferably at least 90%, and more preferably at least 95% (any C-terminal elongation and CDR that may be present in the same amino acid sequence are not considered in determining the degree of sequence identity). and / or - An amino acid difference of seven or fewer, for example, five or fewer, preferably three or fewer, for example, three, two or one, relative to the amino acid sequence of SEQ ID NO: 1 (as defined herein, without regard to any of the above-listed mutations at positions 11, 89, 110 or 112, which may exist, and without regard to any C-terminal elongations which may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR). It has, And, as may be the case (especially if ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention): -C-terminal extension (X) n[In the same extension, 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), and each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)] It has, And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, Here, 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 (and preferably T), and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q (and preferably S). Regarding immunoglobulin single variable domains.

[0065] In another preferred embodiment, the present invention is -The following sequences are selected from GFTFSTGEMA (sequence number: 8) and GFTFDTSSML (sequence number: 13), preferably GFTFDTSSML (sequence number: 13) CDR1 (by Abm) and -The following sequences are selected from SISSSGATTY (sequence number: 9) and VIHQSGTPTY (sequence number: 14), preferably VIHQSGTPTY (sequence number: 14) CDR2 (by Abm) and - The following sequence is selected from PRHPQGGVTFDY (sequence number: 7), FPSTHGKFDY (sequence number: 12), and FPSSRMKFDY (sequence number: 15), preferably FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), and most preferably FPSSRMKFDY (sequence number: 15) CDR3 (according to Abm) It has, and - Sequence identity to the amino acid sequence of Sequence ID No. 1 of at least 85%, preferably at least 90%, and more preferably at least 95% (any C-terminal elongation and CDR that may be present in the same amino acid sequence are not considered in determining the degree of sequence identity). and / or Seven or fewer "amino acid differences" to the amino acid sequence of Sequence ID No. 1, for example, five or fewer, preferably three or fewer, for example, three, two, or one (as defined herein, without regard to any of the above-listed mutations at positions 11, 89, 110, or 112, which may exist, and without regard to any C-terminal elongations which may exist) (where, if such amino acid differences exist, they may be present in the framework and / or CDR, but preferably only in the framework and not in the CDR). It has, And, as may be the case (especially if ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention): -C-terminal extension (X) n [In the same extension, 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), and each X is an independently selected (preferably native) amino acid residue, preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)] It has, And, in some cases (especially when ISVD is present at and / or forms part of the N-terminus of the compound or polypeptide of the present invention), having a D and / or E1D mutation at position 1, Here, 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. Regarding immunoglobulin single variable domains.

[0066] In another specific and non-limiting embodiment, the serum albumin binder of the present invention is: -GFTFDTSSML (Sequence ID: 13) is CDR1 (by Abm) and -VIHQSGTPTY (Sequence ID: 14) is CDR2 (according to Abm) and - The following sequence: CDR3 (by Abm) selected from FPSTHGKFDY (sequence number: 12) or FPSSRMKFDY (sequence number: 15), preferably FPSSRMKFDY (sequence number: 15) In addition, in particular -GFTFDTSSML (Sequence ID: 13) is CDR1 (by Abm) and -VIHQSGTPTY (Sequence ID: 14) is CDR2 (according to Abm) and -FPSSRMKFDY (Sequence ID: 15) is CDR3 (according to Abm) It holds.

[0067] In one specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the indicated positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): -11V or when combined with 89L 11V in combination with -110K or 110Q 11V in combination with -112K or 112Q -11V or in combination with 89L and 110K or 110Q - 11V in combination with 89L and 112K or 112Q Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0068] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the mentioned positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): 89L or in combination with -11V 89L in combination with -110K or 110Q or -89L in combination with 112K or 112Q or -89L or in combination with 11V and 110K or 110Q -89L in combination with -11V and 112K or 112Q Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0069] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the mentioned positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): 110K or 110Q in combination with -11V or -110K or 110Q in combination with 89L or -11K or 110Q when combined with 11V and 89L Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0070] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the mentioned positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): 112K or 112Q in combination with -11V or -112K or 112Q in combination with 89L or 112K or 112Q when combined with -11V and 89L. Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0071] In another specific and non-limiting embodiment, the serum albumin binder of the present invention comprises the following amino acid residues at the mentioned positions (numbered according to Kabat) (i.e., mutations compared to the sequences of SEQ ID NOs: 1-4): -89T Includes, Furthermore, it has a CDR and exhibits an overall degree of sequence identity with respect to the reference sequence described herein.

[0072] In another specific and non-limiting embodiment, the present invention relates to an immunoglobulin single variable domain having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 16 to 99.

[0073] In another specific and non-limiting embodiment, the present invention relates to an immunoglobulin single variable domain having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 44 to 99.

[0074] In another specific and non-limiting embodiment, the present invention relates to an immunoglobulin single variable domain having an amino acid sequence which is one of the following sequences: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99.

[0075] In another specific and non-limiting embodiment, the present invention relates to an immunoglobulin single variable domain having the following amino acid sequence: SEQ ID NO: 50, SEQ ID NO: 64, SEQ ID NO: 78, or SEQ ID NO: 92, and preferably one of SEQ ID NO: 78 or SEQ ID NO: 92.

[0076] The present invention also relates to proteins, polypeptides and other constructs, molecules or chemical components comprising (one or more) the serum albumin binders of the present invention as described herein or essentially derived from such binders; methods for expressing / producing the improved heavy immunoglobulin variable domains of the present invention and / or proteins, polypeptides and other constructs, molecules or chemical components comprising them; compositions and products (e.g., pharmaceutical compositions and products) comprising the improved heavy immunoglobulin variable domains of the present invention and / or proteins, polypeptides and other constructs, molecules or chemical components comprising them; nucleotide sequences and nucleic acids encoding proteins or polypeptides encoding the improved heavy immunoglobulin variable domains of the present invention and / or comprising them; and the use (and in particular therapeutic, prophylactic and diagnostic uses) of the improved heavy immunoglobulin variable domains of the present invention and proteins, polypeptides and other constructs, molecules or chemical components comprising them.

[0077] Further aspects, embodiments, advantages, uses, and applications of the present invention will become apparent from further descriptions herein.

[0078] In this specification, The term “immunoglobulin single variable domain” (also known as “ISV” or “ISVD”) is generally used to mean an immunoglobulin variable 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 (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional quadruple-stranded 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), IgNAR domain (single domain) antibodies (e.g., dAb®) that are VH domains or derived from VH domains, and (single domain) antibodies (e.g., dAb®) that are VL domains or derived from VL domains. Unless otherwise specified, ISVDs based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. Most preferably, unless otherwise specified, the ISVD will be a nanobody.

[0079] The term "nanobody" is generally defined as in WO No. 2008 / 020079 or WO No. 2009 / 138519, and therefore, in specific embodiments, it generally means VHH, humanized VHH, or camelized VH (e.g., camelized human VH), or generally sequence-optimized VHH (e.g., optimization of 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 (registered trademark) or Nanobodies (registered trademark).

[0080] Generally, unless otherwise specified, the ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs referred to herein are intended for use in the prevention or treatment of diseases or disorders in humans (and / or, as may, in homeothermic animals and especially mammals). Therefore, generally, the ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs described herein are preferably such that they can be used as (biological) agents or other pharmaceutically or therapeutically active compounds and / or pharmaceuticals or compositions, and / or are appropriately part of such. Such agents, compounds, or products are preferably such that they are suitable for administration to humans, for example, for the prevention or treatment of subjects requiring such prevention or treatment, or, for example, as part of a clinical trial. As further described herein, for this purpose, such agents or compounds may contain other parts, components or binding units other than the ISVD provided by the present invention (these are as described herein and may be, for example, one or more other further therapeutic parts and / or one or more other parts that affect the pharmacokinetic or pharmacodynamic properties of the ISVD-based biological agent or nanobody-based biological agent, e.g., its half-life). Suitable examples of such further therapeutic agents or other parts will be apparent to those skilled in the art and may generally include any therapeutically active protein, polypeptide or other binding domain or binding unit and, for example, modifications, e.g., those described on pages 149-152 of WO 2009 / 138159. ISVD-based biological agents or nanobody-based biological agents are preferably therapeutic agents or are intended for use as therapeutic agents (including prophylactic and diagnostic agents), and for this purpose preferably contain at least one ISVD targeting a therapeutic-related target (e.g., RANK-L, vWF, IgE, RSV, CXCR4, IL-23, or other interleukins).For specific, non-limiting examples of some such ISVD-based biological agents or nanobody-based biological agents, see Examples 8 to 18, and also see, for example, various applications by Ablynx NV (e.g., not limited to WO 2004 / 062551, 2006 / 122825, 2008 / 020079 and 2009 / 068627) and applications such as (and not limited to) WO 2006 / 038027, 2006 / 059108, 2007 / 063308, 2007 / 063311, 2007 / 066016 and 2007 / 085814. Furthermore, as further described herein, additional parts may be ISVDs or nanobodies described herein that are directed toward (human) serum proteins, such as (human) serum albumin, and such ISVDs or nanobodies may find therapeutic uses, in particular, for the TNF binder described herein and / or for extending the half-life of said binder. See, for example, WO 2004 / 041865, 2006 / 122787 and 2012 / 175400. These documents generally describe the use of serum albumin-binding nanobodies for extending the half-life. Furthermore, unless otherwise specified herein, all terms referred to herein have the meanings provided in WO 2009 / 138519 (or the prior art cited in WO 2009 / 138519) or WO 2008 / 020079 (or the prior art cited in WO 2008 / 020079). Also, if a method or technique is not specifically described herein, it may be carried out as described in WO 2009 / 138519 (or the prior art cited in WO 2009 / 138519) or WO 2008 / 020079 (or the prior art cited in WO 2008 / 020079).Furthermore, as described herein, any pharmaceutical or composition comprising any ISVD or compound of the present invention may also include one or more further components known on their own for use in the pharmaceutical or composition (i.e., depending on the intended pharmaceutical form) and / or one or more other compounds or active principles intended for therapeutic use (i.e., for providing a combination product).

[0081] Furthermore, when used herein or in the claims, the following terms: “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,” “against,” “targeting to (antigen),” “specificity,” and “half-life” have the same meanings as provided on pages 62–75 of WO No. 2009 / 138519, and / or their applicability can be determined in the form described on those pages. In addition, the terms “modulating,” “for modulating,” “interaction site,” “specific to,” “crossblocking,” “crossblocked,” and “crossblocking,” as well as “essentially independent of pH,” are defined (and / or may be determined as described on the same pages) on pages 74-79 of Ablynx NV’s WO 2010 / 130832. Furthermore, when referring to constructs, compounds, proteins, or polypeptides of the present invention, terms such as “monovalent,” “divalent” (or “polyvalent”), “bispecificity” (or “multispecificity”) and “biantigen-binding” (or “multiantigen-binding”) may have the meanings provided in WO 2009 / 138519, 2010 / 130832, or 2008 / 020079.

[0082] With respect to ISVDs, nanobodies, ISVD-based biological preparations, nanobody-based biological preparations, or any other amino acid sequences, compounds, or polypeptides as referred herein, the term “half-life” as used herein may generally be defined as set forth in paragraph o) on page 57 of WO 08 / 020079 and as referred herein, for example, meaning the time it takes for the serum concentration of an amino acid sequence, compound, or polypeptide to decrease by 50% in vivo due to degradation and / or clearance or sequestration by the intrinsic mechanism of the sequence or compound. The in vivo half-life of the amino acid sequences, compounds, or polypeptides of the present invention can be determined in any form known by itself, for example, by pharmacokinetic analysis. Appropriate techniques will be apparent to those skilled in the art and may generally be set forth in paragraph o) on page 57 of WO 08 / 020079. Furthermore, as mentioned in paragraph o) on page 57 of WO No. 08 / 020079, the half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta, and area under the curve (AUC). In this regard, as used herein, the term “half-life” means, in particular, t1 / 2-beta or terminal half-life (where t1 / 2-alpha and / or AUC or both may not be considered). For example, the following experimental sections and standard reference books, e.g., Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook f or Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996), are referred to. Also, “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982) is referred to.Similarly, the terms “extend half-life” or “extended half-life” are defined in paragraph o) on page 57 of WO No. 08 / 020079, and in particular mean the extension of t1 / 2-beta, regardless of whether it is an extension of t1 / 2-alpha and / or AUC or both.

[0083] If a term is not specifically defined herein, it shall have its ordinary meaning in the art as would be obvious to those skilled in the art. For example, standard reference books such as Sambrook et al, "Molecular Cloning: A Lab or at or y Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harb or Lab or at or y Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New Y or k (1987); Lewin, "Genes II", John Wiley & Sons, New Y or k, 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 Ed.), Mosby / Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology, 10th Ed. See Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New Y or K (2005), as well as the general background art cited herein.

[0084] Furthermore, as already shown herein, the amino acid residues of the nanobodies are numbered according to the general numbering for VHH provided by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). Similarly, the numbering applied to VHH domains from camelids in the literature Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195, which is also referred to herein. According to this numbering, FR1 of the nanobodies contains amino acid residues at positions 1-30. CDR1 of the nanobodies contains amino acid residues at positions 31-35. FR2 of the nanobodies contains amino acids at positions 36-49. CDR2 of the nanobodies contains amino acid residues at positions 50-65. FR3 of the nanobodies contains amino acid residues at positions 66-94. The CDR3 of the nanobody contains amino acid residues at positions 95-102. The FR4 of the nanobody 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 the VH domain and VHH domain, the total number of amino acid residues in 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 according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number recognized by Kabat numbering). This generally means that the numbering according to 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 number of amino acid residues in the CDR, position 1 according to Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to Kabat numbering corresponds to the start of FR4 and vice versa.

[0085] Furthermore, alternative methods for numbering the amino acid residues of the VH domain can be applied in a manner similar to that of the VHH domains and nanobodies from camelids, such as the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition." However, in this description, embodiments, and drawings, unless otherwise specified, numbering will be followed according to Kabat, as is applied to the VHH domain by Riechmann and Muyldermans.

[0086] Furthermore, it should be noted that the drawings, any sequence listings, and experimental sections / examples are provided solely to illustrate the present invention and should not be understood or construed as limiting the scope of the present invention and / or the appended claims in any way, unless otherwise specified.

[0087] As further described herein, the serum albumin binder of the present invention can be used with the advantages of being a part, binding unit or fusion partner to extend the half-life of a therapeutic part, such as a polypeptide, protein, compound (including, but not limited to, small molecules) or other therapeutic component.

[0088] Therefore, in another embodiment, the present invention provides polypeptides, proteins, constructs, compounds, or other chemical components comprising, or essentially consisting of, 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 components.

[0089] In particular, the present invention provides polypeptides, proteins, constructs, compounds, or other chemical components comprising the serum albumin binder of the present invention, which are appropriately bound to each other directly or via one or more suitable linkers or spacers, 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 or different targets, and if they are directed to the same target, may be directed to the same or different epitopes, moieties, domains, or subunits of the target). Such polypeptides, proteins, or constructs may, but are not limited to, fusion proteins as further described herein.

[0090] Furthermore, the present invention relates to the therapeutic use of such polypeptides, proteins, constructs, or compounds, and to pharmaceutical compositions comprising such polypeptides, proteins, constructs, or compounds.

[0091] In one embodiment, at least one therapeutic moiety comprises or essentially consists of a therapeutic protein, polypeptide, compound, factor, or other component. In a preferred embodiment, the therapeutic moiety is directional to a desired antigen or target, capable of binding to (and in particular, specifically to) a desired antigen, and / or capable of interacting with a desired target. In another embodiment, at least one therapeutic moiety comprises or essentially consists of a therapeutic protein or polypeptide. In further embodiments, at least one therapeutic moiety comprises or is essentially composed of a binding domain or binding unit, e.g., immunoglobulin or immunoglobulin sequence (including, but not limited to, immunoglobulin fragments), e.g., antibody or antibody fragment (including, but not limited to, ScFv fragments), or another suitable protein scaffold, e.g., protein A domain (e.g., Affibodies®), tendamistat, fibronectin, lipocalin, CTLA-4, T cell receptor, designed ankyrin repeat, 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, DNA or RNA aptamers) (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

[0092] In yet another embodiment, at least one therapeutic portion includes or is essentially derived from antibody-variable domains, such as heavy-chain-variable domains or light-chain-variable domains.

[0093] In a preferred embodiment, at least one therapeutic portion comprises or is essentially composed of at least one immunoglobulin single variable domain, e.g., a domain antibody, a single domain antibody, a "dAb" or nanobody (e.g., VHH, humanized VHH or camelid VH) or an IgNAR domain.

[0094] In specific embodiments, at least one therapeutic portion comprises or is essentially composed of at least one monovalent nanobody or a divalent, polyvalent, bispecific, or multispecific nanobody construct.

[0095] A polypeptide, (fusion) protein, construct, or compound comprising the serum albumin binder and one or more therapeutic moieties of the present invention may generally be used as described in the prior art cited above (e.g., WO No. 04 / 041865 and WO No. 06 / 122787), provided that the serum albumin binder of the present invention is used instead of the half-life extension moieties described in the prior art.

[0096] 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 one or more therapeutic moieties themselves.

[0097] Generally, the constructs or fusion proteins described herein preferably have a half-life at least 1.5 times, preferably at least 2 times, for example, at least 5 times, for example, at least 10 times or more than 20 times, than the half-life of the corresponding therapeutic portion itself (as measured in humans or suitable animals, such as mice or cynomolgus monkeys).

[0098] Preferably, any such fusion protein or construct has a human half-life that is greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, for example greater than 12 hours, compared to the half-life of the corresponding therapeutic portion itself.

[0099] Preferably, any fusion protein or construct has a half-life in humans (defined as 1 / 2 beta) of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, for example more than 12 hours and for example about 1 day, 2 days, 1 week, 2 weeks and at most the half-life of serum albumin in humans (estimated to be about 19 days). However, the latter may be less important.

[0100] Half-life can generally be defined as the time it takes for the serum concentration of a polypeptide to decrease by 50% in vivo, for example, through the degradation and / or clearance or sequestration of the ligand by its original mechanism. In particular, half-life can be as defined in WO 2009 / 068627.

[0101] Methods for pharmacokinetic analysis and half-life determination are common to those skilled in the art. Details can be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). Also, refer to "Pharmacokinetics," M Gibaldi & D Perron, published by Marcel Dekker, 2nd revised edition (1982).

[0102] As mentioned, in one embodiment, the serum albumin binder of the present invention can be used to extend the half-life of (one or more) immunoglobulin single variable 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).

[0103] Therefore, one embodiment of the present invention relates to a polypeptide, construct, or fusion protein comprising one or more (e.g., one or two) immunoglobulin single variable domain sequences and a serum albumin binder of the present invention, which are appropriately bound to each other directly or optionally via one or more suitable linkers or spacers. As referred to herein, each such immunoglobulin single variable domain present in such polypeptide, construct, or fusion protein can 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), and according to one specific and non-limiting embodiment, at least one (and at most all) of these immunoglobulin single variable domains contain two or three disulfide crosslinks.

[0104] As mentioned, if the polypeptide or construct of the fusion protein has a heavy chain ISVD (the ISVD can be an ISVD for the serum albumin binder or therapeutic target of the present invention, e.g., a nanobody for the therapeutic target) at its C-terminus, then the polypeptide or construct of the fusion protein (the ISVD present at its C-terminus) preferably has a C-terminal extension at its C-terminus. Again, the C-terminal extension is given by formula (X) n [wherein 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), and each X is an (preferably natural) amino acid residue, independently selected from natural amino acid residues (however, according to one preferred embodiment, not including any cysteine ​​residue), and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)].

[0105] As mentioned, if the polypeptide or construct of the fusion protein has a heavy chain ISVD (the ISVD can be an ISVD for the serum albumin binder or therapeutic target of the present invention, e.g., a nanobody for the therapeutic target) at its N-terminus, the polypeptide or construct of the fusion protein (the ISVD present at the C-terminus) preferably has a D or E1D mutation at position 1.

[0106] Therefore, in another embodiment, the present invention relates to a protein, polypeptide, or other compound, - comprising or essentially consisting of at least one (and preferably only one) serum albumin binder of the present invention and at least one (e.g., one, two, or three) therapeutic moieties or components (in which the serum albumin binder and one or more therapeutic moieties or components are appropriately bound together, optionally via one or more suitable linkers), -It has a heavy chain ISVD at its C-terminus, and at the C-terminus, the ISVD at the C-terminus is C-terminal extension (X) (as further described herein) n It has, -The same protein, polypeptide, or other compound may also have a heavy chain ISVD at its N-terminus, in which case the N-terminal ISVD has D or E1D at position 1. This relates to proteins, polypeptides, or other compounds.

[0107] In addition, in a preferred embodiment, if one or more other ISVDs are present in addition to the serum albumin binder of the present invention (i.e., if one or more therapeutic portions present are ISVDs), then the (one or all) “therapeutic” ISVDs preferably also have amino acid residues / mutations (combinations) that reduce binding by the existing antibody. If the ISVDs are heavy chain ISVDs, these mutations may be one or more mutations (appropriate combinations) at positions 11, 89, 110, and 112, as described in PCT / EP 2015 / 060643, and in particular may be essentially the same type of mutations (or combinations of mutations) as described herein for the serum albumin binder of the present invention. Preferably, if such other ISVDs are present at the C-terminus, then 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 described herein).

[0108] In one specific embodiment, all therapeutic moieties present in a construct, fusion protein, or polypeptide are ISVDs (i.e., ISVDs for therapeutic targets) and, in particular, heavy chain ISVDs, and especially nanobodies (i.e., nanobodies for therapeutic targets).

[0109] For example, non-limitingly, constructs, fusion proteins, or polypeptides comprising the serum albumin binder of the present invention include: - One copy of the serum albumin binder of the present invention and one therapeutic target with ISVD (and preferably nanobody) or - One copy of the serum albumin binder of the present invention and ISVDs (and preferably two nanobodies) directed to two therapeutic targets (the ISVDs may be the same or different, and if different, may have directionality toward the same target, different epitopes on the same target or different therapeutic targets) or - One copy of the serum albumin binder of the present invention and ISVDs (and preferably three nanobodies) directed to three therapeutic targets (the ISVDs may be the same or different, and if different, they may have directionality toward the same target, different epitopes on the same target, or different therapeutic targets). It can include...

[0110] Some non-limiting examples of constructs, fusion proteins, or polypeptides of the present invention can be schematically represented as follows: where "[Alb]" represents the serum albumin binder of the present invention, "[Therapeutic portion 1]" and "[Therapeutic portion 2]" represent therapeutic portions (which, as mentioned, can each be independently an immunoglobulin single variable domain), "[-]" represents a suitable linker (which is optional, with suitable examples being 9GS and 35GS linkers), the N-terminus being on the left, and the C-terminus being on the right. [Alb]-[Treatment part 1] [Treatment part 1]-[Alb]-X (n) [Alb]-[Treatment part 1]-[Treatment part 1] [Treatment part 1]-[Treatment part 1]-[Alb]-X (n) [Treatment part 1]-[Alb]-[Treatment part 1] [Alb]-[Treatment part 1]-[Treatment part 2] [Treatment part 1]-[Treatment part 2]-[Alb]-X (n) [Treatment part 1]-[Alb]-[Treatment part 2]

[0111] When the therapeutic portion is ISVDs (and preferably nanobodies) against a therapeutic target, a preferably non-limiting construct, fusion protein, or polypeptide of the present invention can be schematically represented as follows: where "[Alb]" represents the serum albumin binder of the present invention, "[Therapeutic ISVD1]" and "[Therapeutic ISVD2]" represent ISVDs against a therapeutic target (the ISVDs may be the same or different, and if different, may have directivity to the same target, different epitopes on the same target, or different therapeutic targets), "[-]" represents a suitable linker (the linker is optional), and X(n) represents the C-terminal extension described herein, with the N-terminus on the left and the C-terminus on the right. [Alb]-[Treatment ISVD1]-X (n) [Treatment ISVD1]-[Alb]-X (n) [Alb]-[Treatment ISVD1]-[Treatment ISVD1]-X (n) [Treatment ISVD1]-[Treatment ISVD1]-[Alb]-X (n) [Treatment ISVD1]-[Alb]-[Treatment ISVD1]-X (n) [Alb]-[Treatment ISVD1]-[Treatment ISVD2]-X (n) [Treatment ISVD1]-[Treatment ISVD2]-[Alb]-X (n) [Treatment ISVD1]-[Alb]-[Treatment ISVD2]-X (n)

[0112] Therefore, in another embodiment, the present invention relates to a multispecific (and in particular, bispecific) nanobody construct comprising the serum albumin binder of the present invention and at least one other nanobody (e.g., one or two other nanobodies, which may be the same or different), wherein the at least one other nanobody is preferably directional to a desired target (which is preferably a therapeutic target) and / or another nanobody useful or suitable for therapeutic, preventive and / or diagnostic purposes. Again, the serum albumin binder and other nanobodies of the present invention may be appropriately bound to each other directly or via one or more suitable linkers or spacers.

[0113] For a general description of polyvalent and multispecific polypeptides containing one or more nanobodies and their preparation, 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, WO Nos. 96 / 34103, 99 / 23221, 04 / 041862, 2006 / 122786, 2008 / 020079, 2008 / 142164 or 2009 / 068627.

[0114] Some specific examples of some of the multispecific and / or polyvalent polypeptides of the present invention can be found in the applications of Ablynx NV mentioned herein. In particular, for 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, preparation of the foregoing, and use thereof, refer to the aforementioned international applications WO 04 / 041865 and 06 / 122787 (the serum albumin binder of the present invention described herein can generally be used in the same way as the half-life-extending nanobody described therein, e.g., Alb-8). And refer to, for example, the general descriptions and specific examples of such constructs given in WO 04 / 041862, 2006 / 122786, 2008 / 020079, 2008 / 142164 or 2009 / 068627.

[0115] In one embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (and 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 the serum albumin binder and the one or more further heavy chain ISVDs all 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. This means that (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 and the 11th position is V and the 110th position is K or Q; or (vi) the 89th position is L and 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.

[0116] In another aspect, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all consist of the following amino acid residues: -89T or 89L or in combination with -11V 89L in combination with -110K or 110Q or -89L in combination with 112K or 112Q or -89L or in combination with 11V and 110K or 110Q -89L or in combination with -11V and 112K or 112Q -11V in combination with 110K or 110Q or 11V in combination with -112K or 112Q It contains.

[0117] In another aspect, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all 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 (and preferably T), and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q (and preferably S).

[0118] In another aspect, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all 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.

[0119] In another aspect, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all consist of the following amino acid residues: -11V or when combined with 89L 11V in combination with -110K or 110Q 11V in combination with -112K or 112Q -11V or in combination with 89L and 110K or 110Q - 11V in combination with 89L and 112K or 112Q It contains.

[0120] In another aspect, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all consist of the following amino acid residues: 89L or in combination with -11V 89L in combination with -110K or 110Q or -89L in combination with 112K or 112Q or -89L or in combination with 11V and 110K or 110Q -89L in combination with -11V and 112K or 112Q It contains.

[0121] In another aspect, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all consist of the following amino acid residues: 110K or 110Q in combination with -11V or -110K or 110Q in combination with 89L or -11K or 110Q when combined with 11V and 89L It contains.

[0122] In another aspect, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all consist of the following amino acid residues: 112K or 112Q in combination with -11V or -112K or 112Q in combination with 89L or 112K or 112Q when combined with -11V and 89L. It contains.

[0123] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (and preferably a fusion protein) comprising the serum albumin binder of the present invention and one or more further heavy chain ISVDs, wherein the serum albumin binder and the one or more further heavy chain ISVDs all contain T at position 89.

[0124] In another embodiment, the present invention relates to a protein, polypeptide, or other compound or construct (and 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 serum albumin binder and the one or more further heavy chain ISVDs contain V at position 11 and L at position 89.

[0125] Again, all these polypeptides preferably contain the C-terminal extension X(n) and D at position 1 (as described herein), and may contain serum albumin-binding ISVD as further described herein. They also have the half-lives further described herein.

[0126] Furthermore, the present invention relates to a nucleotide sequence or nucleic acid encoding an albumin binder, compound, or polypeptide of the present invention. The present invention also includes a gene construct comprising the aforementioned nucleotide sequence or nucleic acid and one or more elements for gene constructs known in themselves. The gene construct may be in the form of a plasmid or vector. Again, such constructs may generally be as described in the published patent applications of Ablynx NV, e.g., WO 04 / 041862, 2006 / 122786, 2008 / 020079, 2008 / 142164, or 2009 / 068627, etc.

[0127] Furthermore, the present invention relates to a host or host cell that contains such nucleotide sequences or nucleic acids and / or expresses (or is capable of expressing) the albumin binder, compound, or polypeptide of the present invention. Again, such host cells can generally be as described in the published patent applications of Ablynx NV, e.g., WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627, etc.

[0128] Furthermore, the present invention relates to a method for producing an albumin binder, compound, or polypeptide of the present invention, comprising culturing or maintaining the host cells described herein under conditions such that the host cells produce or express the albumin binder, compound, or polypeptide of the present invention, and optionally further comprising isolating the albumin binder, compound, or polypeptide of the present invention thus produced. Again, such a method can generally be carried out as described in Ablynx NV's published patent applications, e.g., WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627, etc.

[0129] 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 may generally be as described in Ablynx NV's published patent applications, e.g., WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627, etc.

[0130] However, since the compounds or polypeptides of the present invention have an extended half-life, they are preferably administered circulatingly. They can be administered in any suitable manner that allows the compounds or polypeptides of the present invention to enter circulation, for example, by intravenous injection or infusion, or by any other suitable manner (including oral administration, subcutaneous administration, intramuscular administration, cutaneous administration, intranasal administration, pulmonary administration, etc.). Suitable methods and routes of administration will again be apparent to those skilled in the art from the teachings of the published patent applications of Ablynx NV, for example, WO 04 / 041862, 2006 / 122786, 2008 / 020079, 2008 / 142164, or 2009 / 068627.

[0131] Therefore, 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 compounds or polypeptides of the present invention, the method comprising administering to a subject in need thereof a pharmaceutically active amount of the compounds or polypeptides of the present invention and / or a pharmaceutical composition containing them. The diseases and disorders that can be prevented or treated by the use of the compounds or polypeptides of the present invention as described herein will generally be the same as the diseases and disorders that can be prevented or treated by the use of one or more therapeutic portions present in the compounds or polypeptides of the present invention.

[0132] In the context of the present invention, the term “prevention and / or treatment” includes not only preventing and / or treating a disease, but generally also including preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or delaying the onset of one or more symptoms associated with a disease, reducing and / or mitigating one or more symptoms associated with a disease, reducing the severity and / or duration of any symptoms associated with a disease, and / or preventing further increases in the severity of any symptoms associated with a disease, preventing, reducing or reversing any physiological impairment caused by a disease, and generally also including any pharmacological effects beneficial to the patient being treated.

[0133] The animals being treated may be any warm-blooded animals, particularly mammals and especially humans. As will be apparent to those skilled in the art, the subjects being treated will be, in particular, humans suffering from or at risk of suffering from any of the diseases and disorders referred to herein.

[0134] In another embodiment, the present invention relates to a method for immunotherapy and, in particular, passive immunotherapy, comprising administering a pharmaceutically active amount of the compounds or polypeptides of the present invention and / or a pharmaceutical composition containing them to a subject suffering from or at risk of any of the diseases and disorders described herein.

[0135] The compounds or polypeptides of the present invention and / or compositions comprising 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 the disease or disorder to be prevented or treated, the severity of the disease and / or the severity of its symptoms, the specific polypeptide of the present invention used, the specific route of administration and the pharmaceutical formulation or composition used, the patient's age, sex, weight, diet, overall condition, and other factors well known to clinicians.

[0136] Generally, the treatment plan would involve administering one or more compounds or polypeptides of the present invention, or compositions containing one or more of them, in one or more pharmaceutically effective amounts or doses. The specific amount or dose administered may again be determined by the clinician based on the factors mentioned above.

[0137] Generally, for the prevention and / or treatment of the diseases and disorders referred to herein, and depending on the specific disease or disorder being treated, the activity and / or half-life of the compound or polypeptide of the present invention used, the specific route of administration used, and the specific pharmaceutical formulation or composition, the compound or polypeptide of the present invention will generally be administered in a continuous manner (e.g., by infusion) at a dose of 1 gram to 0.01 micrograms per kg of body weight per day, preferably 0.1 grams to 0.1 micrograms per kg of body weight per day, for example, about 1, 10, 100, or 1000 micrograms per kg of body weight per day, either as a single dose or in multiple doses per day. A clinician may generally determine an appropriate daily dose based on the factors referred to herein. It will also be apparent that in specific cases, a clinician may deviate from these amounts based, for example, the factors cited above and the clinician's professional judgment. Generally, some guidance on the dosage can be derived from the dosages typically administered for comparable conventional antibodies or antibody fragments against the same target, delivered via essentially the same route, but taking into account affinity / binding activity, efficacy, biodistribution, half-life, and other similar factors well known to those skilled in the art.

[0138] Furthermore, if the compounds of the present invention contain the half-life-extending serum albumin binder of the present invention, they do not necessarily need to be administered continuously (e.g., by infusion), but 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 in some cases, once every four weeks, or even less frequently, for example, by injection or infusion.

[0139] One aspect of the present invention relates to a pharmaceutical composition comprising at least one compound or polypeptide of the present invention, wherein the composition is intended to be administered once a week to once every four weeks and, in particular, once every seven days to once every 21 days, for example, at intervals of seven or fourteen days.

[0140] Typically, one polypeptide of the present invention would be used in the above method. However, using two or more polypeptides of the present invention in combination is within the scope of the present invention.

[0141] Furthermore, the polypeptide of the present invention can also be used in combination with one or more further pharmaceutically active compounds or principles, i.e., as a combined treatment plan, which may or may not produce a synergistic effect. Again, clinicians may select such further compounds or principles and appropriate combined treatment plans based on the factors cited above and the clinician's professional judgment.

[0142] In particular, the polypeptides of the present invention can be used or used in combination with other pharmaceutically active compounds or principles for the prevention and / or treatment of diseases and disorders that can be prevented or treated by the fusion proteins or constructs of the present invention. Synergistic effects may or may be obtained as a result of such combinations.

[0143] The effectiveness of the treatment plan used in accordance with the present invention can be determined and / or tracked in any manner known in itself for the relevant disease or disorder, as will be evident to the clinician. Furthermore, the clinician may, where applicable and / or on a case-by-case basis, modify or alter a particular treatment plan to achieve the desired therapeutic effect, avoid, limit or reduce undesirable side effects, and / or achieve 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.

[0144] 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.

[0145] The subjects to be treated may be warm-blooded animals, particularly mammals, and especially humans. As will be apparent to those skilled in the art, the subjects to be treated will particularly be humans suffering from or at risk of any of the diseases and disorders referred to herein.

[0146] Other aspects, embodiments, advantages, and applications of the present invention will become apparent from further descriptions herein.

[0147] From here, the present invention will be further described by the following non-limiting preferred embodiments, examples, and drawings. [Brief explanation of the drawing]

[0148] [Figure 1] Figure 1 is a table listing some amino acid positions that will be specifically referred to herein and their numbering according to some alternative numbering systems (e.g., Aho and IMGT). [Figure 2] Figure 2 shows the alignment of the reference sequences referred to herein. [Figure 3-1] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-2] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-3] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-4] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-5] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-6] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-7] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-8] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-9] Figure 3 lists the amino acid sequences mentioned herein. [Figure 3-10] Figure 3 lists the amino acid sequences mentioned herein. [Figure 4] Figure 4 shows two corresponding plots of data points obtained in Example 1 when serum samples from 96 healthy human subjects were tested for binding to Reference A and two representative variants of Reference A according to the present invention (i.e., [Reference A + L11V + V89L + C-terminal alanine] and [Reference A + L11V + V89L + T110K + C-terminal alanine], respectively). Each dot represents the binding level for one of the 96 samples tested. The data points shown in the right and left panels are the same, and in the right panel, the data points measured for each individual sample for each of the three compounds tested (i.e., Reference A; Reference A + L11V + V89L + 114A; and Reference A + L11V + V89L + T110K + 114A) are connected by lines (consequently, the slope of the lines provides an indicator of the degree to which binding by the existing antibody is reduced when the variants of the present invention and C-terminal alanine are introduced). [Figure 5-1] Figure 5 is a table listing the binding data for the data points corresponding to Figure 4 (three columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 5-2] Figure 5 is a table listing the binding data for the data points corresponding to Figure 4 (three columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 5-3] Figure 5 is a table listing the binding data for the data points corresponding to Figure 4 (three columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 6]Figure 6 shows two corresponding plots of data points obtained in Example 2 when serum samples from 96 healthy human subjects were tested for binding to Reference B and two representative variants of Reference B according to the present invention (i.e., [Reference B + L11V + V89L + C-terminal alanine] and [Reference B + L11V + V89L + T110K + C-terminal alanine], respectively). Each dot represents the binding level for one of the 96 samples tested. The data points shown in the right and left panels are the same, and in the right panel, the data points measured for each individual sample for each of the three compounds tested (i.e., Reference B; Reference B + L11V + V89L + 114A; and Reference B + L11V + V89L + T110K + 114A) are connected by lines (consequently, the slope of the lines provides an indicator of the degree to which binding by the existing antibody is reduced when the variants of the present invention and C-terminal alanine are introduced). [Figure 7-1] Figure 7 is a table listing the binding data for the data points corresponding to Figure 6 (three columns giving the normalized PreAb binding level (700 RU) and two columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 7-2] Figure 7 is a table listing the binding data for the data points corresponding to Figure 6 (three columns giving the normalized PreAb binding level (700 RU) and two columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 7-3] Figure 7 is a table listing the binding data for the data points corresponding to Figure 6 (three columns giving the normalized PreAb binding level (700 RU) and two columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 8]Figure 8 shows two corresponding plots of data points obtained in Example 3 when 96 serum samples (66 from healthy human subjects and 30 from subjects presumed to contain existing antibodies capable of binding in the presence of C-terminal alanine (including 13 samples from SLE patients)) were tested for binding to Reference C, Reference D, and two representative variants of Reference D according to the present invention (i.e., [Reference D+L11V+V89L] and [Reference D+L11V+V89L+T110K], respectively). Each dot represents the binding level for one of the 96 samples tested. The data points shown in the right and left panels are the same, and in the right panel, the data points measured for each individual sample of each of the four compounds tested (i.e., reference C; reference D; reference D+L11V+V89L; and reference D+L11V+V89L+T110K) are connected by lines (consequently, the slope of the lines provides an indicator of the degree to which binding by existing antibodies is reduced when the mutations of the present invention and C-terminal alanine are introduced). [Figure 9] Figure 9 shows a plot of data points obtained for the four SLE samples tested in Example 3. The data points measured for each individual sample (i.e., "SLE25", "SLE37", "SLE39", and "SLE41", respectively) are connected by lines (the slope of each line provides an indicator of the degree to which binding by the existing antibody decreases in each sample when the mutation of the present invention is introduced). [Figure 10-1] Figure 10 is a table listing the binding data for the data points corresponding to Figure 8 (four columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 10-2] Figure 10 is a table listing the binding data for the data points corresponding to Figure 8 (four columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 10-3]Figure 10 is a table listing the binding data for the data points corresponding to Figure 8 (four columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 10-4] Figure 10 is a table listing the binding data for the data points corresponding to Figure 8 (four columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used). [Figure 10-5] Figure 10 is a table listing the binding data for the data points corresponding to Figure 8 (four columns giving the normalized PreAb binding level (700 RU) and three columns giving the percentage decrease in PreAb binding compared to the reference compound used).

[0149] Experimental section The human samples used in the following experimental sections were obtained from either commercial suppliers or human volunteers (all obtained after obtaining the necessary consent and authorization) and were used in accordance with applicable laws and regulations, including but not limited to those relating to medical secrets and patient privacy.

[0150] In the following examples, unless otherwise specified, the binding affinity of existing antibodies present in the samples used (i.e., from healthy volunteers, rheumatoid arthritis (RA) patients, and SLE patients) to the tested nanobodies was determined using ProteOn as follows.

[0151] The nanobodies were captured using either serum albumin or a FLAG3 tag employing monoclonal anti-FLAG M2.

[0152] In the case of binding of existing antibodies to nanobodies captured by human serum albumin (HSA), it was evaluated using a ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate buffered saline, pH 7.4, 0.005% Tween 20) was used as the running buffer, and the experiment was conducted at 25°C. The ligand lane of the ProteOn GLC sensor chip was activated with EDC / NHS (flow rate 30 μl / min), and HSA was injected (flow rate 100 μl / min) at 10 μg / ml in ProteOn acetate buffer pH 4.5 to a fixed level of approximately 3200 RU. After immobilization, the surface was inactivated with ethanolamine HCl (flow rate 30 μl / min). The nanobody was injected onto the HSA surface at 45 μl / min for 2 minutes to a nanobody capture level of approximately 200 RU. Samples containing existing antibodies 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 subsequent 400-second dissociation step. After each cycle (i.e., before the capture of a new nanobody and the injection of the blood sample), the HSA surface was regenerated by injection of 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, Inc.). Sensorgrams showing the binding of existing antibodies were obtained after double referencing by subtracting 1) nanobody-HSA dissociation and 2) non-specific binding to the reference ligand lane. The binding level of existing antibodies was determined by setting the reporting point at 125 seconds (5 seconds after the end of association). The % decrease in binding of existing antibodies was calculated relative to the binding level of the reference nanobody at 125 seconds.

[0153] For the binding of existing monoclonal anti-FLAG M2 (Sigma) antibodies to captured FLAG-tagged nanobodies, we evaluated this using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) was used as the running buffer, and the experiment was conducted at 25°C. The ligand lane of the ProteOn GLC sensor chip was activated by EDC / NHS (flow rate 30 μl / min), and anti-FLAG M2 mAb was injected at 10 μg / ml in ProteOn acetate buffer pH 4.5 (flow rate 100 μl / min) to achieve a fixation level of approximately 4000 RU. After fixation, the surface was inactivated with ethanolamine HCl (flow rate 30 μl / min). Nanobodies were injected onto the anti-FLAG M2 surface at 45 μl / min for 2 minutes to achieve a nanobody capture level of approximately 100 RU. To reduce nonspecific binding of blood samples to the anti-FLAG M2 surface, 100 nM 3×FLAG peptide (Sigma) was added to the blood samples. Samples containing existing antibodies 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 subsequent 600-second dissociation step. After each cycle (i.e., before capturing new nanobodies and injecting blood samples), the anti-FLAG M2 surface was regenerated by injecting glycine pH 1.5 (10 mM) at 150 μl / min for 10 seconds. Sensational gram processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensational gram showing binding of existing antibodies was obtained after double referencing by subtracting 1) nanobodies-anti-FLAG M2 dissociation and 2) nonspecific binding to the reference ligand lane. The binding level of existing antibodies was determined by setting a reporting point at 125 seconds (5 seconds after the end of association). The percentage decrease in binding of existing antibodies was calculated relative to the binding level of the reference nanobody at 125 seconds.

[0154] Example 1: By introducing the mutation of the present invention into reference A (SEQ ID NO: 1), a reduction in binding by existing antibodies is achieved. Reference A (Accession No.: 1) and two representative examples of improved mutants of Reference A with mutations according to the present invention (Accession Nos.: 37 and 38, both having an alanine extension and tested with an N-terminal HIS6-FLAG3 tag. See Accession No.: 100) were tested for binding by existing antibodies present in samples from 96 serum samples from healthy human volunteers. The compound was captured using the FLAG tag, and binding was measured using ProteOn according to the protocol given in the previous part of this experimental section.

[0155] The results are shown in Figure 4. In Figure 5, the results for each of the samples that form one of the data points in Figure 4 are listed.

[0156] For most of the 96 samples tested, it can be seen that introducing the mutations according to the present invention results in a decrease in the binding of existing antibodies. Here, the degree of decrease generally depended on the level at which the existing antibodies in each sample were able to bind to Reference A.

[0157] Example 2: Introduction of mutations of the present invention into Reference B (Accession No.: 2) results in a decrease in binding by existing antibodies Reference B (Accession No.: 2) and two representative examples of improved mutants of Reference B with mutations according to the present invention (Accession Nos.: 65 and 66, both having an alanine extension and tested with an N-terminal HIS6-FLAG3 tag. See Accession No.: 100) were tested for binding by existing antibodies present in samples from 96 serum samples from healthy human volunteers. The compound was captured using the FLAG tag, and binding was measured using ProteOn according to the protocol given in the previous part of this experimental section.

[0158] The results are shown in Figure 6. In Figure 7, the results for each of the samples that form one of the data points in Figure 6 are listed.

[0159] Similar to Example 1, it was found that introducing the mutation according to the present invention resulted in a reduction in the binding of existing antibodies in most of the 96 samples tested. Here, the degree of reduction generally depended on the level at which the existing antibodies in each sample were able to bind to reference B.

[0160] Example 3: By introducing the mutations of the present invention into reference C (SEQ ID NO: 3) and reference D (SEQ ID NO: 4), a reduction in binding by existing antibodies is achieved. Reference compound C (SEQ ID NO: 3), reference compound D (SEQ ID NO: 4), and two representative examples of improved variants of reference compound C and reference compound D possessing the mutation according to the present invention (SEQ ID NOs: 93 and 94, both having the same alanine elongation as present in reference compound D and possessing an N-terminal HIS6-FLAG3 tag; see SEQ ID NO: 100) were tested for binding with existing antibodies present in 66 serum samples from healthy human volunteers and 30 samples presumed to contain existing antibodies capable of binding even in the presence of C-terminal alanine (13 of which were from SLE patients). The compounds were captured in human serum albumin, and binding was measured using ProteOn according to the protocol given in the preceding section of this experimental part.

[0161] The results are shown in Figure 8. Figure 9 provides details for four representative SLE samples. Figure 10 lists the results for each sample that forms one of the data points in Figure 8.

[0162] Similar to Examples 1 and 2, it was found that introducing the mutation according to the present invention resulted in a reduction in the binding of the existing antibody in most of the 96 samples tested. Here, the degree of reduction generally depended on the level at which the existing antibody in each sample was able to bind to reference C or reference D.

[0163] The entirety of all references cited throughout this application (including articles, issued patents, published patent applications and concurrently pending patent applications) is expressly incorporated by reference, in particular for the purposes of the above-referenced teachings.

Claims

1. - CDR1 (by Abm) which is GFTFDTSSML (Sequence ID: 13) and - CDR2 (by Abm) which is VIHQSGTPTY (Sequence ID: 14) and - It has CDR3 (by Abm) which is FPSTHGKFDY (Sequence ID: 12), and, - At least 85% sequence identity to the amino acid sequence of Sequence ID No. 3 (where C-terminal elongation and CDR are not considered in determining sequence identity) and / or The amino acid sequence of Sequence ID No. 3 has seven or fewer "amino acid differences" (as defined here, without considering mutations at positions 11, 89, 110, or 112, and without considering C-terminal elongation), where the amino acid differences are not present in the CDR. Here, The amino acid residue at position 11 is selected from L or V, and The amino acid residue at position 89 is selected from T, V, or L, and The amino acid residue at position 110 is selected from T, K, or Q, and The amino acid residue at position 112 is selected from S, K, or Q. Here, the numbering is done by Kabat. An immunoglobulin single variable domain that binds to serum albumin.

2. The immunoglobulin single variable domain according to claim 1, having a C-terminal extension (X)n (where n is 1 to 10, and each X is independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I).

3. An immunoglobulin single variable domain according to claim 1 or 2, having a mutation in which an amino acid is substituted with D at position 1.

4. The immunoglobulin single variable domain according to claim 3, wherein a mutation at position 1 replaces E with D.

5. - An immunoglobulin single variable domain according to any one of claims 1 to 4, having a difference of five or fewer amino acids from the sequence of SEQ ID NO: 3 (without considering mutations at positions 11, 89, 110, or 112, and without considering C-terminal elongation).

6. The following amino acid residues: -11V or in combination with 89L -11V when combined with 110K or 110Q, -11V when combined with 112K or 112Q, -11V or in combination with 89L and 110K or 110Q An immunoglobulin single variable domain according to any one of claims 1 to 5, comprising -89L and 11V in combination with 112K or 112Q.

7. The following amino acid residues: 89L or in combination with -11V 89L in combination with -110K or 110Q or -89L in combination with 112K or 112Q or -89L or in combination with 11V and 110K or 110Q An immunoglobulin single variable domain according to any one of claims 1 to 6, comprising 89L in combination with -11V and 112K or 112Q.

8. The following amino acid residues: 110K or 110Q in combination with -11V or -110K or 110Q in combination with 89L or An immunoglobulin single variable domain according to any one of claims 1 to 7, comprising 110K or 110Q in combination with -11V and 89L.

9. The following amino acid residues: 112K or 112Q in combination with -11V or -112K or 112Q in combination with 89L or An immunoglobulin single variable domain according to any one of claims 1 to 8, comprising 112K or 112Q in combination with -11V and 89L.

10. The following amino acid residues: An immunoglobulin single variable domain according to any one of claims 1 to 9, comprising -89T.

11. Sequence ID: An immunoglobulin single variable domain having an amino acid sequence selected from amino acid sequences 44 to 71.

12. An immunoglobulin single variable domain having the amino acid sequence one of the following sequences: SEQ ID NO: 50 or SEQ ID NO:

64.

13. A polypeptide comprising the immunoglobulin single variable domain described in claim 1, wherein the immunoglobulin single variable domain is (i) present at and / or forming the N-terminus of a polypeptide, where the immunoglobulin single variable domain has a mutation in which the amino acid at position 1 is substituted with D, or (ii) A polypeptide having a C-terminus and / or forming the C-terminus of the polypeptide, wherein the immunoglobulin single variable domain has a C-terminus extension (X)n (where n is 1 to 10, and each X is an amino acid residue independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)).

14. The polypeptide according to claim 13, wherein a mutation at position 1 causes E to be replaced with D.

15. The polypeptide according to claim 13 or 14, wherein the polypeptide further comprises an immunoglobulin single variable domain against a therapeutic target.

16. The polypeptide according to claim 15, wherein the therapeutic target is selected from the group consisting of TNF, RANK-L, vWF, IgE, RSV, CXCR4, IL-23, and interleukins.

17. A pharmaceutical composition comprising at least one immunoglobulin single variable domain according to any one of claims 1 to 12 or a polypeptide according to any one of claims 13 to 16, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

18. A nucleic acid encoding an immunoglobulin single variable domain according to any one of claims 1 to 12 or a polypeptide according to any one of claims 13 to 16.

19. A host or host cell comprising and capable of expressing the nucleic acid described in claim 18, wherein the host is not human.

20. A method for preparing an immunoglobulin single variable domain according to any one of claims 1 to 12 or a polypeptide according to any one of claims 13 to 16, comprising culturing or maintaining the host cells according to claim 19 under conditions such that the host cells produce or express the immunoglobulin single variable domain.

21. An immunoglobulin single variable domain according to any one of claims 1 to 12 or a polypeptide according to any one of claims 13 to 16, for use in a method for the prevention and / or treatment of a disease or disorder.

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