Improved serum albumin-binding immunoglobulin variable domains

Abstract

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

Description

[0001] This application is a divisional application of Chinese Patent Application No. 2016800662466, filed on October 28, 2016, entitled "Improved Variable Structural Domain of Serum Albumin-Binding Immunoglobulin".

[0002] This invention relates to the amino acid sequence that binds to serum albumin.

[0003] In particular, the present invention relates to modified immunoglobulin single variable domains (also referred to herein as “ISV” or “ISVD”), and more particularly to modified heavy chain immunoglobulin single variable domains that bind to serum albumin, and to proteins, peptides and other constructs, compounds, molecules or chemical entities comprising such modified serum albumin conjugates.

[0004] Other aspects, embodiments, features, uses, and advantages of the invention will be apparent to those skilled in the art based on the disclosure herein.

[0005] The improved serum albumin-binding ISVD provided by this invention is also referred to herein as "the serum albumin conjugate of this invention," "the albumin conjugate of this invention," or "serum albumin conjugate" or "albumin conjugate." Furthermore, proteins, polypeptides, and other constructs, compounds, molecules, or chemical entities comprising at least one serum albumin conjugate of this invention are also referred to herein as "compounds of this invention" or "polypeptides of this invention."

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

[0007] In this application, amino acid residues / positions in the variable domains of the immunoglobulin heavy chain will be indicated using Kabat numbering. For convenience, Figure 1 A table is provided listing the positions of some amino acids that will be specifically mentioned herein and their numbers according to some alternative numbering systems (such as Aho and IMGT. Note: Unless explicitly stated otherwise, the Kabat numbering is definitive for the purposes of this specification and claims; other numbering systems are provided for reference only).

[0008] Regarding CDRs, as is well known in the art, there are various definitions and conventions for describing CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of the structural loop region). See, for example, http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, although reference may be made to CDRs according to Kabat, it is most preferably based on the Abm definition (which is based on the AbM antibody modeling software from Oxford Molecular), as this is considered the best compromise between the Kabat and Chothia definitions. See again http: / / www.bioinf.org.uk / abs / .

[0009] ISVDs that can bind to serum albumin and their uses are well known in the art, for example according to WO2004 / 041865, WO 2006 / 122787, EP 2 139 918, WO 2011 / 006915, WO 2012 / 175400 and WO2014 / 111550, which describe serum albumin-binding ISVDs and their use for prolonging the serum half-life of therapeutic compounds, fractions and entities (as defined in these applications).

[0010] This invention aims to provide improved serum albumin conjugates, particularly compared to those disclosed in WO 2011 / 006915 and WO2014 / 111550. Representative examples of serum albumin conjugates known according to these two PCT applications are given as “Reference A” through “Reference D” in Table A below. Alignment of these reference sequences is... Figure 2 The CDRs (combinations) of these reference compounds (according to Kabat and Abm conventions, respectively) are listed in Table B.

[0011] More specifically, the present invention aims to provide an improved serum albumin-binding ISVD as a variant of the serum albumin-binding ISVD mentioned in Table A and having reduced binding by means of interfering factors (commonly referred to as “pre-existing antibodies”) that may be present in the serum of healthy human subjects as well as patients. References are made to WO 12 / 175741, WO2013 / 024059, and also, for example, to Holland et al. (J. Clin. Immunol. 2013, 33(7):1192-203), and to co-pending non-prepublished PCT application PCT / EP2015 / 060643 entitled “Improved immunoglobulin variable domains” filed by the assignee on 13 May 2015 (published as WO 2015 / 173325 on 19 November 2015).

[0012]

[0013] Table B: CDRs of compounds A through D (based on Kabat and Abm)

[0014]

[0015]

[0016] As further described herein, the serum albumin conjugate of the present invention preferably has a combination of the same CDRs as those present in one of references A, B, C or D (i.e., CDR1, CDR2 and CDR3), and most preferably has a combination of the same CDRs as those present in reference C or reference D (which have the same CDRs).

[0017] Among the serum albumin conjugates listed in Table A, the conjugate of SEQ ID NO:4 has a C-terminal alanine extension, i.e., an alanine residue at the C-terminus of the ISVD sequence (sometimes referred to as "position 114"), compared to the usual C-terminal sequence VTVSS (SEQ ID NO:113, present in conjugates of SEQ ID NO:1 to 3). As described in WO 12 / 175741 (and also, for example, in WO2013 / 024059), this C-terminal alanine extension prevents so-called "pre-existing antibodies" (presumably IgG) from binding to the putative epitope located in the C-terminal region of the ISV. It is speculated that such epitopes particularly include the amino acid residues exposed on the surface of the C-terminal sequence VTVSS, as well as the amino acid residues at position 14 (and amino acid residues adjacent to / close to said amino acid residues in the amino acid sequence, such as positions 11, 13, and 15), and may also include the amino acid residues at position 83 (and amino acid residues adjacent to / close to said amino acid residues in the amino acid sequence, such as positions 82, 82a, 82b, and 84) and / or the amino acid residues at these positions 108 (and amino acid residues adjacent to / close to said amino acid residues in the amino acid sequence, such as position 107).

[0018] However, while the presence of such a C-terminal alanine (or more generally, C-terminal extension) can significantly reduce (and in many cases even substantially prevent) the binding of “pre-existing antibodies” that can be found in the serum of a range of subjects (healthy subjects and patients), it has been found that serum from some subjects (such as from patients with certain immune diseases such as SLE) can contain pre-existing antibodies that can bind to the C-terminal region of ISV (when such a region is exposed), even if the ISV contains such a C-terminal alanine (or more generally, such a C-terminal extension). Again, refer to the co-pending non-prepublished PCT application PCT / EP2015 / 060643 entitled “Improved immunoglobulin variable domains”, filed by the assignee on 13 May 2015.

[0019] Therefore, a specific object of the present invention is to provide a serum albumin conjugate as an improved variant of the serum albumin-bound ISVD listed in Table A and having reduced binding by means of so-called "pre-existing antibodies", and specifically belonging to the class described in PCT / EP2015 / 060643 (i.e., those pre-existing antibodies that can bind to the exposed C-terminal region of ISV even in the presence of C-terminal extension).

[0020] Typically, this invention achieves this objective by providing serum albumin-binding ISVs as variants of the sequences SEQ ID NO:1 to 4 (and especially variants of the sequences SEQ ID NO:3 or SEQ ID NO:4) containing the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO:1 to 4):

[0021] -89T; or

[0022] - 89L combined with 11V; or

[0023] - 89L combined with 110K or 110Q; or

[0024] - 89L combined with 112K or 112Q; or

[0025] - 89L in combination with 11V and 110K or 110Q; or

[0026] - 89L in combination with 11V and 112K or 112Q; or

[0027] - 11V in combination with 110K or 110Q; or

[0028] - 11V in combination with 112K or 112Q.

[0029] In a specific aspect, in the serum albumin conjugate of the present invention:

[0030] - The amino acid residue at position 11 is preferably selected from L, V, or K; and

[0031] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0032] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0033] - The amino acid residue at position 112 is preferably selected from S, K or Q;

[0034] Such that (i) position 89 is T; or (ii) position 110 is K or Q; or (iii) position 112 is K or Q; or (iv) position 89 is L and position 11 is V; or (v) position 89 is L and position 110 is K or Q; or (vi) position 89 is L and position 112 is K or Q; or (vii) position 89 is L and position 11 is V and position 110 is K or Q; or (vii) position 89 is L and position 11 is V and position 112 is K or Q; or (viii) position 11 is V and position 110 is K or Q; or (ix) position 11 is V and position 112 is K or Q.

[0035] In the amino acid sequences provided by this invention, amino acid sequences in which position 89 is T or where position 11 is V and position 89 is L (optionally suitably combined with 110K or 110Q mutations and / or 112K or 112Q mutations, and especially with 110K or 110Q mutations) are particularly preferred. Even more preferred are amino acid sequences in which position 11 is V and position 89 is L, optionally having 110K or 110Q mutations.

[0036] The amino acid sequence of the present invention preferably binds to (human) serum albumin having a better affinity than 100 nM, preferably better than 50 nM. For example, the albumin conjugates of the present invention, as variants of Reference A or Reference B, respectively, may have approximately the same affinity for (human) serum albumin as described in WO 2011 / 006915 for Reference A or Reference B; and similarly, the albumin conjugates of the present invention, as variants of Reference C and / or Reference D, respectively, may have approximately the same affinity for (human) serum albumin as described in WO 2014 / 111550 for Reference C and / or Reference D (and the affinity is measured as described in WO 2011 / 006915 or WO 2014 / 111550, respectively).

[0037] Furthermore, the half-life (defined as t1 / 2β) of the albumin conjugates provided by the present invention, as well as the compounds and peptides comprising them (as further described herein), in humans is preferably greater than 1 hour, more preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, and for example, about one day, two days, one week, two weeks and up to the half-life of serum albumin in humans (estimated to be about 19 days), although the latter may be less important.

[0038] For example, albumin conjugates of the present invention, as variants of reference A or reference B respectively, may have half-lives in humans that are equivalent to (and preferably approximately the same as) reference A or reference B respectively (see again WO 2011 / 006915); and similarly, albumin conjugates of the present invention, as variants of reference C and / or reference D respectively, may have half-lives in humans that are equivalent to (and preferably approximately the same as) reference C and / or reference D respectively (see again WO 2014 / 111550).

[0039] Furthermore, compounds or polypeptides of the present invention comprising albumin conjugates respectively as variants of reference A or reference B may have a half-life in humans comparable to (and preferably approximately the same as) that of the same compound or polypeptide but having reference A or reference B instead of the albumin conjugates of the present invention; and similarly, compounds or polypeptides of the present invention comprising albumin conjugates respectively as variants of reference C or reference D may have a half-life in humans comparable to (and preferably approximately the same as) that of the same compound or polypeptide but having reference C or reference D.

[0040] Table C lists some non-limiting possible combinations of amino acid residues present at positions 11, 89, 110, and 112 in the serum albumin conjugate of the present invention. Particularly preferred combinations are indicated in bold, and the most preferred combinations are indicated in […]. Bold / Underline express.

[0041] Table C: Possible combinations of amino acids at positions 11, 89, 110, and 112.

[0042]

[0043] The serum albumin conjugates of the present invention are further described herein in the specification, examples and figures, namely that they have the CDR as described herein and have an overall sequence identity degree (as defined herein) with one of the sequences disclosed herein (SEQ ID NO: 1 to 4) and / or may have a limited number of “amino acid differences” (as described herein) with these reference sequences (one of them).

[0044] The serum albumin conjugate of the present invention preferably comprises the following CDRs (according to Kabat convention):

[0045] -CDR1 (according to Kabat), wherein CDR1 is selected from the sequences TGEMA (SEQ ID NO:5) and TSSML (SEQ ID NO:10), and wherein CDR1 is preferably TSSML (SEQ ID NO:10); and

[0046] -CDR2 (according to Kabat), wherein CDR2 is selected from the sequences: SISSSGATTYYADSVKG (SEQ ID NO:6) and VIHQSGTPTYYADSVKG (SEQ ID NO:11), and wherein CDR2 is preferably VIHQSGTPTYYADSVKG (SEQ ID NO:11); and

[0047] -CDR3 (according to Kabat), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15) and most preferably FPSSRMKFDY (SEQ ID NO:15).

[0048] More preferably, the CDRs are as follows (again, 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).

[0049] Alternatively, when the CDR is given according to Abm convention, the serum albumin conjugate of the present invention preferably comprises the following CDR:

[0050] -CDR1 (according to Abm), wherein CDR1 is selected from the sequences GFTFSTGEMA (SEQ ID NO:8) and GFTFDTSSML (SEQ ID NO:13), and wherein CDR1 is preferably GFTFDTSSML (SEQ ID NO:13); and

[0051] -CDR2 (according to Abm), wherein CDR2 is selected from the sequences: SISSSGATTY (SEQ ID NO: 9) and VIHQSGTPTY (SEQ ID NO: 14), and wherein CDR2 is preferably VIHQSGTPTY (SEQ ID NO: 14); and

[0052] -CDR3 (according to Abm), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is most preferably FPSSRMKFDY (SEQ ID NO:15).

[0053] 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).

[0054] The serum albumin conjugate of the present invention preferably further comprises:

[0055] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with one of the sequences of SEQ ID NO: 1 to 4 (wherein any possible C-terminal extensions and the CDR are not considered in determining the degree of sequence identity); - and especially at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the sequence of SEQ ID NO: 3 or 4 (wherein, again, any possible C-terminal extensions and the CDR are not considered in determining the degree of sequence identity); and / or

[0056] - No more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with one of the sequences of SEQ ID NO: 1 to 4 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR); and especially no more than 5, preferably no more than 3, such as only 3, 2 or 1 such "amino acid differences" with the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR).

[0057] With regard to various and preferred aspects of the albumin conjugates of the present invention, when referring to the degree of sequence identity with respect to one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4, and / or the number and type of “amino acid differences” that may exist in such conjugates of the present invention (i.e., compared with one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4, respectively), it should be noted that when it is considered that (i) the amino acid sequence of the present invention has at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with respect to one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4, respectively, and / or SEQ ID NO:4, it is considered that When considering the degree of sequence identity of the sequence NO:4 (wherein, for determining the degree of sequence identity, CDR, any possible C-terminal extensions, and mutations at positions 11, 89, 110, and / or 112 required for the specific aspect involved are not taken into account); and / or when it is considered that (ii) the amino acid sequence of the present invention has no more than 7, preferably no more than 5, such as only 3, 2, or 1 “amino acid differences” from the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4 respectively (again, without considering any possible C-terminal extensions and without considering mutations at positions 11, 89, 110, and / or 112 required for the specific aspect involved), it also includes sequences that do not have amino acid differences from the sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4 respectively, except for mutations at positions 11, 89, 110, and / or 112 required for the specific aspect involved and any possible C-terminal extensions.

[0058] Therefore, in one specific aspect of the invention, the albumin conjugate of the invention may have 100% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4, respectively and when applicable (including CDR, but excluding one or more mutations or combinations of mutations at positions 11, 89, 110 and / or 112 disclosed herein and / or any C-terminal extensions that may be present), and / or may not have amino acid differences with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4, respectively and when applicable (i.e., except for one or more mutations or combinations of mutations at positions 11, 89, 110 and / or 112 disclosed herein and any C-terminal extensions that may be present).

[0059] When any amino acid differences are present (i.e., apart from any C-terminal extensions and mutations at positions 11, 89, 110, and / or 112 required for the specific aspects of the invention involved), these amino acid differences may be present in the CDR and / or the framework region, but they are preferably present only in the framework region (as defined by Abm custom, i.e. not in the CDR as defined by Abm custom), i.e., such that the albumin conjugate of the invention has a combination of the same CDRs (as defined by Abm custom) present, respectively and when applicable, in one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and / or SEQ ID NO:4.

[0060] When the serum albumin conjugates of the present invention are present in and / or form the C-terminus of the compounds or peptides of the present invention (or when they additionally have "exposed" C-terminals in the protein, peptide, or other compound or construct in which they are present, meaning that the C-terminus of the ISV is not bound to or connected to a constant structural domain (such as a CH1 domain; see again WO 12 / 175741 and PCT / EP2015 / 06043), they preferably also have formula (X). n The C-terminus of the extension is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue independently selected from naturally occurring amino acid residues (although according to a preferred aspect, it does not contain any cysteine ​​residues), and preferably an amino acid residue independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (preferably naturally occurring).

[0061] Based on this C-end extension X (n) Some preferred, but non-limiting, instances of X and n can be as follows:

[0062] (a) n = 1 and X = Ala;

[0063] (b) n = 2 and each X = Ala;

[0064] (c) n = 3 and each X = Ala;

[0065] (d) n = 2 and at least one X = Ala (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0066] (e) n = 3 and at least one X = Ala (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0067] (f) n = 3 and at least two X = Ala (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0068] (g)n=1 and X=Gly;

[0069] (h)n = 2 and each X = Gly;

[0070] (i)n = 3 and each X = Gly;

[0071] (j) n = 2 and at least one X = Gly (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0072] (k)n = 3 and at least one X = Gly (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0073] (l) n = 3 and at least two X = Gly (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0074] (m)n = 2 and each X = Ala or Gly;

[0075] (n)n = 3 and each X = Ala or Gly;

[0076] (o)n = 3 and at least one X = Ala or Gly (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile); or

[0077] (p)n = 3 and at least two X = Ala or Gly (and one or more of the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0078] Furthermore, aspects (a), (b), (c), (g), (h), (i), (m), and (n) are particularly preferred, and aspects where n = 1 or 2 are preferred, and aspects where n = 1 are particularly preferred.

[0079] It should also be noted that, preferably, any C-terminal extensions present in the serum albumin conjugate of the present invention do not contain (free) cysteine ​​residues (unless said cysteine ​​residues are used or intended for further functionalization, such as for polyethylene glycol oxidative degradation).

[0080] Some specific, but non-limiting, examples of available C-terminal extensions are the following amino acid sequences: A, AA, AAA, g, GG, GGG, AG, GA, AAG, AGG, AGA, GGA, GAA, or GAG.

[0081] When the serum albumin conjugate of the present invention contains a mutation at position 110 or 112 (optionally in combination with mutations at positions 11 and / or 89 as described herein), the C-terminal amino acid residues of the frame 4 (starting from position 109) may be as follows: (i) if no C-terminal extension is present: VTVKS (SEQ ID NO: 101), VTVQS (SEQ ID NO: 102), VKVSS (SEQ ID NO: 103), or VQVSS (SEQ ID NO: 104); or (ii) if a C-terminal extension is present: VTVKSX (n) (SEQ ID NO:105), VTVQSX(n)(SEQ ID NO:106), VKVSSX(n)(SEQ ID NO:107) or VQVSSX (n)(SEQ ID NO:108), such as VTVKSA (SEQ ID NO:109), VTVQSA (SEQ ID NO:110), VKVSSA (SEQ ID NO:111), or VQVSSA (SEQ ID NO:112). When the serum albumin conjugate of the present invention does not contain the mutation at position 110 or 112 (but only the mutation at position 11 and / or 89 as described herein), the C-terminal amino acid residues of the framework 4 (starting from position 109) will generally be: (i) when there is no C-terminal extension: VTVSS (SEQ ID NO:113) (as in the sequence of SEQ ID NO:3); or (ii) when there is a C-terminal extension: VTVSSX (n) (SEQ ID NO:114) as VTVSSA (SEQ ID NO:115) (as in the sequence of SEQ ID NO:4). In these C-terminal sequences, X and n are as defined herein for C-terminal extensions.

[0082] Furthermore, when the serum albumin conjugate of the present invention is present in and / or forms the N-terminus of the compound or polypeptide of the present invention, the serum albumin conjugate preferably has a D at position 1 (i.e., an E1D mutation compared to the sequences given in SEQ ID NO: 1 to 4 and 16 to 99).

[0083] Furthermore, generally, when the compounds or peptides of the present invention have a heavy chain ISVD at their C-terminus (which may be the serum albumin conjugate of the present invention, but may also be, for example, an ISVD that binds to a time-dependent therapeutic target), the C-terminal ISVD (and even the compounds or peptides of the present invention) preferably has a C-terminal extension X(n) as described herein. Similarly, when the compounds or peptides of the present invention have a heavy chain ISVD at their N-terminus (which may be the serum albumin conjugate of the present invention, but may also be, for example, an ISVD that binds to a time-dependent therapeutic target), the N-terminal ISVD (and even the compounds or peptides of the present invention) preferably has a D at position 1.

[0084] Furthermore, preferably, when the compounds or peptides of the present invention contain one or more other ISVDs in addition to one or more albumin conjugates of the present invention (the other ISVDs may be, for example, one or more ISVDs targeting a therapeutic target), preferably, all ISVDs present in the compounds or peptides contain one or more skeletal mutations in their sequences that reduce the binding of a pre-existing antibody. In particular, when these other ISVDs are nanobodies or (single) domain antibodies consisting essentially of a VH domain and / or derived from a VH domain, they may contain amino acid residues / mutations (suitable combinations) at positions 11, 89, 110, and / or 112 as described herein with respect to the albumin conjugates of the present invention.

[0085] As mentioned, the amino acid sequences provided by this invention are proteins that can bind to human serum albumin, and in particular, can bind specifically to human serum albumin (as described herein). Therefore, they can be used as binding units or binding domains for binding to (human) serum albumin, for example, to impart an increase in half-life (as defined herein) to a therapeutic compound, part, or entity. For the use of serum albumin binding domains to increase the half-life of therapeutic compounds, parts, or entities, reference is generally made to WO 2004 / 041865, WO 2006 / 122787, EP 2139918, WO 2011 / 006915, WO 2012 / 175400, and / or WO 2014 / 111550. The albumin conjugates of this invention can generally be used in the same manner and for the same purpose as the serum albumin conjugates described in these references.

[0086] Preferred, but non-limiting, examples of the ISVs of the invention are given in SEQ ID NO: 16 to 99, and each of these sequences constitutes another aspect of the invention (as do proteins, polypeptides, or other compounds or constructs comprising one of these sequences). Among these:

[0087] -SEQ ID NO:16 to 29 are instances of variants of the sequence of SEQ ID NO:1. These sequences have CDR1 (as defined by Kabat) of the sequence of SEQ ID NO:5; CDR2 (as defined by Kabat) of the sequence of SEQ ID NO:6; and CDR3 (as defined by Kabat) of the sequence of SEQ ID NO:7;

[0088] -SEQ ID NO:30 to 43 are examples of variants of the sequence of SEQ ID NO:1 having a C-terminal alanine (as described herein, a preferred, but not limiting, example of a C-terminal extension). These sequences have CDR1 (as defined in Kabat) of the sequence of SEQ ID NO:5; CDR2 (as defined in Kabat) of the sequence of SEQ ID NO:6; and CDR3 (as defined in Kabat) of the sequence of SEQ ID NO:7;

[0089] -SEQ ID NO:44 to 57 are instances of variants of the sequence of SEQ ID NO:2. These sequences have CDR1 (as defined by Kabat) of the sequence of SEQ ID NO:10; CDR2 (as defined by Kabat) of the sequence of SEQ ID NO:11; and CDR3 (as defined by Kabat) of the sequence of SEQ ID NO:12;

[0090] -SEQ ID NO:58 to 71 are examples of variations of the sequence of SEQ ID NO:2 having a C-terminal alanine (as described herein, a preferred, but not limiting, example of a C-terminal extension). These sequences have CDR1 (as defined by Kabat) of the sequence of SEQ ID NO:10; CDR2 (as defined by Kabat) of the sequence of SEQ ID NO:11; and CDR3 (as defined by Kabat) of the sequence of SEQ ID NO:12;

[0091] -SEQ ID NO:72 to 85 are instances of variants of the sequence of SEQ ID NO:3. These sequences have CDR1 (as defined by Kabat) of the sequence of SEQ ID NO:10; CDR2 (as defined by Kabat) of the sequence of SEQ ID NO:11; and CDR3 (as defined by Kabat) of the sequence of SEQ ID NO:15;

[0092] -SEQ ID NO:86 to 99 to 54 are examples of variants of the sequence SEQ ID NO:4 (which is SEQ ID NO:3 with a C-terminal alanine extension). These sequences have CDR1 of the sequence SEQ ID NO:10 (as defined by Kabat); CDR2 of the sequence SEQ ID NO:11 (as defined by Kabat); and CDR3 of the sequence SEQ ID NO:15 (as defined by Kabat).

[0093] Of these variants, sequences SEQ ID NO:72 to 85 (when C-terminal extension is not required) and sequences SEQ ID NO:86 to 99 (when C-terminal extension is required) are the most preferred.

[0094] Therefore, in a first aspect, the present invention relates to an immunoglobulin monovariable domain, said immunoglobulin monovariable domain having:

[0095] -CDR1 (according to Kabat), wherein CDR1 is selected from the sequences TGEMA (SEQ ID NO:5) and TSSML (SEQ ID NO:10), and wherein CDR1 is preferably TSSML (SEQ ID NO:10); and

[0096] -CDR2 (according to Kabat), wherein CDR2 is selected from the sequences: SISSSGATTYYADSVKG (SEQ ID NO:6) and VIHQSGTPTYYADSVKG (SEQ ID NO:11), and wherein CDR2 is preferably VIHQSGTPTYYADSVKG (SEQ ID NO:11); and

[0097] -CDR3 (according to Kabat), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12), and

[0098] And it has:

[0099] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1 (wherein any possible C-terminal extensions and the CDR are not taken into account in determining the degree of sequence identity);

[0100] and / or

[0101] - No more than 7, such as no more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with the amino acid sequence of SEQ ID NO:1 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0102] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0103] -C-end extension (X) n, where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0104] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0105] in:

[0106] - The amino acid residue at position 11 is preferably selected from L or V; and

[0107] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0108] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0109] - The amino acid residue at position 112 is preferably selected from S, K or Q;

[0110] So that (i) position 89 is T; or (ii) position 89 is L and position 11 is V; or (iii) position 89 is L and position 110 is K or Q; or (iv) position 89 is L and position 112 is K or Q; or (v) position 89 is L and position 11 is V and position 110 is K or Q; or (vi) position 89 is L and position 11 is V and position 112 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (vii) position 11 is V and position 112 is K or Q.

[0111] In another aspect, the present invention relates to an immunoglobulin monovariable domain, said immunoglobulin monovariable domain having:

[0112] -CDR1 (according to Kabat), wherein CDR1 is selected from the sequences TGEMA (SEQ ID NO:5) and TSSML (SEQ ID NO:10), and wherein CDR1 is preferably TSSML (SEQ ID NO:10); and

[0113] -CDR2 (according to Kabat), wherein CDR2 is selected from the sequences: SISSSGATTYYADSVKG (SEQ ID NO:6) and VIHQSGTPTYYADSVKG (SEQ ID NO:11), and wherein CDR2 is preferably VIHQSGTPTYYADSVKG (SEQ ID NO:11); and

[0114] -CDR3 (according to Kabat), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15) and most preferably FPSSRMKFDY (SEQ ID NO:15);

[0115] And it has:

[0116] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with one of the sequences of SEQ ID NO: 1 to 4 (wherein any possible C-terminal extensions and the CDR are not considered in determining the degree of sequence identity); - and especially at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the sequence of SEQ ID NO: 3 or 4 (wherein, again, any possible C-terminal extensions and the CDR are not considered in determining the degree of sequence identity); and / or

[0117] - No more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with one of the sequences of SEQ ID NO: 1 to 4 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR); and especially no more than 5, preferably no more than 3, such as only 3, 2 or 1 such "amino acid differences" with the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0118] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0119] -C-end extension (X) n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0120] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0121] The immunoglobulin single variable domain contains the following amino acid residues at the mentioned position (according to the Kabat number) (i.e., the mutation compared to the sequence of SEQ ID NO:1 to 4):

[0122] -89T; or

[0123] - 89L combined with 11V; or

[0124] - 89L combined with 110K or 110Q; or

[0125] - 89L combined with 112K or 112Q; or

[0126] - 89L in combination with 11V and 110K or 110Q; or

[0127] - 89L in combination with 11V and 112K or 112Q; or

[0128] - 11V in combination with 110K or 110Q; or

[0129] - 11V in combination with 112K or 112Q.

[0130] In particular, the serum albumin conjugate of the present invention preferably has no more than 5, preferably no more than 3, such as only 3, 2 or 1, an "amino acid difference" (as defined herein, and without regard to any of the above-listed mutations at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) of one of the sequences of SEQ ID NO: 1 to 4 (wherein, if present, the amino acid difference may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR); and especially no more than 5, preferably no more than 3, such as only 3, 2 or 1, an "amino acid difference" of no more than 5, preferably no more than 3, such as only 3, 2 or 1, of such a sequence as SEQ ID NO: 3 (wherein, if present, the amino acid difference may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR).

[0131] Some specific, but non-limiting examples of such possible mutations / amino acid differences (i.e., compared to the sequences of SEQ ID NO: 1 to 4) are: E1D, P14A; P41A, P41L, P41S, or P41T (and especially P41A); P42E or T87A. Other examples of mutations are one or more suitable “camelizing” substitutions (suitable combinations); for example, refer to Davies and Riechmann, Protein Engineering, vol. 9, no. 6, 531-537, 1996 and Davies and Riechmann, FEBS Letters 399(1004), 285-290, and Tables A-3 to A-8 from WO08 / 020079.

[0132] As mentioned, in this invention, amino acid sequences in which position 89 is T or where position 11 is V and position 89 is L (optionally suitably combined with 110K or 110Q mutations and / or 112K or 112Q mutations, and especially with 110K or 110Q mutations) are particularly preferred. Even more preferred are amino acid sequences in which position 11 is V and position 89 is L, optionally having 110K or 110Q mutations.

[0133] Therefore, in a preferred aspect, the present invention relates to an immunoglobulin monovariable domain, said immunoglobulin monovariable domain having:

[0134] -CDR1 (according to Kabat), wherein CDR1 is selected from the sequences TGEMA (SEQ ID NO:5) and TSSML (SEQ ID NO:10), and wherein CDR1 is preferably TSSML (SEQ ID NO:10); and

[0135] -CDR2 (according to Kabat), wherein CDR2 is selected from the sequences: SISSSGATTYYADSVKG (SEQ ID NO:6) and VIHQSGTPTYYADSVKG (SEQ ID NO:11), and wherein CDR2 is preferably VIHQSGTPTYYADSVKG (SEQ ID NO:11); and

[0136] -CDR3 (according to Kabat), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15) and most preferably FPSSRMKFDY (SEQ ID NO:15);

[0137] And it has:

[0138] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1 (wherein any possible C-terminal extensions and the CDR are not taken into account in determining the degree of sequence identity);

[0139] and / or

[0140] - No more than 7, such as no more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with the amino acid sequence of SEQ ID NO:1 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0141] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0142] -C-end extension (X) n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0143] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0144] in:

[0145] - The amino acid residue at position 11 is preferably selected from L or V; and

[0146] - The amino acid residue at position 89 is T; and

[0147] - The amino acid residue at position 110 is preferably selected from T, K, or Q (and preferably T); and

[0148] - The amino acid residue at position 112 is preferably selected from S, K or Q (and preferably S).

[0149] In another preferred aspect, the present invention relates to an immunoglobulin monovariable domain, said immunoglobulin monovariable domain having:

[0150] -CDR1 (according to Kabat), wherein CDR1 is selected from the sequences TGEMA (SEQ ID NO:5) and TSSML (SEQ ID NO:10), and wherein CDR1 is preferably TSSML (SEQ ID NO:10); and

[0151] -CDR2 (according to Kabat), wherein CDR2 is selected from the sequences: SISSSGATTYYADSVKG (SEQ ID NO:6) and VIHQSGTPTYYADSVKG (SEQ ID NO:11), and wherein CDR2 is preferably VIHQSGTPTYYADSVKG (SEQ ID NO:11); and

[0152] -CDR3 (according to Kabat), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15) and most preferably FPSSRMKFDY (SEQ ID NO:15);

[0153] And it has:

[0154] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1 (wherein any possible C-terminal extensions and the CDR are not taken into account in determining the degree of sequence identity);

[0155] and / or

[0156] - No more than 7, such as no more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with the amino acid sequence of SEQ ID NO:1 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0157] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0158] -C-end extension (X) n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0159] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0160] in:

[0161] - The amino acid residue at position 11 is V; and

[0162] - The amino acid residue at position 89 is L; and

[0163] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0164] - The amino acid residue at position 112 is preferably selected from S, K or Q.

[0165] In a specific, but not limiting, aspect, the serum albumin conjugate of the present invention has:

[0166] -CDR1 (according to Kabat), said CDR1 is TSSML (SEQ ID NO:10); and

[0167] -CDR2 (according to Kabat), said CDR2 is VIHQSGTPTYYADSVKG (SEQ ID NO:11); and

[0168] -CDR3 (according to Kabat), wherein CDR3 is selected from the following sequence: FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15), and preferably FPSSRMKFDY (SEQ ID NO:15);

[0169] And more specifically:

[0170] -CDR1 (according to Kabat), said CDR1 is TSSML (SEQ ID NO:10); and

[0171] -CDR2 (according to Kabat), said CDR2 is VIHQSGTPTYYADSVKG (SEQ ID NO:11); and

[0172] -CDR3 (according to Kabat), wherein CDR3 is FPSSRMKFDY (SEQ ID NO:15).

[0173] In one specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0174] - 11V combined with 89L; or

[0175] - 11V in combination with 110K or 110Q;

[0176] - 11V in combination with 112K or 112Q;

[0177] - 11V in combination with 89L and 110K or 110Q; or

[0178] - 11V in combination with 89L and 112K or 112Q;

[0179] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0180] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0181] - 89L combined with 11V; or

[0182] - 89L combined with 110K or 110Q; or

[0183] - 89L combined with 112K or 112Q; or

[0184] - 89L in combination with 11V and 110K or 110Q; or

[0185] - 89L in combination with 11V and 112K or 112Q;

[0186] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0187] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0188] - 110K or 110Q combined with 11V; or

[0189] - 110K or 110Q combined with 89L; or

[0190] - 110K or 110Q in combination with 11V and 89L;

[0191] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0192] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0193] - 112K or 112Q combined with 11V; or

[0194] - 112K or 112Q combined with 89L; or

[0195] - 112K or 112Q in combination with 11V and 89L;

[0196] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0197] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention contains a T at position 89 and has a CDR and has a degree of overall sequence identity with the reference sequence as described herein.

[0198] In another specific, but non-limiting aspect, the serum albumin conjugate 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 with the reference sequence as described herein.

[0199] In another aspect, the present invention relates to an immunoglobulin monovariable domain, said immunoglobulin monovariable domain having:

[0200] -CDR1 (according to Abm), wherein CDR1 is selected from the sequences GFTFSTGEMA (SEQ ID NO:8) and GFTFDTSSML (SEQ ID NO:13), and wherein CDR1 is preferably GFTFDTSSML (SEQ ID NO:13); and

[0201] -CDR2 (according to Abm), wherein CDR2 is selected from the sequences: SISSSGATTY (SEQ ID NO: 9) and VIHQSGTPTY (SEQ ID NO: 14), and wherein CDR2 is preferably VIHQSGTPTY (SEQ ID NO: 14); and

[0202] -CDR3 (according to Abm), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is most preferably FPSSRMKFDY (SEQ ID NO:15);

[0203] And it has:

[0204] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1 (wherein any possible C-terminal extensions and the CDR are not taken into account in determining the degree of sequence identity);

[0205] and / or

[0206] - No more than 7, such as no more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with the amino acid sequence of SEQ ID NO:1 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0207] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0208] -C-end extension (X) n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0209] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0210] in:

[0211] - The amino acid residue at position 11 is preferably selected from L or V; and

[0212] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0213] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0214] - The amino acid residue at position 112 is preferably selected from S, K or Q;

[0215] So that (i) position 89 is T; or (ii) position 89 is L and position 11 is V; or (iii) position 89 is L and position 110 is K or Q; or (iv) position 89 is L and position 112 is K or Q; or (v) position 89 is L and position 11 is V and position 110 is K or Q; or (vi) position 89 is L and position 11 is V and position 112 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (vii) position 11 is V and position 112 is K or Q.

[0216] In another aspect, the present invention relates to an immunoglobulin monovariable domain, said immunoglobulin monovariable domain having:

[0217] -CDR1 (according to Abm), wherein CDR1 is selected from the sequences GFTFSTGEMA (SEQ ID NO:8) and GFTFDTSSML (SEQ ID NO:13), and wherein CDR1 is preferably GFTFDTSSML (SEQ ID NO:13); and

[0218] -CDR2 (according to Abm), wherein CDR2 is selected from the sequences: SISSSGATTY (SEQ ID NO: 9) and VIHQSGTPTY (SEQ ID NO: 14), and wherein CDR2 is preferably VIHQSGTPTY (SEQ ID NO: 14); and

[0219] -CDR3 (according to Abm), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is most preferably FPSSRMKFDY (SEQ ID NO:15);

[0220] And it has:

[0221] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with one of the sequences of SEQ ID NO: 1 to 4 (wherein any possible C-terminal extensions and the CDR are not considered in determining the degree of sequence identity); - and especially at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the sequence of SEQ ID NO: 3 or 4 (wherein, again, any possible C-terminal extensions and the CDR are not considered in determining the degree of sequence identity); and / or

[0222] - No more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with one of the sequences of SEQ ID NO: 1 to 4 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR); and especially no more than 5, preferably no more than 3, such as only 3, 2 or 1 such "amino acid differences" with the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0223] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0224] -C-end extension (X)n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0225] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0226] The immunoglobulin single variable domain contains the following amino acid residues at the mentioned position (according to the Kabat number) (i.e., the mutation compared to the sequence of SEQ ID NO:1 to 4):

[0227] -89T; or

[0228] - 89L combined with 11V; or

[0229] - 89L combined with 110K or 110Q; or

[0230] - 89L combined with 112K or 112Q; or

[0231] - 89L in combination with 11V and 110K or 110Q; or

[0232] - 89L in combination with 11V and 112K or 112Q; or

[0233] - 11V in combination with 110K or 110Q; or

[0234] - 11V in combination with 112K or 112Q.

[0235] In particular, the serum albumin conjugate of the present invention preferably has no more than 5, preferably no more than 3, such as only 3, 2 or 1, an "amino acid difference" (as defined herein, and without regard to any of the above-listed mutations at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) of one of the sequences of SEQ ID NO: 1 to 4 (wherein, if present, the amino acid difference may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR); and especially no more than 5, preferably no more than 3, such as only 3, 2 or 1, such an "amino acid difference" of the sequence of SEQ ID NO: 3 (wherein, if present, the amino acid difference may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR).

[0236] As mentioned, in this invention, amino acid sequences in which position 89 is T or where position 11 is V and position 89 is L (optionally suitably combined with 110K or 110Q mutations and / or 112K or 112Q mutations, and especially with 110K or 110Q mutations) are particularly preferred. Even more preferred are amino acid sequences in which position 11 is V and position 89 is L, optionally having 110K or 110Q mutations.

[0237] Therefore, in a preferred aspect, the present invention relates to an immunoglobulin monovariable domain, said immunoglobulin monovariable domain having:

[0238] -CDR1 (according to Abm), wherein CDR1 is selected from the sequences GFTFSTGEMA (SEQ ID NO:8) and GFTFDTSSML (SEQ ID NO:13), and wherein CDR1 is preferably GFTFDTSSML (SEQ ID NO:13); and

[0239] -CDR2 (according to Abm), wherein CDR2 is selected from the sequences: SISSSGATTY (SEQ ID NO: 9) and VIHQSGTPTY (SEQ ID NO: 14), and wherein CDR2 is preferably VIHQSGTPTY (SEQ ID NO: 14); and

[0240] -CDR3 (according to Abm), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is most preferably FPSSRMKFDY (SEQ ID NO:15);

[0241] And it has:

[0242] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1 (wherein any possible C-terminal extensions and the CDR are not taken into account in determining the degree of sequence identity);

[0243] and / or

[0244] - No more than 7, such as no more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with the amino acid sequence of SEQ ID NO:1 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0245] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0246] -C-end extension (X) n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0247] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0248] in:

[0249] - The amino acid residue at position 11 is preferably selected from L or V; and

[0250] - The amino acid residue at position 89 is T; and

[0251] - The amino acid residue at position 110 is preferably selected from T, K, or Q (and preferably T); and

[0252] - The amino acid residue at position 112 is preferably selected from S, K or Q (and preferably S).

[0253] In another preferred aspect, the present invention relates to an immunoglobulin monovariable domain having:

[0254] -CDR1 (according to Abm), wherein CDR1 is selected from the sequences GFTFSTGEMA (SEQ ID NO:8) and GFTFDTSSML (SEQ ID NO:13), and wherein CDR1 is preferably GFTFDTSSML (SEQ ID NO:13); and

[0255] -CDR2 (according to Abm), wherein CDR2 is selected from the sequences: SISSSGATTY (SEQ ID NO: 9) and VIHQSGTPTY (SEQ ID NO: 14), and wherein CDR2 is preferably VIHQSGTPTY (SEQ ID NO: 14); and

[0256] -CDR3 (according to Abm), wherein CDR3 is selected from the following sequences: PRHPQGGVTFDY (SEQ ID NO:7), FPSTHGKFDY (SEQ ID NO:12) and FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is preferably FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15), and wherein CDR3 is most preferably FPSSRMKFDY (SEQ ID NO:15);

[0257] And it has:

[0258] - At least 85%, preferably at least 90%, more preferably at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1 (wherein any possible C-terminal extensions and the CDR are not taken into account in determining the degree of sequence identity);

[0259] and / or

[0260] - No more than 7, such as no more than 5, preferably no more than 3, such as only 3, 2 or 1 "amino acid differences" with the amino acid sequence of SEQ ID NO:1 (as defined herein, and without regard to any mutations listed above at positions 11, 89, 110 or 112 and without regard to any possible C-terminal extensions) (wherein, if present, the amino acid differences may be present in the frame and / or CDR, but preferably only in the frame and not in the CDR);

[0261] And optionally, it has (especially when the ISVD is present at and / or forms the C-terminus of the compound or polypeptide of the present invention):

[0262] -C-end extension (X) n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue (preferably naturally occurring) independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0263] And optionally, it has (in particular, when the ISVD is present at and / or forms the N-terminus of the compound or polypeptide of the present invention) a D and / or E1D mutation at position 1;

[0264] in:

[0265] - The amino acid residue at position 11 is V; and

[0266] - The amino acid residue at position 89 is L; and

[0267] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0268] - The amino acid residue at position 112 is preferably selected from S, K or Q.

[0269] In a specific, but not limiting, aspect, the serum albumin conjugate of the present invention has:

[0270] -CDR1 (according to Abm), said CDR1 is GFTFDTSSML (SEQ ID NO:13); and

[0271] -CDR2 (according to Abm), said CDR2 is VIHQSGTPTY (SEQ ID NO:14); and

[0272] -CDR3 (according to Abm), wherein CDR3 is selected from the following sequences: FPSTHGKFDY (SEQ ID NO:12) or FPSSRMKFDY (SEQ ID NO:15), and preferably FPSSRMKFDY (SEQ ID NO:15).

[0273] And more specifically:

[0274] -CDR1 (according to Abm), said CDR1 is GFTFDTSSML (SEQ ID NO:13); and

[0275] -CDR2 (according to Abm), said CDR2 is VIHQSGTPTY (SEQ ID NO:14); and

[0276] -CDR3 (according to Abm), wherein CDR3 is FPSSRMKFDY (SEQ ID NO:15).

[0277] In one specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0278] - 11V combined with 89L; or

[0279] - 11V in combination with 110K or 110Q;

[0280] - 11V in combination with 112K or 112Q;

[0281] - 11V in combination with 89L and 110K or 110Q; or

[0282] - 11V in combination with 89L and 112K or 112Q;

[0283] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0284] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0285] - 89L combined with 11V; or

[0286] - 89L combined with 110K or 110Q; or

[0287] - 89L combined with 112K or 112Q; or

[0288] - 89L in combination with 11V and 110K or 110Q; or

[0289] - 89L in combination with 11V and 112K or 112Q;

[0290] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0291] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0292] - 110K or 110Q combined with 11V; or

[0293] - 110K or 110Q combined with 89L; or

[0294] - 110K or 110Q in combination with 11V and 89L;

[0295] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0296] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0297] - 112K or 112Q combined with 11V; or

[0298] - 112K or 112Q combined with 89L; or

[0299] - 112K or 112Q in combination with 11V and 89L;

[0300] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0301] In another specific, but non-limiting aspect, the serum albumin conjugate of the present invention comprises the following amino acid residues (i.e., mutations compared to the sequences of SEQ ID NO: 1 to 4) at the mentioned positions (according to the Kabat number):

[0302] -89T;

[0303] It also has a CDR and a degree of sequence identity with the overall sequence as described herein.

[0304] In another specific, but non-limiting aspect, the present invention relates to an immunoglobulin monovariable domain having an amino acid sequence selected from the amino acid sequences of SEQ ID NO:16 to 99.

[0305] In another specific, but non-limiting aspect, the present invention relates to an immunoglobulin monovariable domain having an amino acid sequence selected from the amino acid sequences of SEQ ID NO:44 to 99.

[0306] In another specific, but non-limiting aspect, the present invention relates to an immunoglobulin monovariable domain having an amino acid sequence as 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.

[0307] In another specific, but non-limiting aspect, the present invention relates to an immunoglobulin monovariable domain having an amino acid sequence as one of the following sequences: SEQ ID NO:50, SEQ ID NO:64, SEQ ID NO:78 or SEQ ID NO:92, and preferably SEQ ID NO:78 or SEQ ID NO:92.

[0308] The present invention also relates to proteins, peptides, and other constructs, molecules, or chemical entities comprising or substantially composed of serum albumin conjugates of the present invention as described herein; methods for expressing / preparing the modified heavy chain immunoglobulin variable domains of the present invention and / or for expressing / preparing proteins, peptides, and other constructs, molecules, or chemical entities comprising thereof; compositions and products (such as pharmaceutical compositions and products) containing the modified heavy chain immunoglobulin variable domains of the present invention and / or proteins, peptides, and other constructs, molecules, or chemical entities comprising thereof; nucleotide sequences and nucleic acids encoding the modified heavy chain immunoglobulin variable domains of the present invention and / or encoding proteins or peptides comprising thereof; and uses of the modified heavy chain immunoglobulin variable domains of the present invention and proteins, peptides, and other constructs, molecules, or chemical entities comprising thereof (and especially therapeutic, preventative, and diagnostic uses).

[0309] Further aspects, embodiments, advantages, applications, and uses of the invention will become clear from the further description herein.

[0310] In this instruction manual:

[0311] The term "immunoglobulin single variable domain" (also known as "ISV" or "ISVD") is generally used to refer to an immunoglobulin variable domain (which can be a heavy or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with another variable domain (e.g., in the absence of the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of ISVDs will be clear to those skilled in the art and include, for example, nanobodies (including VHH, humanized VHH, and / or camel-derived VH such as camel-derived human VH), IgNARs, domains, and antibodies (such as dAbs) that are (single domains) that are VH domains or derived from VH domains. TM ) and antibodies that are VL domains or derived from VL domains (single domain) (such as dAbs) TM Unless otherwise expressly stated herein, ISVDs based on and / or derived from variable domains of the heavy chain (such as VH or VHH domains) are generally preferred. Most preferably, unless otherwise expressly stated herein, the ISVD will be a nanobody.

[0312] The term "nanobody" is generally as defined in WO 2008 / 020079 or WO 2009 / 138519, and therefore in specific contexts generally refers to a VHH, a humanized VHH, or a camel-derived VH (such as a camel-derived human VH), or generally a sequence-optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). It should be noted that the term nanobody (Nanobody or Nanobodies) is a registered trademark of Ablynx NV and is therefore also referred to as... and / or

[0313] - Generally, unless otherwise stated herein, the ISVDs, nanobodies, peptides, proteins, and other compounds and constructs mentioned herein are intended for the prevention or treatment of diseases or conditions in humans (and / or optionally, also in warm-blooded animals, and especially mammals). Therefore, the ISVDs, nanobodies, peptides, proteins, and other compounds and constructs described herein are preferably intended to be used as, and / or suitably used as, part of a (biological) pharmaceutical or other pharmaceutically or therapeutically active compound and / or part of a pharmaceutical product or composition. Such pharmaceuticals, compounds, or products are preferably intended to be administered to humans, for example, to prevent or treat subjects requiring such prevention or treatment, or, for example, as part of a clinical trial. As further described herein, for this purpose, such pharmaceuticals or compounds may contain portions, entities, or binding units other than the ISVD provided by the present invention (which, as also stated herein, may be, for example, one or more other additional therapeutic portions and / or one or more other portions affecting the pharmacokinetic or pharmacodynamic properties of ISVD-based or nanobody-based biological products, such as their half-life). Suitable examples of such additional therapeutic or other components will be apparent to those skilled in the art, and may typically include, for example, any therapeutically active protein, peptide, or other binding domain or binding unit, and, for example, modifications, such as those described on pages 149 to 152 of WO 2009 / 138159. ISVD-based or nanobody-based bioproducts are preferably therapeutic agents or intended for use as therapeutic agents (including prevention and diagnostics), and for this purpose preferably contain at least one ISVD targeting a therapeutically relevant target (e.g., RANK-L, vWF, IgE, RSV, CXCR4, IL-23, or other interleukins). For specific, but non-limiting examples of such ISVD-based or nanobody-based bioproducts, reference is made to Examples 8 through 18, and also, for example, to several applications of Ebolingx Inc. (e.g., but not limited to, WO2004 / 062551, WO 2006 / 122825, WO 2008 / 020079 and WO 2009 / 068627), and, for example (but not limited to) applications such as WO 2006 / 038027, WO 2006 / 059108, WO 2007 / 063308, WO 2007 / 063311, WO 2007 / 066016 and WO 2007 / 085814.Furthermore, as further described herein, additional portions may be ISVDs or nanobodies as described herein for (human) serum proteins such as (human) serum albumin, and such ISVDs or nanobodies may also be found to have therapeutic uses, particularly in prolonging the TNF conjugates described herein and / or for prolonging the half-life of the TNF conjugates described herein. For example, refer to WO 2004 / 041865, WO 2006 / 122787 and WO 2012 / 175400, which broadly describe the use of serum albumin-bound nanobodies for half-life prolongation. Furthermore, in this specification, unless otherwise expressly stated herein, all terms used herein have the meanings given in WO 2009 / 138519 (or in the prior art referenced in WO 2009 / 138519) or in WO 2008 / 020079 (or in the prior art referenced in WO 2008 / 020079). Furthermore, where a method or technique is not specifically described herein, it may be carried out in accordance with the description in WO 2009 / 138519 (or in the prior art referenced in WO 2009 / 138519) or in WO 2008 / 020079 (or in the prior art referenced in WO 2008 / 020079). Additionally, as described herein, any pharmaceutical product or composition comprising any ISVD or compound of the present invention may also comprise one or more additional components known per se for use in pharmaceutical products or compositions (i.e., depending on the intended pharmaceutical form) and / or one or more other compounds or active ingredients, for example, for therapeutic use (i.e., for providing a combination product).

[0314] Furthermore, when used in this specification or claims, the following terms have the same meaning as given on pages 62-75 of WO 2009 / 138519, and / or may be determined, where applicable, in the manner described on pages 62-75 of WO 2009 / 138519: “agonist,” “antagonist,” “reverse agonist,” “nonpolar uncharged amino acid residue,” “polar uncharged amino acid residue,” “polar charged amino acid residue,” “sequence identity,” “identical,” and “amino acid difference” (when referring to a sequence comparison of two amino acid sequences), “(in) substantially separate (form),” “domain,” “binding domain,” “antigenic determinant,” “epitope,” “against” or “directed against” (antigen), “specificity,” and “half-life.” Furthermore, the terms “regulation” and “for regulation”, “interacting site”, “specific to”, “cross-blocking”, “cross-blocked” and “cross-blocking”, and “substantially pH-independent” are as defined (and / or may be determined as described therein) in pages 74-79 of Ebolings Inc.’s WO 2010 / 130832. Additionally, when referring to the constructs, compounds, proteins, or peptides of the present invention, terms such as “monovalent”, “divalent” (or “multivalent”), “bispecific” (or “multispecific”), and “bicomplementary” (or “multicomplementary”) may have the meanings given in WO 2009 / 138519, WO 2010 / 130832, or WO 2008 / 020079.

[0315] The term "half-life," as used herein in connection with ISVD, nanobody, ISVD-based biologic, nanobody-based biologic, or any other amino acid sequence, compound, or peptide mentioned herein, may generally be defined according to the definition described in paragraph o) on page 57 of WO 2008 / 020079, and as mentioned therein, refers to the time taken for the serum concentration of an amino acid sequence, compound, or peptide to decrease by 50% in vivo, for example, due to degradation and / or sequestration of the sequence or compound caused by natural mechanisms. The in vivo half-life of the amino acid sequence, compound, or peptide of the present invention may be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may be, for example, generally as described in paragraph o) on page 57 of WO 2008 / 020079. As also mentioned in paragraph o) on page 57 of WO 2008 / 020079, the half-life may be expressed using parameters such as t1 / 2-α, t1 / 2-β, and area under the curve (AUC). In this regard, it should be noted that the term "half-life" as used herein specifically refers to t1 / 2-β or terminal half-life (where t1 / 2-α and / or AUC or both may be disregarded). See, for example, the experimental section below, and standard manuals such as Kenneth, A. et al., *Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists* and Peters et al., *Pharmacokinetic Analysis: A Practical Approach* (1996). Also see *Pharmacokinetics*, M. Gibaldi & D. Perron, by Marcel Dekker, 2nd revised edition (1982). Similarly, the terms “increase in half-life” or “increased half-life” are also defined as in paragraph o) on page 57 of WO 2008 / 020079, and specifically refer to an increase in t1 / 2-β, while t1 / 2-α and / or AUC or both increase or do not increase.

[0316] When a term is not specifically defined herein, it has its usual meaning in the art, which will be clear to a person skilled in the art. For example, refer to standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, New York, NY (1985); Old et al., “Principles of Gene Manipulation: An Introduction to Genetic Engineering”, 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., “Immunology” (6th edition), Mosby / Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential Immunology (Roitt Basic Immunology), 10th edition, Blackwell Publishing, UK (2001); and Janeway et al., Immunobiology (6th edition), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background techniques cited herein.

[0317] Furthermore, as already noted in this paper, the amino acid residues of the nanobodies are numbered according to the general numbering system for VH provided by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), such as that applied to the VHH domain of camels in the article Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240(1-2):185-195; or as mentioned in this paper. According to this numbering, FR1 of the nanobody contains amino acid residues at positions 1-30, CDR1 of the nanobody contains amino acid residues at positions 31-35, FR2 of the nanobody contains amino acid residues at positions 36-49, CDR2 of the nanobody contains amino acid residues at positions 50-65, FR3 of the nanobody contains amino acid residues at positions 66-94, CDR3 of the nanobody contains amino acid residues at positions 95-102, and FR4 of the nanobody contains amino acid residues at positions 103-113. [In this regard, it should be noted—as is known in the art regarding the VH domain and the VHH domain—that the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not occupy the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, generally, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence.] However, it is generally accepted that, based on the Kabat number and without considering the number of amino acid residues in the CDR, position 1 of the Kabat number corresponds to the start of FR1 and vice versa, position 36 of the Kabat number corresponds to the start of FR2 and vice versa, position 66 of the Kabat number corresponds to the start of FR3 and vice versa, and position 103 of the Kabat number corresponds to the start of FR4 and vice versa.

[0318] An alternative method for numbering the amino acid residues of the VH domain is the method described by Chothia et al. (Nature 342, 877-883 (1989)), namely the so-called "AbM definition" and the so-called "contact definition," which can also be applied in a similar manner to VHH domains from camelids and to nanobodies. However, in this specification, various aspects, and figures, the numbering of the VHH domains according to Kabat, as applied by Riechmann and Muyldermans, will be followed unless otherwise indicated.

[0319] It should also be noted that the accompanying drawings, any sequence listings, and experimental sections / exemplary embodiments are provided only for further illustrating the invention and should not be construed or understood as limiting the scope of the invention and / or the appended claims in any way, unless otherwise expressly stated herein.

[0320] As further described herein, the serum albumin conjugate of the present invention can be advantageously used as a part, binding unit, or fusion partner to increase the half-life of therapeutic parts such as polypeptides, proteins, compounds (including, but not limited to, small molecules) or other therapeutic entities.

[0321] Therefore, in another aspect, the present invention provides a serum albumin conjugate comprising the present invention and one or more other amino acid sequences, (binding) domains, binding units or other portions or chemical entities or substantially composed therefrom, including polypeptides, proteins, constructs, compounds or other chemical entities.

[0322] In particular, the present invention provides a polypeptide, protein, construct, compound, or other chemical entity comprising a serum albumin conjugate of the present invention suitably linked to each other directly or via one or more suitable linkers or spacers, and one or more (e.g., one or two) therapeutic portions (which may be the same or different, and may, for example, target the same or different targets, and when they target the same target, may target the same or different epitopes, sites, domains, or subunits of said targets). Such polypeptides, proteins, or constructs may, for example, and are not limited to, fusion proteins, as further described herein.

[0323] The present invention further relates to the therapeutic use of such polypeptides, proteins, constructs or compounds and to pharmaceutical compositions comprising such polypeptides, proteins, constructs or compounds.

[0324] In one aspect, at least one therapeutic portion comprises or is substantially composed of a therapeutic protein, peptide, compound, factor, or other entity. In a preferred embodiment, the therapeutic portion is directed against a desired antigen or target, capable of binding to the desired antigen (and particularly capable of specifically binding to the desired antigen), and / or capable of interacting with the desired target. In another embodiment, at least one therapeutic portion comprises or is substantially composed of a therapeutic protein or peptide. In yet another embodiment, at least one therapeutic portion comprises a binding domain or binding unit, such as an immunoglobulin or immunoglobulin sequence (including but not limited to fragments of immunoglobulins), such as an antibody or antibody fragment (including but not limited to ScFv fragments), or is substantially composed of, or is substantially composed of another suitable protein scaffold, such as a protein A domain (e.g., Affibodies). TM ), amylase aprotinin (tendamistat), fibronectin, lipid transport protein, CTLA-4, T cell receptor, designed ankyrin repeat sequence, high affinity multimer (avimer) and PDZ domain (Binz et al., Nat. Biotech 2005, Vol 23:1257) and DNA or RNA-based binding moieties, including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 20069(8):619-32).

[0325] In yet another aspect, at least one therapeutic component comprises, or is substantially composed of, an antibody variable domain, such as a heavy chain variable domain or a light chain variable domain.

[0326] In a preferred aspect, at least one therapeutic portion comprises, or is substantially composed of, at least one immunoglobulin monovariable domain, such as a domain antibody, a single-domain antibody, a “dAb” or nanobody (e.g., VHH, humanized VHH or camel-derived VH) or an IgNAR domain.

[0327] In a specific implementation, at least one therapeutic component comprises, or is substantially composed of, at least one monovalent nanobody or a bivalent, multivalent, bispecific, or multispecific nanobody construct.

[0328] Peptides, (fusion) proteins, constructs, or compounds comprising the serum albumin conjugate of the present invention and one or more therapeutic portions can generally be prepared and used as described in the prior art cited above (such as WO04 / 041865 and WO 06 / 122787), but the serum albumin conjugate of the present invention is used instead of the portion with increased half-life described in the prior art.

[0329] Compared to one or more therapeutic components on their own, polypeptides, (fusion) proteins, constructs, or compounds comprising the serum albumin conjugate of the present invention and one or more therapeutic components will generally and preferably have an increased half-life.

[0330] Typically, the constructs or fusion proteins described herein preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times greater than the half-life of the corresponding therapeutic part itself (as in humans or suitable animals such as mice or cynomolgus monkeys).

[0331] Furthermore, preferably, any such fusion protein or construct has an increased half-life in humans of more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, compared to the half-life of the corresponding therapeutic portion itself.

[0332] Furthermore, preferably, the half-life (defined as t1 / 2β) of any fusion protein or construct in humans is greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours, such as greater than 12 hours, and for example about one day, two days, one week, two weeks and up to the half-life of serum albumin in humans (estimated to be about 19 days), although the latter may be less important.

[0333] 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, due to ligand degradation and / or ligand clearance or chelation caused by natural mechanisms. In particular, half-life can be as defined in WO2009 / 068627.

[0334] Methods for pharmacokinetic analysis and determination of half-life are well known to those skilled in the art. Details can be found in Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Also see “Pharmacokinetics,” M. G. Babaldi & D. Perron, by Marcel Dekker, 2nd revised edition (1982).

[0335] As mentioned, in one aspect, the serum albumin conjugate of the present invention can be used to increase the half-life of (one or more) immunoglobulin monovariable domains such as domain antibodies, single-domain antibodies, "dAb", VHH, or nanobodies (such as VHH, humanized VHH, or camel-derived VH such as camel-derived human VH).

[0336] Therefore, one embodiment of the invention relates to a polypeptide, construct, or fusion protein comprising a serum albumin conjugate of the invention suitably linked to each other directly or optionally via one or more suitable adapters or spacers, and one or more (e.g., one or two) immunoglobulin monovariable domain sequences. As mentioned herein, each such immunoglobulin monovariable domain present in such polypeptides, constructs, or fusion proteins can independently be a domain antibody, a single-domain antibody, a “dAb”, or a nanobody (e.g., VHH, humanized VHH, or camel-derived VH, such as camel-derived human VH); and according to a specific, but non-limiting aspect, at least one (and up to all) of these immunoglobulin monovariable domains comprises two or three disulfide bonds.

[0337] As mentioned, when the construct of the polypeptide or fusion protein has a heavy chain ISVD (the IVSD can be the serum albumin conjugate of the present invention or an ISVD targeting a therapeutic target, such as a nanobody targeting a therapeutic target) at its C-terminus, the construct of the polypeptide or fusion protein (the ISVD present at its C-terminus) preferably has a C-terminal extension. Similarly, the C-terminal extension will have the form of formula (X). n , where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an amino acid residue independently selected from naturally occurring amino acid residues (although according to a preferred aspect, it does not contain any cysteine ​​residues), and preferably an amino acid residue independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (preferably naturally occurring).

[0338] As mentioned, when the construct of the polypeptide or fusion protein has a heavy chain ISVD at its N-terminus (the IVSD may be the serum albumin conjugate of the present invention or an ISVD for a therapeutic target, such as a nanobody for a therapeutic target), the construct of the polypeptide or fusion protein (the ISVD present at its C-terminus) preferably has a D or E1D mutation at position 1.

[0339] Therefore, in another aspect, the present invention relates to proteins, polypeptides, or other compounds, wherein the proteins, polypeptides, or other compounds are:

[0340] - Contains at least one (and preferably only one) serum albumin conjugate of the present invention and at least one (such as one, two or three) therapeutic portion or entity ( wherein the serum albumin conjugate and one or more therapeutic portions or entities are optionally suitably connected by one or more suitable connectors);

[0341] - It has a heavy chain ISVD at its C-terminus, wherein the ISVD at the C-terminus has a C-terminal extension (X). n (as further described herein);

[0342] The protein, polypeptide or other compound may also have a heavy chain ISVD at its N-terminal end, in which case the N-terminal ISVD preferably has a D or E1D at position 1.

[0343] Furthermore, in a preferred aspect, when one or more other ISVDs are present in addition to the serum albumin conjugate of the present invention (i.e., when one or more of the present therapeutic portions are ISVDs), the “therapeutic” ISVDs (one or more or all of them) preferably also have a combination of amino acid residues / mutations that reduce the binding of pre-existing antibodies. When the ISVD is a heavy chain ISVD, these mutations can be as described in PCT / EP2015 / 060643, and in particular, can be suitable combinations of one or more mutations at positions 11, 89, 110, and 112 of substantially the same kind as those described herein with respect to the serum albumin conjugate of the present invention. Preferably, if such other ISVDs are present at the C-terminus, then at least the therapeutic ISVD contains such mutations at positions 11, 89, 110, and / or 112 (i.e., in addition to the C-terminal extension as described herein).

[0344] According to a specific aspect, all therapeutic portions present in constructs, fusion proteins, or peptides are ISVDs (i.e., ISVDs targeting therapeutic targets), and especially heavy chain ISVDs, and even more so nanobodies (i.e., nanobodies targeting therapeutic targets).

[0345] For example, and without limitation, a construct, fusion protein, or polypeptide comprising the serum albumin conjugate of the present invention may include:

[0346] -A copy of the serum albumin conjugate of the present invention and an ISVD (and preferably a nanobody) targeting a therapeutic target; or

[0347] - The serum albumin conjugate of the present invention and a copy of two ISVDs (and preferably two nanobodies) targeting a therapeutic target (the ISVDs may be the same or different, and when different, may target the same target, different epitopes on the same target, or different therapeutic targets); or

[0348] - The serum albumin conjugate of the present invention and a copy of three ISVDs (and preferably three nanobodies) targeting therapeutic targets (the ISVDs may be the same or different, and when different, they may target the same target, different epitopes on the same target, or different therapeutic targets).

[0349] Some non-limiting examples of the constructs, fusion proteins, or peptides of the present invention can be schematically represented as follows, wherein “[Alb]” represents the serum albumin conjugate of the present invention, “[Therapeutic part 1]” and “[Therapeutic part 2]” represent therapeutic parts (which, as mentioned, may each be an immunoglobulin monovariable domain independently), “-” represents a suitable linker (which is optional; suitable examples are 9GS and 35GS linkers), and the N-terminus is on the left-hand side and the C-terminus is on the right-hand side:

[0350] [Alb] - [Therapeutic Part 1]

[0351] [Therapeutic Part 1]-[Alb]-X (n)

[0352] [Alb]-[Healing Part 1]-[Healing Part 1]

[0353] [Therapeutic Part 1]-[Therapeutic Part 1]-[Alb]-X (n)

[0354] [Healing Part 1] - [Alb] - [Healing Part 1]

[0355] [Alb]-[Healing Part 1]-[Healing Part 2]

[0356] [Therapeutic Part 1]-[Therapeutic Part 2]-[Alb]-X (n)

[0357] [Healing Section 1] - [Alb] - [Healing Section 2]

[0358] When the therapeutic portion is an ISVD (and preferably a nanobody) targeting a therapeutic target, preferred, but non-limiting constructs, fusion proteins, or peptides of the present invention can be schematically represented as follows, wherein “[Alb]” represents the serum albumin conjugate of the present invention, “[Therapeutic ISVD 1]” and “[Therapeutic ISVD 2]” represent ISVDs targeting a therapeutic target (the ISVDs may be the same or different, and when different, may target the same target, different epitopes on the same target, or different therapeutic targets), “-” represents a suitable linker (which is optional), X(n) represents a C-terminal extension as described herein, with the N-terminus on the left-hand side and the C-terminus on the right-hand side:

[0359] [Alb]-[Therapeutic ISVD 1]-X (n)

[0360] [Therapeutic ISVD 1]-[Alb]-X (n)

[0361] [Alb]-[Therapeutic ISVD 1]-[Therapeutic ISVD 1]-X (n)

[0362] [Therapeutic ISVD 1]-[Therapeutic ISVD 1]-[Alb]-X (n)

[0363] [Therapeutic ISVD 1]-[Alb]-[Therapeutic ISVD 1]-X (n)

[0364] [Alb]-[Therapeutic ISVD 1]-[Therapeutic ISVD 2]-X (n)

[0365] [Therapeutic ISVD 1]-[Therapeutic ISVD 2]-[Alb]-X (n)

[0366] [Therapeutic ISVD 1]-[Alb]-[Therapeutic ISVD 2]-X (n)

[0367] Therefore, in another aspect, the present invention relates to a multispecific (and especially bispecific) nanobody construct comprising the serum albumin conjugate of the present invention and at least one other nanobody (such as one or two other nanobodies that may be the same or different), wherein the at least one other nanobody is preferably targeted at a desired target (which is preferably a therapeutic target) and / or another nanobody is available or suitable for therapeutic, preventive and / or diagnostic purposes. Similarly, the serum albumin conjugate and other nanobodies of the present invention can be directly or optionally suitably linked to each other via one or more suitable linkers or spacers.

[0368] For a general description of multivalent and multispecific polypeptides containing one or more nanobodies and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology, 74 (2001), 277-302; and see, for example, WO 96 / 34103, WO 99 / 23221, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO2008 / 142164 or WO 2009 / 068627.

[0369] Other examples of specific multispecific and / or multivalent peptides of the present invention can be found in the applications of Eberlinx Inc. mentioned herein. In particular, for a general description of multivalent and multispecific constructs for increasing half-life comprising at least one nanobody targeting a serum protein, the nucleic acid encoding therein, compositions comprising therein, preparation of the foregoing, and use of the foregoing, reference is made to the aforementioned international applications WO 04 / 041865 and WO 06 / 122787 (the serum albumin conjugates of the present invention described herein can generally be used similarly to the extended half-life nanobodies such as Alb-8 described therein), and to the general descriptions and specific examples of such constructs given in, for example, WO 04 / 041862, WO 2006 / 122786, WO2008 / 020079, WO 2008 / 142164 or WO 2009 / 068627.

[0370] In one aspect, the present invention relates to proteins, peptides, or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy-chain ISVDs (e.g., nanobodies comprising or derived from the VH domain or (single) domain antibodies), wherein the serum albumin conjugate and the one or more additional heavy-chain ISVDs each contain the following amino acid residues:

[0371] - The amino acid residue at position 11 is preferably selected from L or V; and

[0372] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0373] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0374] - The amino acid residue at position 112 is preferably selected from S, K or Q;

[0375] So that (i) position 89 is T; or (ii) position 89 is L and position 11 is V; or (iii) position 89 is L and position 110 is K or Q; or (iv) position 89 is L and position 112 is K or Q; or (v) position 89 is L and position 11 is V and position 110 is K or Q; or (vi) position 89 is L and position 11 is V and position 112 is K or Q; or (vii) position 11 is V and position 110 is K or Q; or (vii) position 11 is V and position 112 is K or Q.

[0376] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0377] -89T; or

[0378] - 89L combined with 11V; or

[0379] - 89L combined with 110K or 110Q; or

[0380] - 89L combined with 112K or 112Q; or

[0381] - 89L in combination with 11V and 110K or 110Q; or

[0382] - 89L in combination with 11V and 112K or 112Q; or

[0383] - 11V in combination with 110K or 110Q; or

[0384] - 11V in combination with 112K or 112Q.

[0385] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0386] - The amino acid residue at position 11 is preferably selected from L or V; and

[0387] - The amino acid residue at position 89 is T; and

[0388] - The amino acid residue at position 110 is preferably selected from T, K, or Q (and preferably T); and

[0389] - The amino acid residue at position 112 is preferably selected from S, K or Q (and preferably S).

[0390] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0391] - The amino acid residue at position 11 is V; and

[0392] - The amino acid residue at position 89 is L; and

[0393] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0394] - The amino acid residue at position 112 is preferably selected from S, K or Q.

[0395] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0396] - 11V combined with 89L; or

[0397] - 11V in combination with 110K or 110Q;

[0398] - 11V in combination with 112K or 112Q;

[0399] - 11V in combination with 89L and 110K or 110Q; or

[0400] - 11V in combination with 89L and 112K or 112Q.

[0401] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0402] - 89L combined with 11V; or

[0403] - 89L combined with 110K or 110Q; or

[0404] - 89L combined with 112K or 112Q; or

[0405] - 89L in combination with 11V and 110K or 110Q; or

[0406] - 89L in combination with 11V and 112K or 112Q.

[0407] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0408] - 110K or 110Q combined with 11V; or

[0409] - 110K or 110Q combined with 89L; or

[0410] - 110K or 110Q combined with 11V and 89L.

[0411] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain the following amino acid residues:

[0412] - 112K or 112Q combined with 11V; or

[0413] - 112K or 112Q combined with 89L; or

[0414] - 112K or 112Q in combination with 11V and 89L.

[0415] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs both contain a T at position 89.

[0416] In another aspect, the present invention relates to proteins, polypeptides or other compounds or constructs (and preferably fusion proteins) comprising the serum albumin conjugate of the present invention and one or more additional heavy chain ISVDs, wherein the serum albumin conjugate and the one or more additional heavy chain ISVDs each contain V at position 11 and L at position 89.

[0417] Similarly, all these polypeptides preferably contain a C-terminal extension X(n) (as described herein) and a D at position 1, and may contain serum albumin-binding ISVD, as further described herein. They also have a half-life as further described herein.

[0418] This invention also relates to nucleotide sequences or nucleic acids encoding albumin conjugates, compounds, or polypeptides of the present invention. The invention also includes genetic constructs comprising the aforementioned nucleotide sequences or nucleic acids and one or more elements known per se for use in genetic constructs. Genetic constructs may be in the form of plasmids or vectors. Similarly, such constructs may be typically as described in the published patent applications of Ebolings Inc., such as, for example, WO 04 / 041862, WO2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0419] The present invention also relates to a host or host cell containing such nucleotide sequences or nucleic acids and / or expressing (or being able to express) the albumin conjugate, compound, or polypeptide of the present invention. Similarly, such host cells can generally be as described in the published patent applications of Eberlinx Inc., such as, for example, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0420] The present invention also relates to a method for preparing the albumin conjugate, compound, or polypeptide of the present invention, said method comprising culturing or maintaining host cells as described herein under conditions that cause said host cells to produce or express the albumin conjugate, compound, or polypeptide of the present invention, and optionally further comprising isolating the albumin conjugate, compound, or polypeptide of the present invention thus generated. Similarly, such methods can generally be carried out according to the patent applications published by Eberlinx Inc., such as, for example, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0421] The present invention also relates to pharmaceutical compositions comprising at least one compound or polypeptide of the present invention and optionally at least one pharmaceutical carrier, diluent, or excipient. Such formulations, carriers, excipients, and diluents may generally be as described in the published patent applications of Ebolings Inc., such as, for example, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0422] However, because the compounds or peptides of the present invention have an increased half-life, they are preferably administered into circulation. Therefore, they can be administered in any suitable manner that allows the compounds or peptides of the present invention to enter circulation, such as intravenous, by injection or infusion, or in any other suitable manner (including oral, subcutaneous, intramuscular, transdermal, intranasal, transpulmonary, etc.). Similarly, suitable methods and routes of administration will be apparent to those skilled in the art, for example, based on the teachings of published patent applications by Ebolings Inc., such as, for example, WO 04 / 041862, WO 2006 / 122786, WO 2008 / 020079, WO 2008 / 142164, or WO 2009 / 068627.

[0423] Therefore, in another aspect, the present invention relates to a method for preventing and / or treating at least one disease or condition that can be prevented or treated by using a compound or polypeptide of the present invention, said method comprising administering to a subject in need of it a pharmaceutically active amount of the compound or polypeptide of the present invention and / or a pharmaceutical composition comprising thereto. Diseases and conditions that can be prevented or treated by using a compound or polypeptide of the present invention as described herein will generally be the same as diseases and conditions that can be prevented or treated by using one or more therapeutic portions present in a compound or polypeptide of the present invention.

[0424] In the context of this invention, the term "prevention and / or treatment" includes not only the prevention and / or treatment of disease, but also generally includes preventing the onset of disease, delaying or reversing the progression of disease, preventing or delaying the onset of one or more symptoms associated with disease, reducing and / or alleviating one or more symptoms associated with disease, reducing the severity and / or duration of disease and / or any symptoms associated with it and / or preventing further increase in the severity of disease and / or any symptoms associated with it, preventing, reducing or reversing any physiological damage caused by disease, and any pharmacological effects that are generally beneficial to the patient being treated.

[0425] The subjects to be treated can be any warm-blooded animal, but especially mammals, and even more so humans. As will be clear to the technicians, the subjects to be treated will especially be those suffering from or at risk of the diseases and conditions mentioned herein.

[0426] In another embodiment, the present invention relates to a method for immunotherapy, and in particular for passive immunotherapy, the method comprising administering a pharmaceutically active amount of a compound or polypeptide of the present invention and / or a pharmaceutical composition comprising the present invention to a subject suffering from or at risk of any of the diseases and conditions mentioned herein.

[0427] The compounds or peptides of the present invention and / or compositions comprising them are administered according to a treatment regimen suitable for the prevention and / or treatment of a disease or condition to be prevented or treated. Clinicians will generally be able to determine a suitable treatment regimen based on various factors, such as the disease or condition to be prevented or treated, the severity of the disease to be treated and / or the severity of its symptoms, the specific peptide of the present invention to be used, the specific route of administration and pharmaceutical preparation or composition to be used, age, sex, weight, diet, the patient's general condition, and similar factors well known to clinicians.

[0428] Typically, the treatment regimen will involve administering one or more pharmaceutically effective amounts or doses of one or more compounds or peptides of the present invention, or one or more compositions comprising them. The specific amount or dose to be administered can be determined again by the clinician based on the factors cited above.

[0429] Typically, for the prevention and / or treatment of the diseases and conditions mentioned herein, and depending on the specific disease or condition to be treated, the potency and / or half-life of the compound or peptide of the present invention to be used, the specific route of administration, and the specific pharmaceutical formulation or composition, the compound or peptide of the present invention will generally be administered continuously throughout the day (e.g., by infusion), either as a single daily dose or as multiple fractions, in an amount between 1 gram and 0.01 micrograms / kg body weight / day, preferably between 0.1 gram and 0.1 micrograms / kg body weight / day, such as about 1, 10, 100, or 1000 micrograms / kg body weight / day. Clinicians will generally be able to determine the appropriate daily dose based on the factors mentioned herein. It will also be clear that, in specific cases, clinicians may choose amounts different from these, for example, based on the factors cited above and their professional judgment. Typically, some guidelines for the amount to be applied can be derived from the amounts typically applied to comparable conventional antibodies or antibody fragments targeting the same target via substantially the same route, but differences in affinity / antibody tropism, efficacy, biodistribution, half-life, and similar factors known to the technician must be taken into account.

[0430] Furthermore, because the compounds of the present invention contain the extended half-life serum albumin conjugates of the present invention, they do not need to be administered substantially continuously (e.g., by infusion), but they can be administered at suitable intervals (determined by a person skilled in the art). For example, they can be administered, for instance, by injection or infusion, once every two days (at a suitable dose), once every four days, once weekly, once every two weeks, and in some cases, once every four weeks, or even less frequently.

[0431] One aspect of the invention relates to a pharmaceutical composition comprising at least one compound or polypeptide of the invention, wherein the composition is intended for administration at intervals between once a week and once every four weeks, and particularly between once every seven days and once every 21 days, such as once every seven days or 14 days.

[0432] Typically, the above methods will use a single polypeptide of the present invention. However, the use of two or more polypeptides of the present invention in combination is also within the scope of the present invention.

[0433] The polypeptides of the present invention can also be used in combination with one or more other pharmaceutically active compounds or ingredients, i.e., as a combination therapy, which may or may not induce a synergistic effect. Similarly, clinicians will be able to select such additional compounds or ingredients, and suitable combination therapy regimens, based on the factors cited above and their professional judgment.

[0434] In particular, the polypeptides of the present invention can be used in combination with other pharmaceutically active compounds or ingredients that are used for or can be used to prevent and / or treat diseases and conditions that can be prevented or treated by the fusion proteins or constructs of the present invention, and as a result, a synergistic effect may or may not be obtained.

[0435] As clinicians will understand, the effectiveness of the treatment regimens used according to the present invention can be determined and / or tracked in any manner known to the disease or condition in question. Clinicians will also be able to modify or improve specific treatment regimens where appropriate and / or on a case-by-case basis to achieve the desired therapeutic effect, to avoid, limit, or reduce undesirable side effects, and / or to strike an appropriate balance between achieving the desired therapeutic effect on one hand and avoiding, limiting, or mitigating undesirable side effects on the other.

[0436] Typically, the treatment plan will be monitored until the desired therapeutic effect is achieved and / or simply to maintain the desired therapeutic effect. This, too, can be determined by the clinician.

[0437] The subjects to be treated can be any warm-blooded animal, especially mammals, and even more so humans. As will be clear to those skilled in the art, the subjects to be treated will especially be those suffering from or at risk of the diseases and conditions mentioned herein.

[0438] Other aspects, embodiments, advantages, and applications of the invention will become clear from the further description herein.

[0439] The invention will now be further described with reference to the following non-limiting preferred aspects, embodiments, and accompanying drawings, wherein:

[0440] - Figure 1 This is a table listing some of the amino acid positions that will be specifically mentioned in this article and their numbers according to some alternative numbering systems (such as Aho and IMGT);

[0441] - Figure 2 The alignment of the reference sequence mentioned in this paper is shown.

[0442] - Figure 3 The amino acid sequences mentioned in this article are listed;

[0443] - Figure 4Two corresponding graphs are shown showing the data points obtained in Example 1 when 96 serum samples from healthy human subjects were tested for binding to Reference A and two representative variants of Reference A according to the invention (i.e., [Reference A+L11V+V89L+C-terminal alanine] and [Reference A+L11V+V89L+T110K+C-terminal alanine], respectively). Each point represents the binding level of one of the 96 samples tested. The data points shown in the right and left graphs are the same; in the right graph, the data points measured for each of the three tested compounds (i.e., Reference A; Reference A+L11V+V89L+114A; and Reference A+L11V+V89L+T110K+114A) for each individual sample are connected by lines (as a result, the slant of the lines provides an indication of the degree of reduction in binding of the pre-existing antibody when the mutation of the invention and C-terminal alanine are introduced);

[0444] - Figure 5 It is listed in Figure 4 The data points compiled in the table contain the binding data (in three columns, the normalized PreAb binding level (RU at 700) is provided, and in three columns, the percentage reduction in PreAb binding compared to the reference compound used is provided).

[0445] - Figure 6 Two corresponding graphs are shown in Example 2 of data points obtained when 96 serum samples from healthy human subjects were tested for binding to Reference B and two representative variants of Reference B according to the invention (i.e., [Reference B+L11V+V89L+C-terminal alanine] and [Reference B+L11V+V89L+T110K+C-terminal alanine], respectively). Each point represents the binding level of one of the 96 samples tested. The data points shown in the right and left graphs are the same; in the right graph, the data points measured for each of the three tested compounds (i.e., Reference B; Reference B+L11V+V89L+114A; and Reference B+L11V+V89L+T110K+114A) for each individual sample are connected by lines (as a result, the slant of the lines provides an indication of the degree of reduction in binding of the pre-existing antibody when the mutation of the invention and C-terminal alanine are introduced);

[0446] - Figure 7 It is listed in Figure 6 The data points compiled in the table contain the binding data (in three columns, the normalized PreAb binding level (RU at 700) is provided, and in two columns, the percentage reduction in PreAb binding compared to the reference compound used is provided).

[0447] - Figure 8Two corresponding graphs are shown in Example 3 of data points obtained when testing 96 serum samples (66 from healthy human subjects and 30 from subjects presumed to contain pre-existing antibodies that can bind in the presence of C-terminal alanine, including 13 samples from SLE patients) for binding to reference C, reference D, and two representative variants of reference D according to the invention (i.e., [reference D+L11V+V89L] and [reference D+L11V+V89L+T110K], respectively). Each point represents the binding level of one of the 96 samples tested. The data points shown in the right and left graphs are the same; in the right graph, the data points measured for each of the four tested compounds (i.e., reference C; reference D; reference D+L11V+V89L; and reference D+L11V+V89L+T110K) for each individual sample are connected by lines (as a result, the slant of the lines provides an indication of the degree to which binding of the pre-existing antibody is reduced when the mutation of the invention and C-terminal alanine are introduced);

[0448] - Figure 9 A graph is shown for four data points obtained in the SLE samples tested in Example 3. The data points measured for individual samples (i.e., “SLE 25”, “SLE 37”, “SLE 39”, and “SLE 41”, respectively) are connected by lines (as a result, the slant of each line provides an indication of the degree of reduction in binding of the pre-existing antibody in each sample when the mutation of the present invention is introduced);

[0449] - Figure 10 It is listed in Figure 8 The data points compiled in the table contain the binding data (individually, columns 4 provide the normalized PreAb binding level (RU at 700) and columns 3 provide the percentage reduction in PreAb binding compared to the reference compound used).

[0450] Experimental Section

[0451] The human samples used in the following experimental sections were obtained from commercial sources or from human volunteers (after obtaining all necessary permission or approval) and used in accordance with applicable laws and regulations (including, but not limited to, those concerning medical secrets and patient privacy).

[0452] In the following embodiments, unless otherwise explicitly stated, ProteOn is used to determine the binding of pre-existing antibodies present in the samples used (i.e., from healthy volunteers, patients with rheumatoid arthritis (RA), and SLE patients) to the tested nanobodies:

[0453] Nanobodies were captured on serum albumin using monoclonal anti-FLAG M2 or via FLAG3 tagging.

[0454] In cases where pre-existing antibodies bind to nanobodies captured on human serum albumin (HSA), evaluation was performed using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) was used as the run buffer, and experiments were conducted at 25°C. The ligand channels of the ProteOn GLC sensor chip were activated with EDC / NHS (30 μl / min) and HSA was injected at 10 μg / ml in ProteOn acetate buffer pH 4.5 (100 μl / min) to provide a fixation level of approximately 3200 RU. After fixation, the surface was inactivated with ethanolamine HCl (30 μl / min). Nanobodies were injected onto the HSA surface at 45 μl / min over 2 min to provide a nanobodies capture level of approximately 200 RU. Samples containing pre-existing antibodies were centrifuged at 14,000 rpm for 2 minutes, and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%) and injected at 45 μl / min over 2 minutes, followed by a subsequent 400-second dissociation step. After each cycle (i.e., before a new nanobody capture and blood sample injection step), the HSA surface was regenerated by injecting HCl (100 mM) at 45 μl / min for 2 minutes. Sensing map processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensing maps showing the binding of the pre-existing antibody were obtained after double referencing by subtracting 1) nanobody-HSA dissociation and 2) non-specific binding to the reference ligand pathway. The binding level of the pre-existing antibody was determined by setting a reporter point at 125 seconds (5 seconds after the end of binding). The percentage decrease in binding of the pre-existing antibody relative to the binding level of the reference nanobody at 125 seconds was calculated.

[0455] Evaluation was performed using ProteOn XPR36 (Bio-Rad Laboratories, Inc.) in cases where a pre-existing antibody bound to a FLAG-labeled nanobody on a monoclonal anti-FLAG M2 (Sigma) nanobody. Experiments were conducted using PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) as the run buffer and at 25°C. The ligand channels of the ProteOn GLC sensor chip were activated with EDC / NHS (30 μl / min) and anti-FLAG M2 mAb was injected at 10 μg / ml in ProteOn acetate buffer pH 4.5 (100 μl / min) to provide a fixation level of approximately 4000 RU. After fixation, the surface was inactivated with ethanolamine HCl (30 μl / min). The nanobody was injected onto the anti-FLAG M2 surface at 45 μl / min over 2 min to provide a nanobody capture level of approximately 100 RU. To reduce nonspecific binding of blood samples to the anti-FLAG M2 surface, 100 nM 3xFLAG peptide (Sigma) was added to the blood sample. The sample containing the pre-existing antibody was centrifuged at 14,000 rpm for 2 min, and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%) and injected at 45 μl / min over 2 min, followed by a subsequent 600-second dissociation step. After each cycle (i.e., before a new nanobody capture and blood sample injection step), the anti-FLAG M2 surface was regenerated by injecting 150 μl / min with glycine at pH 1.5 (10 mM) for 10 seconds. Sensing image processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). After double referencing by subtracting 1) dissociation of the nanobody-anti-FLAG M2 and 2) nonspecific binding to the reference ligand pathway, a sensor map showing the binding of the pre-existing antibody was obtained. The binding level of the pre-existing antibody was determined by setting a reporter point at 125 seconds (5 seconds after binding ended). The percentage decrease in binding of the pre-existing antibody relative to the binding level of the reference nanobody at 125 seconds was calculated.

[0456] Example 1: Introducing the mutation of the present invention into reference A (SEQ ID NO:1) causes the formation of a pre-existing antibody. The decrease in the combination.

[0457] Binding to pre-existing antibodies present in 96 serum samples from healthy volunteers was tested using reference A (SEQ ID NO: 1) and two representative examples of modified variants of reference A carrying mutations according to the invention (SEQ ID NO: 37 and 38, both with alanine extension and tested with an N-terminal HIS6-FLAG3 tag (see SEQ ID NO: 100). Compounds were captured using a FLAG tag, and binding was measured using ProteOn according to the protocol given in the preface of this experimental section.

[0458] The result is Figure 4 As shown in the image. Figure 5 List of components Figure 4 The result for each sample from one of the data points.

[0459] It can be seen that for most of the 96 samples tested, the introduction of the mutation according to the invention caused a reduction in the binding of the pre-existing antibody, and the extent of the reduction generally depended on the level at which the pre-existing antibody in each sample was able to bind to reference A.

[0460] Example 2: Introducing the mutation of the present invention into reference B (SEQ ID NO:2) causes the formation of a pre-existing antibody. The decrease in the combination.

[0461] Binding to pre-existing antibodies present in 96 serum samples from healthy human volunteers was tested using reference B (SEQ ID NO:2) and two representative examples of modified variants of reference B carrying mutations according to the invention (SEQ ID NO:65 and 66, both with alanine extension and tested with an N-terminal HIS6-FLAG3 tag (see SEQ ID NO:100)). Compounds were captured using a FLAG tag, and binding was measured using ProteOn according to the protocol given in the preface of this experimental section.

[0462] The result is Figure 6 As shown in the image. Figure 7 List of components Figure 6 The result for each sample from one of the data points.

[0463] Similar to Example 1, it can be seen that for most of the 96 samples tested, the introduction of the mutation according to the invention caused a reduction in the binding of the pre-existing antibody, and the extent of the reduction generally depended on the level at which the pre-existing antibody in each sample was able to bind to reference B.

[0464] Example 3: Introducing the mutation index of the present invention in references C (SEQ ID NO:3) and D (SEQ ID NO:4) The binding of pre-existing antibodies is reduced.

[0465] Binding of the pre-existing antibodies in 66 serum samples from healthy volunteers and 30 samples (13 of which were from SLE patients) presumed to contain antibodies that could bind even in the presence of C-terminal alanine was tested against reference C (SEQ ID NO:3), reference D (SEQ ID NO:4), and two representative examples of modified variants of reference C and reference D carrying mutations according to the invention (SEQ ID NO:93 and 94, both having the alanine extension as present in reference D and being tested with an N-terminal HIS6-FLAG3 tag (see SEQ ID NO:100)). The compounds were captured on human serum albumin and binding was measured using ProteOn according to the protocol given in the preface of this experimental section.

[0466] The result is Figure 8 As shown in [the image]. Figure 9 The article provides details of four representative SLE samples. Figure 10 List of components Figure 8 The result for each sample from one of the data points.

[0467] Similar to Examples 1 and 2, it can be seen that for most of the 96 samples tested, the introduction of the mutation according to the invention caused a reduction in the binding of the pre-existing antibody for the vast majority of the samples, and the extent of the reduction generally depended on the level at which the pre-existing antibody in each sample was able to bind to reference C or reference D.

[0468] All references cited throughout this application (including bibliographic references, granted patents, published patent applications, and co-pending patent applications) are expressly linked by reference, especially for the teachings cited above.

Claims

1. An immunoglobulin monovariable domain that binds to human serum albumin, said immunoglobulin monovariable domain having an amino acid sequence of one of the following sequences: SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:93 or SEQ ID NO:94.

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