Improved immunoglobulin variable domains

Abstract

a VH domain, wherein: (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and wherein the VH domain comprises a C-terminal extension (X) n wherein 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 (preferably naturally occurring) amino acid residue, which is independently selected, and preferably independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

Description

[0001] This invention relates to an improved variable domain of heavy chain immunoglobulins.

[0002] This invention particularly relates to such modified heavy chain immunoglobulin variable domains having exposed C-terminal regions or ends (as further described herein; see also WO 12 / 175741) or used (or intended for) such applications, wherein they have exposed C-terminal regions or ends (again, as further described herein). Some preferred, non-limiting examples of the former are immunoglobulin single variable domains (also referred to herein as “ISV” or “ISVD”) such as nanobodies (including VHH, humanized VHH, and camel-derived VH such as camel-derived human VH), antibodies as VH domains or derived from VH domains (single-domain) antibodies, and dAbs as VH domains or derived from VH domains. Some preferred, non-limiting examples of the latter are VH domains used (or intended for) single-chain FV (ScFv) or biantibodies.

[0003] This invention also relates to proteins, peptides, and other constructs, molecules, or chemical entities comprising or substantially composed of one or more of the improved heavy chain immunoglobulin variable domains of the present invention as described herein; to methods for expressing / preparing the improved 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; to compositions and products (e.g., pharmaceutical compositions and products) comprising the improved heavy chain immunoglobulin variable domains of the present invention and / or containing proteins, peptides, and other constructs, molecules, or chemical entities containing the improved heavy chain immunoglobulin variable domains of the present invention; to nucleotide sequences and nucleic acids encoding the improved heavy chain immunoglobulin variable domains of the present invention and / or encoding proteins or peptides comprising the improved heavy chain immunoglobulin variable domains of the present invention; and to uses of the improved heavy chain immunoglobulin variable domains of the present invention and proteins, peptides, and other constructs, molecules, or chemical entities comprising thereof (and particularly therapeutic, preventive, and diagnostic uses).

[0004] Further aspects, embodiments, advantages, applications, and uses of the invention will become clear from the further description herein.

[0005] In this application, the amino acid residues / positions in the variable domains of the immunoglobulin heavy chain will be indicated according to the Kabat number. For convenience, Figure 1A table is provided listing the positions of some amino acids that will be specifically mentioned herein and their numbers according to several alternative numbering systems (such as Aho and IMGT. Note: Kabat numbering is definitive for the specification and claims of this application; other numbering systems are provided for reference only).

[0006] Furthermore, in this invention, when the variable domain of an immunoglobulin is not associated with a constant domain (such as C...), H When a structural domain is combined or connected, it is said to have an "exposed C-terminal end or region". Refer to the relevant prior art cited herein.

[0007] Specifically, as described in WO 12 / 175741, the C-terminal region (when the term is used herein) is part of a putative epitope on the ISV, which, among other residues, includes an amino acid residue at position 14 (and adjacent / near amino acid residues in the amino acid sequence, such as positions 11, 13, and 15) and may also include an amino acid residue at position 83 (and adjacent / near amino acid residues in the amino acid sequence, such as positions 82, 82a, 82b, and 84) and / or an amino acid residue at position 108 (and adjacent / near amino acid residues in the amino acid sequence, such as position 107). As in WO 12 / 17574, this putative epitope is also referred to herein as the “C-terminal region”. It is to be understood that the C-terminal region includes at least the C-terminal sequence VTVSS (i.e., each of positions 109, 110, 111, 112, and 113) and amino acid residues at position 14, and may also include amino acid residues at positions 83 and 108, and may also include amino acid residues at positions 13, 15, 82b, 83, 84, and 107.

[0008] As a result of studies on single-chain Fv or "ScFv" (which are constructs containing immunoglobulin variable domains that, similar to ISVD, do not bind to a constant domain), it has been described in the art that the C-terminus of an immunoglobulin variable domain contains a hydrophobic patch that is embedded in the interface between the variable and constant domains in a conventional full-size antibody but is exposed to a solvent when the variable domain does not bind to the constant domain (see, for example, Nieba et al., Protein Engineering, 10, 435-444 (1997) and Harmsen et al., Molecular Immunology (2000), 579-590).

[0009] It is also known that epitopes (also referred to as “neo-epitopes” or “cryptic epitopes”) that are typically embedded within protein structures can trigger the immune system upon exposure to solvents (e.g., due to degradation, misfolding, or aggregation of the protein involved). For example, in the case of embedded hydrophobic portions of biomolecules (so-called “hyppos”), it has been suggested that upon exposure to solvents, the hydrophobic portions form part of a molecular pattern associated with overall damage leading to an innate immune system response (see, for example, Seong and Matzinger, Nature Reviews 2004, 469), and numerous examples of previously embedded hydrophobic sheets triggering an immune response have been described in the art (see, for example, David et al., JBC, 2001, 6370-6377; Matsuura et al., International Immunology, 2000, 1183-1192; Rasheed et al., Life Sciences 79 (2000), 2320-2328). More generally, it is also known in the art that hydrophobic amino acids tend to be part of B-cell epitopes (see, for example, WO 11 / 07586, page 10; and Kolaskar, FEBS 276, 172-174 (1990)). Similarly, it has been described that hydrophobic sheets at the C-terminus of the heavy chain variable domain (as described above by Nieba et al. and Harmsen et al.) can form B-cell epitopes that can induce and / or interact with (new and / or pre-existing) anti-drug antibodies (WO 11 / 07586). For this reason, it has been proposed to mutate some of the amino acid residues forming the C-terminal portion of the variable domain to reduce hydrophobicity and / or remove the B-cell epitope. For example, Nieba et al. proposed mutation positions 11, 14, 41, 84, 87, and / or 89 of the VH region (according to Kabat numbering), while WO 11 / 07586 suggests mutation positions 99, 101, and / or 148 of the VL domain (AHo numbering) or positions 12, 97, 98, 99, 103, and / or 144 of the VH domain (again, AHo numbering—these positions correspond to positions 11, 83, 84, 85, 89, and 103 according to Kabat). Similarly, Harmsen et al. suggested mutation positions 12 and 101 (IMGT numbering; these are positions 11 and 89 according to Kabat) to compensate for C. H The absence of the 1 domain; and it also identified a specific subfamily of VHH (referred to as "VHH4") containing amino acids that are suitable candidates for substitution at these positions.

[0010] Prior art (see, for example, WO 12 / 175741 and the references cited in the following paragraphs) has described how biological samples obtained from human subjects may contain (original) proteins or factors capable of binding to exposed C-terminal regions or ends of immunoglobulin variable domains (e.g., C-terminal regions or ends of VH or VL domains in ISVD or ScFv or biantibodies).

[0011] For example, WO 2013 / 024059 describes “the presence of pre-existing anti-VH autoantibodies that can bind to VH domain antibodies and VHH molecules, as well as anti-VL (e.g., Vκ(VK)) autoantibodies that can bind to VL molecules” in the serum of some healthy, first-time experimental human subjects, and “the similarity between pre-existing ADA binding to VH dAb and anti-hinge antibodies is that they bind to IgG fragments but not to those same sequences found in situ on intact IgG.”

[0012] Holland et al., J. Clin. Immunol. 2013, 33(7): 1192-203, described a novel class of anti-IgG autoantibodies present at varying levels in the blood of approximately half of normal healthy humans, which can bind to full-length human V. H The framework sequence of the domain antibody (also referred to by Holland et al. as "HAVH autoantibodies"). Holland et al. also mentioned that these autoantibodies are predominantly IgG isotypes, showing high resistance to V. H Sequence affinity (approximately 10) -10 M), and the free C-terminus shows resistance to these HAVH autoantibodies and V. H The combination of structural domains is important.

[0013] Xue et al., AAPS J. 2013; 15(3): 852-5 also generally discussed issues related to pre-existing biotherapeutic reactive antibodies against biotherapeutic molecules and their regulatory effects.

[0014] The aforementioned prior art also focuses on such a method, in which the sequence of the immunoglobulin variable domain can be modified to prevent or reduce the binding of such pre-existing antibodies / factors to the variable domain. In this regard, WO 2011 / 07586 proposes one or more mutations in the amino acid sequence of the variable domain at certain specific locations (which are exposed on the surface) of the domain. WO 12 / 175741 describes a method to reduce the binding of such pre-existing antibodies / factors by adding several amino acid residues (and as few as one alanine residue) to the C-terminus of the VH-domain and / or by making one or more specific substitutions or deletions in the C-terminal region of the variable domain, which is described in WO 12 / 175741 as containing at least the C-terminal amino acid sequence VTVSS and an amino acid residue at position 14 (for which WO 12 / 175741 teaches that the presence of an alanine residue provides reduced binding of pre-existing antibodies compared to the presence of the “human” amino acid residue proline), and equally likely the amino acid residues at positions 108 and 83 and the amino acid residues adjacent to said positions (WO 2013 / 024059 provides substantially the same teaching as WO 12 / 175741).

[0015] For example, in the studies conducted by the applicant / assignee leading to the filing of WO 12 / 175741, it has been found that adding a single alanine residue to the C-terminal region or end of the exposed VH domain generally prevents / removes (substantially all) binding to pre-existing antibodies / factors present in samples obtained from most human subjects (see, for example, pages 62, lines 20-25 and pages 57, lines 30-58, lines 3 of WO 12 / 175741); and these findings have been confirmed by additional results (data not shown) obtained by the applicant / assignee after filing WO 12 / 175741 when applying the C-terminal alanine substitution of WO 12 / 175741 to other nanobodies.

[0016] Furthermore, in the applicant's / assignee's WO 12 / 175741 and WO 12 / 175400, the C-terminal extension described in WO 12 / 175741 has been applied to certain serum-albumin-binding nanobodies (see, for example, the constructs shown in WO 12 / 175741: SEQ ID NO: 37, 51-53 and 55-64 and SEQ ID NO: 41, 43 and 44; and WO 12 / 175400: SEQ ID NO: 6 to 11).

[0017] Figure 9 of WO 12 / 175741 also describes two albumin-binding sequences, which are used as reference sequences in the following experimental section. These two sequences are SEQ ID NO:37 of WO 12 / 175741 (also referred to herein as "Reference B"; its sequence is given herein as SEQ ID NO:45) and "SEQ ID NO:37 without the added C-terminal amino acid residue" of WO 12 / 175741 (also referred to herein as "Reference A"; its sequence is given in SEQ ID NO:44). Both Reference A and Reference B are derived from the sequence of the humanized anti-albumin nanobody "Alb-8" given in WO 06 / 122787 as SEQ ID NO:62 (and also referred to herein as "Alb-11"); however, compared to the sequence of Alb-11, Reference A contains an N-terminal His tag; and Reference B contains an N-terminal His tag and a C-terminal alanine residue. Reference A, Reference B and Alb-8 / Alb-11 all contain the CDRs given in SEQ ID NO:41 to 43, respectively.

[0018] Other examples of nanobodies and other immunoglobulin monovariable domains having C-terminal extensions and / or mutations in their C-terminal regions can be found, for example, in the following prior art: WO 06 / 129843 (see, for example, SEQ ID NO: 4, 6, 8 and 10); WO 03 / 035695 (see, for example, some sequences listed on pages 61-64); Vu et al., Molecular Immunology, 1121-1131, 1997 (see, for example...) Figure 2 Some of the sequences listed in the document); WO 11 / 003622 (see, for example, sequences given as SEQ ID NO: 10 to 27); WO09 / 058383 (see, for example, sequence TAR2h-10-27 mentioned on page 51); WO 10 / 042815 (see, for example, sequences SEQ ID NO: 15, 17, 27 and 30); and WO 04 / 044204 (see, for example, sequences SEQ ID NO: 31, 35, 37, 47 and 49).

[0019] Some of the references cited in this article also provide examples of ISVD sequences in which the last C-terminal amino acid of the ISVD is an amino acid other than serine (S), for example because the serine at position 113 has been replaced by another amino acid and / or because the serine at position 113 has been deleted and a C-terminal amino acid has been added (in fact, the final result will be the same for the C-terminal amino acid sequence).

[0020] Several references cited in this article also provide examples of nanobodies where position 112 is an amino acid other than serine, and other immunoglobulin single variable domains. For example, WO 12 / 175741 describes a nanobodies where position 112 is glycine (G); Vu et al. (ibid.) describe nanobodies where position 112 is alanine (A) or isoleucine (I); WO13 / 024059 illustrates S112A substitution; and WO 08 / 020079, cited below, illustrates S112F substitution and also generally states that the nanobodies described therein may contain a limited number of amino acid residues added to the carboxyl terminus of the amino acid sequence of the nanobodies.

[0021] In the research leading to this invention, after it was determined that adding a C-terminal extension (which can be as simple as a single C-terminal alanine residue, see also WO 12 / 175741, Example 3) to the C-terminal region or end of a nanobody substantially prevents / removes the binding of pre-existing antibodies / factors in most human subject / patient samples, it was investigated whether samples obtained from human subjects (healthy volunteers and / or subjects with diseases or conditions) might contain (other) pre-existing antibodies or factors that could still bind to the exposed C-terminal region of the nanobody (or other VH domains) even in the presence of the C-terminal extension. In doing so, the inventors discovered that while such pre-existing antibodies binding to the C-terminal extended VH domain were substantially not found in the blood or serum of healthy volunteers or in the blood or serum of patients with one of a variety of different diseases (including some inflammatory diseases or autoimmune diseases – data not shown), some blood or serum samples obtained from some (not all) subjects with certain severe (autoimmune) diseases (such as systemic lupus erythematosus; also abbreviated herein as “SLE”) showed the presence of some pre-existing antibodies / factors that could still bind to the nanobody when the nanobody included the C-terminal extension.

[0022] Therefore, in general, the object of the present invention is to provide improved heavy chain immunoglobulin variable domains (and in particular improved heavy chain ISVDs and more particularly improved nanobodies), which are less susceptible to binding by pre-existing antibodies / factors (such as those found in blood or serum samples obtained from human subjects) when they have exposed C-terminal regions or ends.

[0023] In particular, the object of the present invention is to provide an improved heavy chain immunoglobulin variable domain that, when it has an exposed C-terminal region or end, is less susceptible to binding by pre-existing antibodies / factors (as well as those found in blood or serum samples obtained from human subjects) that can still bind to the exposed C-terminal region or end of the heavy chain variable domain when the domain includes a C-terminal extension (e.g., as described in WO 12 / 175741, WO 13 / 024059 and other prior art cited herein).

[0024] As mentioned herein, the inventors have discovered that such pre-existing antibodies capable of binding to a C-terminal extended heavy chain variable domain are present in blood or serum samples obtained from human subjects suffering from certain (auto)immune diseases or conditions (such as SLE) that severely affect / activate the immune system.

[0025] Therefore, more specifically, the object of the present invention is to provide improved heavy chain immunoglobulin variable domains (and particularly improved heavy chain ISVDs and more particularly improved nanobodies), which, when having exposed C-terminal regions or ends, are less susceptible to binding by pre-existing antibodies / factors (such as those found in blood or serum samples obtained from human subjects suffering from certain (auto)immune diseases or conditions (such as SLE) that severely affect / activate the immune system).

[0026] More specifically, the object of the present invention is to provide improved heavy chain immunoglobulin variable domains (and particularly improved heavy chain ISVDs and even more particularly improved nanobodies) that, when having an exposed C-terminal region or terminus, are less susceptible to binding by those pre-existing antibodies / factors found in blood or serum samples obtained from human subjects suffering from certain (auto)immune diseases or conditions, and which, when the VH domain includes a C-terminal extension, can still bind to the exposed C-terminal region or terminus of the VH domain.

[0027] It has now been found that mutating the serine residue at position 112 (Kabat number) to lysine (K) or glutamine (Q) can (further) reduce the binding of pre-existing antibodies / factors to heavy chain variable domains with exposed C-termini. Specifically, such S112K or S112Q mutations have been found to (further) reduce or substantially prevent / remove the binding of pre-existing antibodies / factors (such as those found in blood or serum samples from subjects with severe autoimmune diseases such as SLE) that can bind to heavy chain variable domains containing C-terminal extensions (but without S112K or S112Q mutations).

[0028] It was also found that introducing specific mutations disclosed herein (and in particular the following mutation: L11V, which in combination with V89L, and optionally further in combination with T110K) can improve, or contribute to (further) improve, the solubility of immunoglobulin single variable domains (such as ISVD) disclosed herein in general and specific form (data not shown).

[0029] Therefore, in a first aspect, the present invention relates to a variable domain (VH domain) of the immunoglobulin heavy chain, wherein the amino acid residue at position 112 (Kabat number) is a lysine (K) residue or a glutamine (Q) residue. This type of variable domain of the immunoglobulin heavy chain is also referred to herein as the "VH domain of the present invention". When the VH domain of the present invention is an immunoglobulin monovariable domain (as preferred), it will also be referred to herein as the "ISVD of the present invention". Similarly, when the VH domain of the present invention is a nanobody (as even more preferred), it will also be referred to herein as the "nanobody of the present invention".

[0030] Generally, the VH domain of the present invention has an exposed C-terminal end or region, and / or is present in proteins, peptides, compounds, entities or constructs having an exposed C-terminal end or region, and / or is intended for use in which it has an exposed C-terminal region (e.g., for proteins, peptides, compounds, entities or constructs where it is intended to form a C-terminal end or region).

[0031] In one aspect of the invention, the VH domain of the present invention is a (heavy chain) immunoglobulin monovariable domain, meaning that a heavy chain variable domain that can form a functional antigen-binding site without interacting with the VL domain can be formed. For example, the VH domain of the present invention can be a nanobody (including VHH, humanized VHH, and / or camel-derived VH such as camel-derived human VH), a (mono)domain antibody as a VH domain or derived from a VH domain, or a dAb as a VH domain or derived from a VH domain. Preferably, the VH domain of the present invention is a nanobody (e.g., a VHH domain, a humanized VHH domain, or a camel-derived VH domain such as a camel-derived human VH domain).

[0032] According to another aspect of the invention, the VH domain of the invention can be a heavy chain variable domain that, in the protein, polypeptide, protein, or construct in which it is present, needs to interact with the VL domain to form an antigen-binding site and has or forms an exposed C-terminal or region. For example, the VH domain according to this aspect of the invention can be a VH domain present in and / or used in ScFv and / or biantibodies.

[0033] According to a more specific aspect of the invention, the VH domain of the invention has a C-terminal sequence of VTVKS (SEQ ID NO:1) or VTVQS (SEQ ID NO:2) (according to positions 109 to 113 of Kabat), or has a sequence that differs from the VTVKS and / or VTVQS sequences by one amino acid (i.e. at one of positions 109, 110, 111 or 113) while still having a lysine (K) or glutamine (Q) at position 112.

[0034] Furthermore, as further described herein, in a particularly preferred aspect of the invention, the VH domain of the invention further includes a C-terminal extension at its C-terminus (e.g., as described in WO 12 / 175741 and / or WO 13 / 024059) (i.e., linked to a serine residue at the end of a VTVKS-, VTVQS-, or similar motif), particularly the C-terminal extension further defined herein. However, also as further described herein, it is possible that a VTVKS-, VTVQS-, or similar motif forms the C-terminus of the VH domain (although this is generally less preferred), or that the VH domain of the invention is linked (optionally with a suitable linker) at its C-terminus to another amino acid sequence, moiety, domain, or binding unit. For example, when the VH domain of the invention is an ISVD, the VH domain may optionally be linked at its C-terminus to another ISVD (and said other ISVD may also be the VH domain of the invention) via a linker.

[0035] In summary, as is generally known regarding immunoglobulin variable domains, the VH domain of this invention will comprise four frame regions (FW1, FW2, FW3, and FW4) and three CDRs (CDR1, CDR2, and CDR3). Regarding immunoglobulin variable domains, the sequence of the CDRs will generally depend on the antigen / target to which the VH domain of this invention is targeted and / or intended to bind. The frame regions can generally be any frame region suitable for the VH domain (although positions 112 and / or 89 will be as further described herein). If the VH domain of this invention is an ISVD, the VH domain will have a frame sequence suitable for the ISVD (optionally binding one or more CDRs). For example, if the VH domain of this invention is a nanobody, the frame regions will generally contain a suitable number of VHH marker residues (see, for example, WO 08 / 020079 and several other applicant / assignee patent applications cited herein).

[0036] Therefore, for example, when the VH domain of the present invention is a nanobody, the nanobody of the present invention may contain one or more “marker residues” that are characteristic of the VHH / nanobody (e.g., at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and / or 108; see, for example, Tables A-3 and A-5 to A-8 of WO 08 / 020079); one or more other amino acid residues that may be present in the VHH / nanobody (such as one or more humanization substitutions known to be useful for VHH and nanobody; refer to, for example, the teachings in WO 08 / 020079; see also the previously mentioned Tables A-3 and A-5 to A-8) and / or one or more other amino acid residues or substitutions suitable for the VHH / nanobody; or any suitable combination of the amino acid residues / substitutions.

[0037] The nanobody of the present invention with position 112 being K or Q (i.e., with or without C-terminal extension) preferably contains an amino acid selected from L (the most frequently occurring amino acid residue in VHH), E, ​​K, M, S, V, W, or Y; more preferably selected from L, E, K, V, or Y; and even more preferably selected from L, K, or V (of which V is the most preferred). For example, and without limitation, it may contain an L11K or L11V mutation compared to the leucine residue most frequently occurring in VHH. It may also, but is not limited to, contain, for example, a Q108L mutation (a known humanization substitution for VHH / nanobody). Other amino acid residues that may be present (again, not limited to, and for example, other amino acid residues naturally occurring at this position in human VH or VHH may also be present at these positions) include, for example, alanine (A) at position 14 (which is a very common amino acid residue at this position in naturally occurring VHH), proline at position 14 (which is the most common amino acid at this position in the human VH domain), and one or more of the mutations suggested by Harmsen et al. (particularly those based on VHH4-like VHH sequences suggested by Harmsen et al., such as V89M or V89T) and / or (other) mutations at positions suggested by Nieba (e.g., one or more at positions 11, 87, and / or 89, see Nieba, page 437, right column). Another suitable mutation is, for example, T110K or T110Q. Furthermore: (i) Position 41 may, for example, be one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) furthermore, Position 42 may, for example, be one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) Position 87 may, for example, be WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0038] According to a more specific aspect of the invention, the VH domain of the invention (which is further described herein) has a frame 4 (FW4 sequence), which is:

[0039] a) is one of the FW4 sequences SEQ ID NO:3 to 20 mentioned in Table 1 below.

[0040] Table 1: FW4 Sequences

[0041] WGKGTLVTVKS(SEQ ID NO:4) RGQGTRVTVKS(SEQ ID NO:5) WGLGTQVTISS(SEQ ID NO:6) GSQGTQVTVKS(SEQ ID NO:7) LRGGTQVTVKS(SEQ ID NO:8) RGQGTLVTVKS(SEQ ID NO:9) RSRGIQVTVKS(SEQ ID NO:10) WGKGTQVTVKS(SEQ ID NO:11) WGQGTQVTVQS(SEQ ID NO:12) WGKGTLVTVQS(SEQ ID NO:13) RGQGTRVTVQS(SEQ ID NO:14) WGLGTQVTISS(SEQ ID NO:15) GSQGTQVTVQS(SEQ ID NO:16) LRGGTQVTVQS(SEQ ID NO:17) RGQGTLVTVQS(SEQ ID NO:18) RSRGIQVTVQS(SEQ ID NO:19) WGKGTQVTVQS(SEQ ID NO:20)

[0042] or:

[0043] b) is a sequence having no more than three, preferably no more than two, amino acid differences compared to at least one of the FW4 sequences of SEQ ID NO 3 to 20, wherein (i) the amino acid residue corresponding to position 112 of the Kabat number is K or Q; and wherein (ii) the amino acid residue corresponding to position 103 of the Kabat number is preferably W or R; (iii) the amino acid residue corresponding to position 104 of the Kabat number is preferably G; (iv) the amino acid residue corresponding to position 106 of the Kabat number is preferably G; (v) the amino acid residue corresponding to position 107 of the Kabat number is preferably T; (vi) the amino acid residue corresponding to position 108 of the Kabat number is preferably Q or L (and preferably L in humanized nanobodies); (vii) the amino acid residue corresponding to position 109 of the Kabat number is preferably V; (viii) the amino acid residue corresponding to position 110 of the Kabat number is preferably T (or alternatively K or Q); and (ix) the amino acid residue corresponding to position 111 of the Kabat number is preferably V. Table 2 below provides some non-limiting examples of amino acid residues that can be present at different positions (according to Kabat numbering) in this FW4 sequence.

[0044] Table 2: Examples of amino acid residues that may be present in the FW4 sequence of the VH domain of this invention.

[0045]

[0046] Preferably, the VH domain of the present invention has the following frame 4 (FW4 sequence):

[0047] a) WGQGTQVTVKS (SEQ ID NO:3) or WGQGTQVTVQS (SEQ ID NO:12); or

[0048] b) A sequence having no more than three, preferably no more than two, amino acid differences (e.g., only one amino acid difference) compared to SEQ ID NO:3 and / or SEQ ID NO:12, wherein (i) the amino acid residue corresponding to position 112 of the Kabat number is K or Q; and wherein (ii) the amino acid residue corresponding to position 103 of the Kabat number is preferably W or R; (iii) the amino acid residue corresponding to position 104 of the Kabat number is preferably G; (iv) the amino acid residue corresponding to position 106 of the Kabat number is preferably G; (v) the amino acid residue corresponding to position 107 of the Kabat number is preferably T; (vi) the amino acid residue corresponding to position 108 of the Kabat number is preferably Q or L (and preferably L in humanized nanobodies); (vii) the amino acid residue corresponding to position 109 of the Kabat number is preferably V; (viii) the amino acid residue corresponding to position 110 of the Kabat number is preferably T (or alternatively K or Q); and (ix) the amino acid residue corresponding to position 111 of the Kabat number is preferably V. Similarly, Table 2 provides some non-limiting examples of amino acid residues that can be present at different positions (according to Kabat numbering) in this FW4 sequence.

[0049] As further described herein, according to a preferred aspect of the invention, the VH domain of the invention comprising the FW4 sequence as described above preferably further comprises a C-terminal extension (as further described herein). However, as also further described herein, it is also possible that the FW4 sequence forms the C-terminus of the VH domain (although this is generally less preferred) or that the VH domain of the invention is linked (optionally via a suitable linker) at its C-terminus to another amino acid sequence, portion, domain, or binding unit. For example, when the VH domain of the invention is an ISVD, the VH domain may optionally be linked to another ISVD (and said other ISVD may also be the VH domain of the invention) via a linker at its C-terminus.

[0050] As noted herein, according to a preferred but non-limiting aspect of the invention, the VH domain of the invention contains a C-terminal extension, such as the C-terminal extension described in WO 12 / 175741 and / or WO 13 / 024059, and particularly the C-terminal extension described in WO 12 / 175741.

[0051] Therefore, according to this aspect, the VH domain of the present invention is an immunoglobulin heavy chain variable domain (VH domain), wherein: (i) the amino acid residue at position 112 (Kabat number) is not a serine residue, and preferably a lysine (K) residue or a glutamine (Q) residue; and (ii) at its C-terminus (i.e., connected to an amino acid residue at position 113 according to the Kabat number or at a position corresponding to position 113 according to the Kabat number) is connected to another amino acid sequence (i.e., “C-terminal extension”), the other amino acid sequence comprising 1 to 5 (e.g., 1, 2, 3, 4 or 5, and preferably 1, 2 or 3, and most preferably only 1 or 2, such as only 1) amino acid residues, each of which is independently selected from suitable amino acid residues, and preferably each of which is independently selected from naturally occurring amino acids, and more preferably each of which is independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (however, as by WO As can be seen from the data provided in 12 / 17574, other amino acid residues such as serine, proline, threonine, and / or lysine can also be used as part of the C-terminal extension.

[0052] In particular, according to this aspect of the invention, the VH structural domain of the invention preferably has a C-terminal sequence of VTVKS(X). n (SEQ ID NO:21) or VTVQS(X) n (SEQ ID NO:22) (or an amino acid sequence that has an amino acid difference at the position of the VTVKS motif or VTVQS-motif compared to the sequence VTVKS and / or VTVQS, while still having lysine (K) or glutamine (Q) at position 112), wherein (i) the amino acid residues of the VTVKS- or VTVQS-motif (or VTVKS- or VTVQS-like motif) correspond to positions 109 to 113 of the VH domain according to the Kabat number; (ii) 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 (iii) each X is (preferably naturally occurring) such amino acid residues that are independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0053] More specifically, according to this aspect of the invention, the VH domain of the invention may have one of the FW4 sequences of SEQ ID NO 3 to 20 (or an amino acid sequence having no more than three, preferably no more than two, amino acid differences compared to at least one of the FW4 sequences of SEQ ID NO 3 to 20, wherein the amino acid residue corresponding to position 112 of the Kabat number is K or Q) as its FW4 sequence, wherein the FW4 sequence is connected at its C-terminus to a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).

[0054] Therefore, in this aspect of the invention, the VH domain of the invention may have the following characteristics at its C-terminus:

[0055] a) One of the amino acid sequences given in SEQ ID NO:23 to 40 in Table 3;

[0056] Table 3: FW4 sequences with C-terminal extension

[0057] <![CDATA[WGKGTLVTVKS(X) n (SEQ ID NO:24)]]> <![CDATA[RGQGTRVTVKS(X) n (SEQ ID NO:25)]]> <![CDATA[WGLGTQVTISS(X) n (SEQ ID NO:26)]]> <![CDATA[GSQGTQVTVKS(X) n (SEQ ID NO:27)]]> <![CDATA[LRGGTQVTVKS(X) n (SEQ ID NO:28)]]> <![CDATA[RGQGTLVTVKS(X) n (SEQ ID NO:29)]]> <![CDATA[RSRGIQVTVKS(X) n (SEQ ID NO:30)]]> <![CDATA[WGKGTQVTVKS(X) n (SEQ ID NO:31)]]> <![CDATA[WGQGTQVTVQS(X) n (SEQ ID NO:32)]]> <![CDATA[WGKGTLVTVQS(X) n (SEQ ID NO:33)]]> <![CDATA[RGQGTRVTVQS(X) n (SEQ ID NO:34) <!-- 9 -->]]> <![CDATA[WGLGTQVTISS(X) n (SEQ ID NO:35)]]> <![CDATA[GSQGTQVTVQS(X) n (SEQ ID NO:36)]]> <![CDATA[LRGGTQVTVQS(X) n (SEQ ID NO:37)]]> <![CDATA[RGQGTLVTVQS(X) n (SEQ ID NO:38)]]> <![CDATA[RSRGIQVTVQS(X) n (SEQ ID NO:39)]]> <![CDATA[WGKGTQVTVQS(X) n (SEQ ID NO:40)]]>

[0058] Among them, (i) (X) in SEQ ID NO:23 to 40 n The amino acid residues of the FW4 sequence prior to the C-terminal extension correspond to the amino acid positions of the FW4 in the VH domain (i.e., positions 103 to 113 according to Kabat numbering); (ii) 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 (iii) each X is (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I); or

[0059] b) An amino acid sequence having no more than three, preferably no more than two, amino acid differences compared to at least one of the amino acid sequences SEQ ID NO 23 to 40 (wherein the amino acid difference is at the position corresponding to the amino acid position of FW4 of the VH domain, i.e., at positions 103 to 113 according to the Kabat number, wherein the C-terminal extension (X) is ignored). n(any amino acid difference within), wherein: (i) the amino acid residue at position 112 corresponding to Kabat number is K or Q; (ii) 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 (iii) each X is (preferably naturally occurring) such amino acid residues that are independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). Similarly, further regarding features (i) to (iii) mentioned in the previous statements, in this amino acid sequence: (iv) the amino acid residue corresponding to position 103 of the Kabat number is preferably W or R; (v) the amino acid residue corresponding to position 104 of the Kabat number is preferably G; (vi) the amino acid residue corresponding to position 106 of the Kabat number is preferably G; (vii) the amino acid residue corresponding to position 107 of the Kabat number is preferably T; (viii) the amino acid residue corresponding to position 108 of the Kabat number is preferably Q or L (and preferably L in humanized nanobodies); (ix) the amino acid residue corresponding to position 109 of the Kabat number is preferably V; (x) the amino acid residue corresponding to position 110 of the Kabat number is preferably T (or alternatively K or Q); (xi) the amino acid residue corresponding to position 111 of the Kabat number is preferably V; and for the possible amino acid residues that may be present at each position, refer again to Table 2.

[0060] Preferably, according to this aspect of the invention, the VH domain of the invention has the following characteristics at its C-terminus:

[0061] a) Amino acid sequence WGQGTQVTVKS(X) n (SEQ ID NO:23) or WGQGTQVTVQS(X) n (SEQ ID NO:32), wherein (ii)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 (iii) each X is (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I); or

[0062] b) With amino acid sequence WGQGTQVTVKS(X) n (SEQ ID NO:23) or WGQGTQVTVQS(X) nAt least one of (SEQ ID NO: 32) has an amino acid sequence with no more than three, preferably no more than two, amino acid differences (wherein the amino acid difference is at the position corresponding to the amino acid position of FW4 of the VH domain, i.e., at positions 103 to 113 according to the Kabat number, where the C-terminal extension (X) is ignored). n Any amino acid differences within the sequence, wherein: (i) the amino acid residue at position 112 corresponding to Kabat number is K or Q; (ii) 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 (iii) each X is (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). Similarly, for such amino acid sequences, features (vi) to (xi) as described in the preceding paragraphs are also preferably applicable.

[0063] Furthermore, as mentioned herein, the VH domain of the present invention may also contain other amino acid residues or substitutions at relevant positions that are known in the art for use in the VH domain and, in particular, in ISVDs (and more particularly, nanobodies). Some non-limiting examples may include, for example, one or more “marker residues” characteristic of VHH / nanobodies (including, for example, leucine (L) at position 11), other amino acid residues naturally present in VHH (such as alanine (A) at position 14), humanized substitutions known in VHH / nanobodies (such as Q108L and A14P), one or more mutations suggested by Harmsen (such as V89M or V89T) and / or one or more mutations at positions suggested by Nieba (such as 11, 87, or 89); or any suitable combination thereof; and / or, for example, T110K, T110Q, or V89L mutations.

[0064] When the VH structural domain of the present invention includes a C-terminal extension (X) n In some preferred, but not limiting, instances of this extension, X and n can be as follows:

[0065] (a) n = 1 and X = Ala;

[0066] (b) n = 2 and each X = Ala;

[0067] (c) n = 3 and each X = Ala;

[0068] (d) n = 2 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0069] (e) n = 3 and at least one X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0070] (f) n = 3 and at least two X = Ala (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0071] (g)n=1 and X=Gly;

[0072] (h)n = 2 and each X = Gly;

[0073] (i)n = 3 and each X = Gly;

[0074] (j) n = 2 and at least one X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0075] (k)n = 3 and at least one X = Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

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

[0077] (m)n = 2 and each X = Ala or Gly;

[0078] (n)n=3 and each X=Ala or Gly;

[0079] (o)n = 3 and at least one X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile); or

[0080] (p)n = 3 and at least two X = Ala or Gly (the remaining amino acid residues X are independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and / or Ile);

[0081] Aspects (a), (b), (c), (g), (h), (i), (m), and (n) are particularly preferred, aspects with n = 1 or 2 are preferred, and aspects with n = 1 are particularly preferred.

[0082] It should also be noted that, preferably, any C-terminal extension present in the VH domain of the present invention does not contain (free) cysteine ​​residues (unless said cysteine ​​residues are used or intended for other functionalizations, such as for polyethylene glycolation).

[0083] Furthermore, the preferred selection of C-terminal extensions for use according to WO 12 / 175741 (which is also the preferred C-terminal extension for the VH structural domain according to the invention) as indicated on pages 35 to 41 of WO 12 / 175741 also applies to C-terminal extensions for the VH structural domain according to the invention, and these preferred selections according to WO 12 / 175741 are also incorporated herein by reference.

[0084] Preferably, when the VH structural domain of the present invention includes a C-terminal extension (X) n In this case, n = 1, 2, or 3, and each X is Ala or Gly. More preferably, each X is Ala, and n = 1 or 2, and preferably 1.

[0085] When the VH domain of the present invention contains a C-terminal extension, it will typically be present (and typically form) at the C-terminus of the protein, polypeptide, compound, construct, or other chemical entity in which it is present. Similarly, such a protein, polypeptide, compound, construct, or other chemical entity may contain one or more other VH domains of the present invention (i.e., not at the C-terminus); in this case, said other VH domains of the present invention will contain lysine (K) or glutamine (Q) at position 112 (and as further described herein), but will not contain a C-terminal extension (instead, it may be linked at its C-terminus (optionally via one or more suitable linkers) to one or more other amino acid sequences, portions, binding domains, or binding units present in the protein, polypeptide, compound, construct, or other chemical entity, such as the VH domain of the present invention having a C-terminal extension present at the C-terminus).

[0086] Because the VH domain of the present invention (and proteins, peptides, compounds, constructs and other chemical entities comprising it, as further described herein) is particularly useful for (and intended for) pharmaceutical uses (such as the prevention, treatment and / or diagnosis of diseases and conditions in human subjects in need of such use), it preferably has a high degree of sequence homology in its frame region to the frame sequence of the human VH domain. In particular, the VH domain of the present invention preferably has an overall sequence identity of at least 80%, preferably at least 85%, such as more than 90%, with at least one human germline sequence (such as DP-47, DP-51 or DP-29) (as determined as further described herein, and considering only the frame region and not the CDR, and not considering any possible substitutions at position 112 and any C-terminal extensions). More particularly, the VH domain of the present invention preferably has an overall sequence identity of at least 80%, preferably at least 85%, such as more than 90%, with at least one of the following phylogenetic sequences: DP-47, DP-51 and / or DP-29 (as determined as further described herein, and taking into account only the frame region and not the CDR, and not taking into account any possible substitutions at position 112 and any C-terminal extensions).

[0087] As further described herein, according to one aspect of the invention, the VH domain of the invention can be a heavy chain variable domain that interacts with / binds to (or is intended to interact with / bind to) the VL domain in the protein, polypeptide, protein, or construct in which it is present to form an antigen-binding site, wherein at least the VH domain has an exposed C-terminal or region. For example, the VH domain according to this aspect of the invention can be a VH domain present and / or used in ScFv and / or biantibodies, wherein it will bind to the VL domain to form an antigen-binding site.

[0088] However, according to a preferred aspect of the invention, the VH domain of the invention is a (heavy chain) immunoglobulin single variable domain or "ISVD," meaning a heavy chain variable domain that can form a functional antigen-binding site without interacting with the VL domain. For example, the VH domain of the invention can be a nanobody (including VHH, humanized VHH, and / or camel-derived VH, such as camel-derived human VH), and the (single domain) antibody is a VH domain or derived from a VH domain, or is a dAb as a VH domain or derived from a VH domain. The VH domain of the invention is preferably a nanobody (and more preferably a VHH domain, a humanized VHH domain, or a camel-derived VH domain such as camel-derived human VH domain).

[0089] In this instruction manual:

[0090] The term "nanobody" is generally as defined in WO 08 / 020079 or WO 09 / 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 refers to a sequence-optimized VHH (such as optimized for, for example, chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). It should be noted that the term nanobody or nanobodies is a registered trademark of Ebolings Inc. and can therefore also be referred to as... and / or );

[0091] The term "ISVD" (or "ISV"), as used in its broadest sense herein, also includes "ISVD-based biologics" and, when the ISVD is a nanobody, includes "nanobody-based biologics." "ISVD-based biologics" is defined herein as containing at least one (e.g., one, two, or three) ISVDs or proteins, peptides, or other biological agents substantially composed of them. Similarly, "nanobody-based biologics" is defined as containing at least one (e.g., one, two, or three) nanobodies or proteins, peptides, or other biological agents substantially composed of them. Regarding the term "ISVD," when using the term "ISVD-based biologics," it should be understood that such ISVD-based biologics are preferably nanobody-based biologics. In the context of this invention, "ISVD-based biologics" and "nanobody-based biologics" can be, for example, monovalent, bivalent (or multivalent), bispecific (or multispecific), and biantibody determinant (or "multiantibody determinant") ISVD constructs or nanobody constructs. Furthermore, any ISVD-based or nanobody-based biologic may, for example, optionally include, in addition to one or more (e.g., one, two, or three) ISVDs or nanobodies, one or more (e.g., one or two) other additional therapeutic components and / or one or more (e.g., one or two) other components affecting the pharmacokinetic or pharmacodynamic properties (e.g., its half-life) of the ISVD-based or nanobody-based biologic. Suitable examples of such additional therapeutic agents or other components are apparent to those skilled in the art and, for example, typically include any protein, peptide, or other binding domain or binding unit having therapeutic activity, and, for example, modifications such as WO3. The examples described on pages 149 to 152 of 09 / 138159. ISVD-based or nanobody-based biologics 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 (such as, for example, RANK-L, vWF, IgE, RSV, CXCR4, IL-23, or other interleukins). For some specific, but not limiting, examples of such ISVD-based or nanobody-based biologics can be found in Examples 8 to 18 and, for example, in various applications of Ebolingx Inc. (such as, for example, but not limited to, WO 2004 / 062551, WO 2006 / 122825, WO 2008 / 020079 and WO 2009 / 068627), and, for example (but not limited to) applications such as WO 06 / 038027, WO 06 / 059108, WO 07 / 063308, WO 07 / 063311, WO07 / 066016 and WO 07 / 085814.Furthermore, as further described herein, the ISVD or nanobody used herein may be against (human) serum proteins such as (human) serum albumin, and such ISVD or nanobody may also have therapeutic uses, particularly in extending the half-life of therapeutic fractions and compounds and / or for extending the half-life of therapeutic fractions and compounds (such as in extending the half-life of ISV-based biologics described herein or for extending the half-life of ISV-based biologics described herein). References, for example, to WO 2004 / 041865, WO 2006 / 122787 and WO2012 / 175400, generally describe the use of serum-albumin-bound nanobodies for half-life extension. Furthermore, in this specification, unless expressly otherwise stated herein, all terms used herein have the meanings given in WO 09 / 138519 (or the prior art referenced in WO 09 / 138519) or WO 08 / 020079 (or the prior art referenced in WO 08 / 020079). Additionally, when a method or technique is not specifically described herein, it may be performed as described in WO 09 / 138519 (or the prior art referenced in WO 09 / 138519) or WO 08 / 020079 (or the prior art referenced in WO 08 / 020079).

[0092] Furthermore, when used in this specification or claims, the following terms have the same meaning as given on pages 62-75 of WO 09 / 138519 and / or, where applicable, can be determined in the manner described on pages 62-75 of WO 09 / 138519: “agonist,” “antagonist,” “antiagonist,” “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 sequence comparisons of two amino acid sequences), “(for) substantially separate (forms),” “domain,” “binding domain,” “antigenic determinant,” “epitope,” “targeted” or “targeted” (antigen), “specificity,” and “half-life.” Furthermore, the terms “mediated” and “mediating,” “interaction site,” “specific to,” “cross-blocking,” “cross-blocked,” “cross-blocking,” and “substantially pH-independent” are as defined (and / or may be determined as described therein) on pages 74-79 of the applicant’s WO 10 / 130832. Additionally, when referring to constructs, compounds, proteins, or peptides of the present invention, terms such as “monovalent,” “divalent” (or “multivalent”), “bispecific” (or “multispecific”), and “biantibody determinant” (or “multiantibody determinant”) may have the meanings given in WO 09 / 138.519, WO 10 / 130832, or WO 08 / 020079.

[0093] The term "half-life" as used herein with respect to ISVD, nanobody, ISVD-based biologic, nanobody-based biologic, or any other amino acid sequence, compound, or peptide mentioned herein may generally be defined as described in paragraph o) on page 57 of WO 08 / 020079 and, as mentioned therein, refers to the time required for a 50% reduction in serum concentration of the amino acid sequence, compound, or peptide in vivo (e.g., due to degradation and / or clearance or chelation of the sequence or compound 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 described as described in paragraph o) on page 57 of WO 08 / 020079. As also mentioned in paragraph o) on page 57 of WO 08 / 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 the terminal half-life (where t1 / 2-α and / or AUC or both may be disregarded). See, for example, the following experimental sections, 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). See also “Pharmacokinetics”, M. Gibaldi & D. Perron, Marcel Dekker, 2nd Rev. edition (1982). Similarly, the terms “increased half-life” or “increased half-life” are as defined in paragraph o) on page 57 of WO 08 / 020079 and specifically refer to an increase in t1 / 2-β (regardless of whether t1 / 2-α and / or AUC or both are increased).

[0094] When a term is not specifically defined herein, it has its usual meaning in the art, which will be clear to those skilled in the art. References include standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd ed.), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., ed., "Current protocols in molecular biology", Green Publishing & 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 ed., University of California Press, Berkeley, CA (1981); Roitt et al., "Immunology" (6th ed.), Mosby / Elsevier, Edinburgh (2001); Roitt et al., "Roitt's Essential Immunology", 10th ed., Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th ed.), Garland Science. Publishing / Churchill Livingstone, New York (2005), and the general background techniques cited in this paper.

[0095] Furthermore, as already noted in this paper, the amino acid residues of the nanobodies are numbered according to the general numbering given for the VH domain by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIHBethesda, MD, Publication No. 91), as in the article by Riechmann and Muyldermans (J. Immunol. Methods 2000 Jun 23; 240(1-2):185-195); or as referred to herein. According to this numbering, FR1 of the nanobody 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 acids 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. [It should be noted that—as is known in the art regarding the VH domain and the VHH domain—the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat 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, generally speaking, it can be said that, according to the Kabat number and regardless of the number of amino acid residues in the CDR, position 1 of the Kabat number corresponds to the beginning of FR1 and vice versa; position 36 of the Kabat number corresponds to the beginning of FR2 and vice versa; position 66 of the Kabat number corresponds to the beginning of FR3 and vice versa; and position 103 of the Kabat number corresponds to the beginning of FR4 and vice versa.

[0096] Alternative methods for numbering the amino acid residues of the VH domain (which can also be applied in a similar manner to VHH domains and nanobodies from the Camelidae family) are those described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in this specification, aspects and figures, unless otherwise indicated, the numbering according to Kabat applied by Riechmann and Muyldermans to the VHH domain will be followed.

[0097] It should also be noted that, unless expressly indicated otherwise herein, the accompanying drawings, any sequence listings, and experimental sections / examples are provided only to further illustrate the invention and should in no way be construed as limiting the scope of the invention and / or the appended claims.

[0098] The VH domain of the present invention can be targeted at any suitable or desired antigen or target, including any drug and / or treatment-related targets as described herein.

[0099] According to one specific method of the invention, the VH domain of the invention targets serum proteins, and particularly human serum proteins. According to a preferred aspect, when the VH domain of the invention targets serum proteins, it targets serum albumin, and particularly human serum albumin. Therefore, the invention also relates to the VH domain of the invention (including, as defined herein, preferred options defined for the VH domain of the invention), which is capable of specifically binding to serum proteins, particularly human serum proteins, and can preferably specifically bind to serum albumin, and more preferably specifically bind to human serum albumin. Likewise, such a VH domain is preferably an ISVD (as described herein) and more preferably a nanobody.

[0100] For example, the VH domain of the present invention targeting serum albumin may be one of the nanobodies targeting (human) serum albumin described in WO 2004 / 041865 and, in particular, WO 2006 / 122787 and WO 2012 / 175400 (both applications of the applicant / assignee), wherein the amino acid at position 112 is substituted with K or Q, and it is optionally provided with a C-terminal extension as described herein (and may also contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K and / or T110K; although when position 112 is K or Q, it is generally not necessary to have an additional substitution at position 110, in which case position 110 is preferably T). Furthermore, it is anticipated that the present invention can also be applied to other serum-albumin-binding heavy chain ISVDs, such as those described in WO 03 / 035694, WO 04 / 003019, WO 05 / 118642, WO 06 / 059106, WO 08 / 096158, WO 09 / 121804, WO 10 / 108937, or US 2013 / 0129727, i.e., by suitably introducing the substitutions described herein (i.e., at least one of S112K, S112Q, and / or V89T, and optionally one or more of other amino acid residues / substitutions described herein, such as L11V) and optionally (and, generally preferably, as indicated herein) adding C-terminal extensions (as further described herein).Some preferred, and not limiting, examples of this type of serum albumin-binding nanobody of the present invention are humanized variants of the amino acid sequence of SEQ ID NO:52 of WO 2006 / 122787 (referred to as “Alb-1” in WO 2006 / 122787), wherein the amino acid at position 112 is substituted with K or Q (and optionally has a C-terminal extension as described herein), such as the humanized variants of Alb-1 given in SEQ ID NO:57 to 64 of WO 2006 / 122787 (in each case having an S112K or S112Q substitution and optionally a C-terminal extension) or the humanized variants of Alb-1 given in SEQ ID NO:3 to 11 of WO 2012 / 175400 (again, in each case having an S112K or S112Q substitution), wherein SEQ ID NO:3, 4, and 5 may optionally contain a C-terminal extension, and SEQ ID NO:57, 4, and 5 may optionally contain a C-terminal extension. 6 to 11 already contain C-terminal extensions (and similarly, this variant may contain one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K and / or T110K; although when position 112 is K or Q, it is generally not necessary to have an additional substitution at position 110, in which case position 110 is preferably T). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0101] Therefore, in another aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), wherein:

[0102] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0103] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0104] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0105] -The amino acid residue at position 112 is K or Q;

[0106] And it optionally includes a C-terminal extension (X) at its C-terminus. 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly this nanobody is also It may contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K and / or T110K; although when position 112 is K or Q, it is generally not necessary to have an additional substitution at position 110, in which case position 110 is preferably T). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0107] In a particular aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), said nanobodies being humanized variants of SEQ ID NO:52 of WO 2006 / 122787, wherein:

[0108] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0109] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0110] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0111] -The amino acid residue at position 112 is K or Q;

[0112] And it optionally includes a C-terminal extension (X) at its C-terminus. nWhere 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly this nanobody is also It may contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K and / or T110K; although when position 112 is K or Q, it is generally not necessary to have an additional substitution at position 110, in which case position 110 is preferably T). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0113] In a particular aspect, the present invention relates to nanobodies of the present invention (as defined herein), said nanobodies being capable of binding (and particularly specifically binding) serum albumin (and particularly human serum albumin), said nanobodies having at least 80%, preferably at least 85%, more preferably at least 90% (e.g., at least 95%) sequence identity (considering both the framework sequence and CDR, but not any C-terminal extension), wherein the amino acid residue at position 112 is K or Q, and optionally it includes a C-terminal extension (X) at its C-terminus. 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly this nanobody is also It may contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K and / or T110K; although when position 112 is K or Q, it is generally not necessary to have an additional substitution at position 110, in which case position 110 is preferably T). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0114] The nanobody of the present invention is again preferably a humanized variant of Alb-1 (but with S112K or S112Q substitution), and more preferably has at least one, particularly any two, and more particularly all three of CDR1, CDR2 and / or CDR3 respectively given in SEQ ID NO 41 to 43.

[0115] According to one particular aspect, any serum albumin-binding nanobody of the present invention may also have the characteristic amino acid residues of Alb-23 and its variants as described in WO12 / 175400 (i.e., amino acid motif GP at positions 44 and 45, amino acid motif SKN at positions 74 to 76, and preferably G at position 16 and R at position 83, as well as optionally G at position 83).

[0116] Some preferred, non-limiting examples of the nanobodies of the present invention targeting human serum albumin are given in Table 4 and Example 20.

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] In another aspect, the present invention relates to VH domains, and particularly to ISVD, and even more particularly to nanobodies having:

[0126] a) An FW4 sequence that is one of the following FW4 sequences:

[0127] Table 5: FW4 Sequences

[0128] SSQGTLVTVQS (SEQ ID NO:100) SSQGTLVKVSS(SEQ ID NO:101) SSQGTLVQVSS(SEQ ID NO:102) <![CDATA[SSQGTLVTVKS(X) n (SEQ ID NO:103)]]> <![CDATA[SSQGTLVTVQS(X) n (SEQ ID NO:104)]]> <![CDATA[SSQGTLVKVSS(X) n (SEQ ID NO:105)]]> <![CDATA[SSQGTLVQVSS(X) n (SEQ ID NO:106)]]>

[0129] Among them, (i) (X) in SEQ ID NO:23 to 40 nThe amino acid residues of the FW4 sequence prior to C-terminal extension correspond to the amino acid positions of the FW4 in the VH domain (i.e., positions 103 to 113 according to Kabat numbering); (ii) 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 (iii) each X is (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I); or

[0130] c) Having no more than three, preferably no more than two, amino acid differences from at least one of the amino acid sequences SEQ ID NO: 99 to 106 (wherein the amino acid difference is at the position corresponding to the amino acid position of FW4 in the VH domain, i.e., at positions 103 to 113 according to the Kabat number, ignoring the C-terminal extension (X). n The amino acid sequence of any amino acid difference within the Kabat number 112, wherein: (i) when the amino acid residue at position 112 corresponding to Kabat number 112 is K or Q, the amino acid difference is at another amino acid position different from 112, and the amino acid residue at position 89 of the VH domain is preferably selected from V, T or L (and most preferably V); (ii) when the amino acid residue at position 110 corresponding to Kabat number 110 is K or Q, the amino acid difference is at another amino acid position different from 110, and position 89 is VH domain is L; (iii) 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 (iv) each X is (preferably naturally occurring) such amino acid residues that are independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). Similarly, for features (i) to (iv) mentioned in the previous statements, further, in this amino acid sequence: (v) the amino acid residue corresponding to position 103 of the Kabat number is preferably S; (vi) the amino acid residue corresponding to position 104 of the Kabat number is preferably S; (vii) the amino acid residue corresponding to position 106 of the Kabat number is preferably G; (viii) the amino acid residue corresponding to position 107 of the Kabat number is preferably T; (ix) the amino acid residue corresponding to position 108 of the Kabat number is preferably Q or L (and preferably L in humanized nanobodies); (ix) the amino acid residue corresponding to position 109 of the Kabat number is preferably V; and for the possible amino acid residues that may be present at each position, refer again to Table 2.

[0131] Similarly, in this VH domain of the present invention: (a) the amino acid residue at position 11 is one of L, V, or K (and more preferably V); the amino acid residue at position 14 is preferably one of A or P; and the amino acid residue at position 41 is preferably one of A or P. Furthermore, this VH domain of the present invention is preferably an ISVD, and more preferably a nanobody; and can also be for human serum albumin (in which case it preferably has CDR1, CDR2, and CDR3 corresponding to SEQ ID NO41, 42, and 43, respectively). Furthermore, when the VH domain is an ISVD or nanobody for human serum albumin, it can further be as described herein for the nanobody of the present invention for human serum albumin.

[0132] The VH domain of the present invention targeting serum albumin (and particularly the serum-albumin-binding ISVD of the present invention) can be used to increase the half-life of therapeutically active compounds, (poly)peptides, proteins, binding domains, binding units, or other therapeutically active entities or portions, essentially in the manner described in WO 2004 / 041865, WO 2006 / 122787, and / or WO 2012 / 175400 for the use of serum albumin-binding nanobodies disclosed in the aforementioned documents (i.e., by suitably linking the serum albumin-binding ISVD to a protein, peptide, compound, or other entity, optionally via a suitable linker. For example, pages 12 and 13 of WO 12 / 175400 give some examples of this manner in which suitable fusion proteins can be constructed).

[0133] In another aspect, the present invention relates to immunoglobulin heavy chain variable domains (VH domains) wherein the amino acid residue at position 89 (Kabat number) is threonine (T) and the amino acid residue at position 112 is serine (S), lysine (K), or glutamine (Q). Such immunoglobulin heavy chain variable domains (VH domains) with a T at position 89 are also included in the term "VH domain of the invention" as used herein in its broadest sense and may further be as described herein for VH domains of the invention containing K or Q at position 112. Therefore, such immunoglobulin heavy chain variable domains (VH domains) may have a C-terminal extension as further described herein (including indicated preferred options for such C-terminal extension); may be ISVDs and particularly nanobodies, as further described herein.

[0134] Similarly, if the VH domain of this invention has an exposed C-terminal region (e.g., because it forms the C-terminus of a protein, polypeptide, or other construct therein), it preferably includes a C-terminal extension (see data shown in Table C below).

[0135] Furthermore, the amino acid contained at position 11 of the nanobody of the present invention (i.e., with or without C-terminal extension) where position 89 is T is preferably selected from: L (the most frequently occurring amino acid residue in VHH), E, ​​K, M, S, V, W, or Y; more preferably selected from L, E, K, V, or Y, and even more preferably selected from L, K, or V (where V is the most preferred). For example, it may contain L11K or L11V substitution, and for example P14A or A14P substitution, Q108L substitution, and / or T110K, T110Q, S112K, and / or S112Q substitution (although generally, when position 89 is T, it is not necessary to have one or two additional substitutions at positions 110 and / or 112, in which case position 110 is preferably T and position 112 is preferably S).

[0136] Specifically, the immunoglobulin heavy chain variable domain (VH domain) of the present invention according to this particular aspect has a T and a C-terminus at position 89 (Kabat), said C-terminus being one of the following: VTVSS (SEQ ID NO:77), VTVSS(X)n (SEQ ID NO:78), VTVKS (SEQ ID NO:1), VTVKS(X)n (SEQ ID NO:21), VTVQS (SEQ ID NO:2), VTVQS(X)n (SEQ ID NO:22), VKVSS (SEQ ID NO:95), VKVSS(X)n (SEQ ID NO:97), VQVSS (SEQ ID NO:96), VQVSS(X)n (SEQ ID NO:98), VZVZS (SEQ ID NO:107, wherein each amino acid residue Z is independently K or Q) or VZVZSX(n) (SEQ ID NO:107). NO:108, wherein each amino acid residue Z is independently K or Q (and in particular one of the following: VTVKS (SEQ ID NO:1), VTVQS (SEQ ID NO:2), VTVSS (SEQ ID NO:77), VTVKS(X)). n (SEQ ID NO:21), VTVQS(X) n (SEQ ID NO:22) or VTVSS(X) n(SEQ ID NO:78), and more particularly VTVSS (SEQ ID NO:77) or VTVSS(X)n (SEQ ID NO:78)), where n and X are as further described herein for the VH domain of the invention where position 112 is Q or K (and where any C-terminal extension is preferably as further described herein for the VH domain of the invention where position 112 is Q or K). Furthermore, similar to the VH domain of the invention where position 112 is Q or K, when such a VH domain with position 89 being T is a nanobody, position 11 is preferably leucine (L), position 14 may particularly be alanine (A) or proline (P), and position 108 may particularly be Q or L (and preferably L in humanized nanobodies); and such nanobodies with position 89 being T may contain one or more nanobody marker residues and / or may be suitably humanized. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0137] The VH domain of the present invention with a T at position 89 can also be a serum albumin nanobody as further described herein. For example, such a serum albumin-binding nanobody can be one of sequences SEQ ID NO:46 to 75 (but with a T at position 89); or another serum albumin-binding nanobody with a T at position 89 having at least 80%, preferably at least 85%, more preferably at least 90% (e.g., at least 95%) sequence identity (considering both the frame sequence and CDR, but not any C-terminal extension) to at least one of Alb-1 (SEQ ID NO:52 of WO 2006 / 122787), Alb-8 (SEQ ID NO:46 herein), and / or Alb-23 (SEQ ID NO:61 herein). Other examples of nanobodies with a T at position 89 are given in SEQ ID NO:78 to 91 (these are further variants of Alb-1 / Alb-8 or Alb-23 with a T at position 89 and an S at position 112).

[0138] More generally, the VH domain for serum albumin according to this aspect of the invention can be WO 2004 / 041865, and in particular WO 2006 / 122787 and WO One of the nanobodies targeting (human) serum albumin described in 2012 / 175400 (both applications of the applicant / assignee), wherein the amino acid at position 89 is threonine (T), and it is optionally provided with a C-terminal extension as described herein (and may also suitably contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, S112K and / or S112Q; although generally, when T is present at position 89, it is not generally necessary to have additional substitutions at positions 110 and / or 112, in which case position 110 is preferably T and position 112 is preferably S). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO The amino acid residue mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) may be, in particular (or selected from) threonine (T) or alanine (A). Furthermore, it is contemplated that the invention may also apply other heavy chain ISVDs that bind serum albumin, such as those described in WO 03 / 035694, WO 04 / 003019, WO 05 / 118642, WO 06 / 059106, WO 08 / 096158, WO 09 / 121804, WO 10 / 108937 or US 2013 / 0129727, i.e., by suitably introducing threonine (T) at position 89, and optionally introducing one or more other amino acid residues / substitutions described herein, and optionally (and generally preferred, as described herein) adding a C-terminal extension (as further described herein).

[0139] Some preferred, and not limiting, examples of the serum albumin-binding nanobodies of this invention are humanized variants of the amino acid sequence of SEQ ID NO:52 of WO2006 / 122787 (referred to as “Alb-1” in WO 2006 / 122787), wherein the amino acid at position 89 is T (and it is optionally provided with a C-terminal extension as described herein), such as the humanized variants of Alb-1 given in SEQ ID NO:57 to 64 of WO 2006 / 122787 (in each case having a V89T substitution and optionally a C-terminal extension) or the humanized variants of Alb-1 given in SEQ ID NO:3 to 11 of WO 2012 / 175400 (again, in each case having a V89T substitution), wherein SEQ ID NO:3, 4, and 5 may optionally contain a C-terminal extension, and SEQ ID NO:57, 4, and 5 may optionally contain a C-terminal extension. 6 to 11 already contain C-terminal extensions (and similarly, this variant may contain one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q and / or S112K; although generally, when position 89 is T, it is not necessary to have one or two additional substitutions at positions 110 and / or 112, in which case position 110 is preferably T and position 112 is preferably S). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0140] Therefore, in another aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), wherein:

[0141] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0142] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0143] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0144] -The amino acid residue at position 89 is T;

[0145] And it optionally includes a C-terminal extension (X) at its C-terminus. nWhere 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also contain, for example, other substances mentioned herein). One or more of the amino acid residues / substitutions, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q and / or S112K; although generally, when position 89 is T, it is not usually necessary to have one or two additional substitutions at positions 110 and / or 112, in which case position 110 is preferably T and position 112 is preferably S). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A) or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, WO The amino acid residue mentioned at position 42 in Table A-6 of WO08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) may be, for example, one of the amino acid residues mentioned at position 87 in Table A-7 of WO08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) and may be, for example, threonine (T) or alanine (A).

[0146] In a particular aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), said nanobodies being humanized variants of SEQ ID NO:52 of WO 2006 / 122787, wherein:

[0147] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0148] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0149] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0150] -The amino acid residue at position 89 is T;

[0151] And it optionally includes a C-terminal extension (X) at its C-terminus. 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also contain, for example, other substances mentioned herein). One or more of the amino acid residues / substitutions, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q and / or S112K; although generally, when position 89 is T, it is not usually necessary to have one or two additional substitutions at positions 110 and / or 112, in which case position 110 is preferably T and position 112 is preferably S). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A) or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, WO The amino acid residue mentioned at position 42 in Table A-6 of WO08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) may be, for example, one of the amino acid residues mentioned at position 87 in Table A-7 of WO08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) and may be, for example, threonine (T) or alanine (A).

[0152] In a particular aspect, the present invention relates to nanobodies of the present invention (as defined herein), said nanobodies being capable of binding (and particularly specifically binding) serum albumin (and particularly human serum albumin), said nanobodies having at least 80%, preferably at least 85%, more preferably at least 90% (e.g., at least 95%) sequence identity (considering both the framework sequence and CDR, but not any C-terminal extension), wherein the amino acid residue at position 89 is T, and it optionally includes a C-terminal extension (X) at its C-terminus. nWhere 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also contain, for example, other substances mentioned herein). One or more of the amino acid residues / substitutions, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q and / or S112K; although generally, when position 89 is T, it is not usually necessary to have one or two additional substitutions at positions 110 and / or 112, in which case position 110 is preferably T and position 112 is preferably S). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0153] According to one particular aspect, any of the present invention’s serum albumin-binding nanobodies with a T at position 89 may also have the characteristic amino acid residues of Alb-23 and its variants as described in WO 12 / 175400 (i.e., amino acid motifs GP at positions 44 and 45, amino acid motifs SKN at positions 74 to 76, and preferably G at position 16 and R at position 83, as also optionally).

[0154] Similar to the VH domain of the present invention containing Q or K at position 112, the VH domain of the present invention containing T at position 89 (optionally together with Q or K at position 112 and / or together with the C-terminal extension) shows reduced binding by pre-existing antibodies, and in particular reduced binding by pre-existing antibodies (such as those found, for example, in samples obtained from SLE patients) capable of binding the VH domain and nanobodies in the presence of the C-terminal extension.

[0155] On the other hand, the present invention relates to immunoglobulin heavy chain variable domains (VH domains) wherein the amino acid residue at position 89 (Kabat number) is leucine (L) and the amino acid residue at position 110 is either a lysine (K) residue or a glutamine (Q) residue. Such immunoglobulin heavy chain variable domains (VH domains) with L at position 89 and K or Q at position 110 are also included in the term "VH domain of the present invention" as used herein in its broadest sense and may further be as described herein for other VH domains of the present invention (i.e., those containing K or Q at position 112 or T at position 89). Therefore, such immunoglobulin heavy chain variable domains (VH domains) may have a C-terminal extension as further described herein (including indicated preferred options for such C-terminal extension); may be ISVDs and particularly nanobodies, as further described herein.

[0156] Similarly, if the VH domain of this invention has an exposed C-terminal region (e.g., because it forms the C-terminus of the protein, polypeptide or other construct in which it is present), it preferably includes a C-terminal extension (see data shown in Table C below).

[0157] Furthermore, the amino acid contained at position 11 of the present invention, where position 89 is L and position 110 is K or Q (i.e., with or without C-terminal extension), is preferably selected from L (the most common amino acid residue in VHH), E, ​​K, M, S, V, W, or Y; more preferably from L, E, K, V, or Y; and even more preferably from L, K, or V (where V is the most preferred). For example, it may contain L11K or L11V substitution, and for example P14A or A14P substitution and / or Q108L substitution (it may also suitably contain S112K and / or S112Q mutations, although generally, when position 89 is L and position 110 is K or Q, it is not generally necessary to have additional substitution at position 112, in which case position 112 is preferably S). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0158] Furthermore, in one aspect, in this type of VH domain of the invention where 89 is L and 110 is K or Q, the amino acid residue at position 112 is serine (S). More particularly, the C-terminus of this VH domain can be (and preferably is) VKVSS (SEQ ID NO: 95), VQVSS (SEQ ID NO: 96), or VKVSS (X). n (SEQ ID NO:97) or VQVSS(X) nOne of (SEQ ID NO: 98), wherein n and X are as further described herein for the VH domain of the invention where position 112 is Q or K (and wherein any C-terminal extension is preferably as further described herein for the VH domain of the invention where position 112 is Q or K). Furthermore, similar to the VH domain of the invention where position 112 is Q or K or position 89 is T, when such a VH domain where position 89 is L and position 110 is K or Q is a nanobody, position 11 is preferably leucine (L), position 14 may particularly be alanine (A) or proline (P), and position 108 may particularly be Q or L (and preferably L in humanized nanobodies); and such nanobodies where position 89 is L and position 110 is K or Q may contain one or more nanobody marker residues and / or may be suitably humanized. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0159] The VH domain of this invention, with L at position 89 and K or Q at position 110, can also be a serum albumin nanobody, as further described herein. For example, such a serum albumin-binding nanobody can be one of sequences SEQ ID NO:46 to 75, but with L at position 89 and K or Q at position 110; or another serum albumin-binding nanobody with L at position 89 and K or Q at position 110, having at least 80%, preferably at least 85%, more preferably at least 90% (e.g., at least 95%) sequence identity (considering both the frame sequence and CDR, but not any C-terminal extension) to at least one of Alb-1 (SEQ ID NO:52 of WO2006 / 122787), Alb-8 (SEQ ID NO:46 herein), and / or Alb-23 (SEQ ID NO:61 herein).

[0160] More generally, the VH domain for serum albumin according to this aspect of the invention can be one of the nanobodies for (human) serum albumin described in WO 2004 / 041865, and in particular WO 2006 / 122787 and WO 2012 / 175400 (both applications of the applicant / assignee), wherein the amino acid at position 89 is leucine (L) and the amino acid residue at position 110 is K or Q, and it is optionally provided with a C-terminal extension as described herein (and may also contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L and / or Q108L; and S112K or S112Q, although generally, when position 89 is L and position 110 is K or Q, it is not generally necessary to have an additional substitution at position 112, in which case position 112 is preferably S). Furthermore, it is anticipated that the present invention can also be applied to other heavy chain ISVDs binding to serum albumin, such as those described in WO 03 / 035694, WO 04 / 003019, WO 05 / 118642, WO 06 / 059106, WO 08 / 096158, WO 09 / 121804, WO 10 / 108937, or US 2013 / 0129727, i.e., by suitably introducing leucine (L) at position 89 and K or Q at position 110, and optionally introducing one or more other amino acid residues / substitutions described herein) and optionally (and generally preferred, as described herein) adding C-terminal extensions (as further described herein). In these VH domains targeting serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NO: 95 to 98.Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0161] Some preferred, and not limiting, examples of this invention’s nanobody that binds to serum albumin are humanized variants of the amino acid sequence of SEQ ID NO:52 of WO2006 / 122787 (referred to as “Alb-1” in WO 2006 / 122787), wherein the amino acid at position 89 is L and the amino acid at position 110 is K or Q (and optionally it is provided with a C-terminal extension as described herein), such as the humanized variants of Alb-1 given in SEQ ID NO:57 to 64 of WO 2006 / 122787 (in each case having a V89L substitution and a T110Q or T110K substitution, and optionally having a C-terminal extension) or the humanized variants of Alb-1 given in SEQ ID NO:3 to 11 of WO 2012 / 175400 (again, in each case having a V89L substitution and a T110Q or T110K substitution), wherein SEQ ID NO 3, 4, and 5 may optionally contain a C-terminal extension, and SEQ ID NOs 6 to 11 already contain a C-terminal extension (and similarly, such variants may contain one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, and / or Q108L; and S112K or S112Q, although generally, when position 89 is L and position 110 is K or Q, it is not generally necessary to have an additional substitution at position 112, in which case position 112 is preferably S). In these VH domains targeting serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NOs: 95 to 98. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0162] Therefore, in another aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), wherein:

[0163] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0164] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0165] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0166] -The amino acid residue at position 89 is L;

[0167] -The amino acid residue at position 110 is K or Q;

[0168] And it optionally includes a C-terminal extension (X) at its C-terminus. 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L and / or Q108L; and S112K or S112Q, although generally, when position 89 is L and position 110 is K or Q, it is not generally necessary to have an additional substitution at position 112, in which case position 112 is preferably S). In these VH domains targeting serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NO: 95 to 98. Furthermore: (i) position 41 can be, for example, one of the amino acid residues mentioned for position 41 in Table A-6 of WO 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues), and can be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at this position in humans or camels, and can be more particularly proline (P) or alanine (A); and / or (ii) position 42 can be, for example, WO The amino acid residue mentioned at position 42 in Table A-6 of WO08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) may be, for example, one of the amino acid residues mentioned at position 87 in Table A-7 of WO08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) and may be, for example, threonine (T) or alanine (A).

[0169] In a particular aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), said nanobodies being humanized variants of SEQ ID NO:52 of WO 2006 / 122787, wherein:

[0170] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0171] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0172] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0173] -The amino acid residue at position 89 is L;

[0174] -The amino acid residue at position 110 is K or Q;

[0175] And it optionally includes a C-terminal extension (X) at its C-terminus. nWhere 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L and / or Q108L; and S112K or S112Q, although generally, when position 89 is L and position 110 is K or Q, it is not generally necessary to have an additional substitution at position 112, in which case position 112 is preferably S)). In these VH domains targeting serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NO: 95 to 98. Furthermore: (i) position 41 can be, for example, one of the amino acid residues mentioned for position 41 in Table A-6 of WO 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues), and can be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at this position in humans or camels, and can be more particularly proline (P) or alanine (A); and / or (ii) position 42 can be, for example, WO08 / 02 The amino acid residue mentioned at position 42 in Table A-6 of 0079 (i.e., human VH3 residue and / or camelid VHH residue) may be, for example, one of the amino acid residues mentioned at position 87 in Table A-7 of WO08 / 020079 (i.e., human VH3 residue and / or camelid VHH residue) and may be, for example, threonine (T) or alanine (A).

[0176] In a particular aspect, the present invention relates to nanobodies (as defined herein) capable of binding (and particularly specifically binding) serum albumin (and particularly human serum albumin), said nanobodies having at least 80%, preferably at least 85%, more preferably at least 90% (e.g., at least 95%) sequence identity with at least one of Alb-1 (SEQ ID NO:52 of WO 2006 / 122787), Alb-8 (SEQ ID NO:46 herein), and / or Alb-23 (SEQ ID NO:61 herein) (considering both the framework sequence and the CDR, but not any C-terminal extension), wherein the amino acid residue at position 89 is L and the amino acid residue at position 110 is K or Q, and optionally includes a C-terminal extension (X) at its C-terminus. 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L and / or Q108L; and S112K or S112Q, although generally, when position 89 is L and position 110 is K or Q, it is not generally necessary to have an additional substitution at position 112, in which case position 112 is preferably S). The nanobodies of this invention are also preferably humanized variants of Alb-1 (but with V89L and T110K or T110Q substitutions), and more preferably have at least one, particularly any two, and more particularly all three of CDR1, CDR2, and / or CDR3 respectively given in SEQ ID NOs 41 to 43. In these VH domains targeting serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NOs: 95 to 98. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0177] According to one specific aspect, any serum albumin-binding nanobody of the present invention with L at position 89 and K or Q at position 110 may also have the characteristic amino acid residues of Alb-23 and its variants as described in WO 12 / 175400 (i.e., amino acid motif GP at positions 44 and 45, amino acid motif SKN at positions 74 to 76, and preferably G at position 16 and R at position 83, as also optionally). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0178] Similar to the VH domain of the present invention containing Q or K at position 112 or T at position 89, the VH domain of the present invention containing L at position 89 and K or Q at position 110 shows reduced binding of pre-existing antibodies, and in particular reduced binding of pre-existing antibodies (such as those found, for example, in samples obtained from SLE patients) capable of binding the VH domain and nanobodies in the presence of C-terminal extension.

[0179] Some non-limiting examples of the VH structural domain of the present invention having L at position 89 and K at position 110 (and furthermore, V at position 11) are shown in Figure 2 The following are given as SEQ ID NO:123-136. These are VH domains that bind to human serum albumin and have the CDRs indicated herein for the preferred serum albumin-binding VH domains of the present invention.

[0180] On the other hand, the present invention relates to a variable domain (VH domain) of the immunoglobulin heavy chain, wherein the amino acid residue at position 89 (Kabat number) is leucine (L) and the amino acid residue at position 11 is valine (V). Such a variable domain (VH domain) of the immunoglobulin heavy chain with L at position 89 and V at position 11 is also included in the term "VH domain of the present invention" as used herein in its broadest sense and may further be as described herein for other VH domains of the present invention described herein (i.e., containing K or Q at position 112, T at position 89, or L at position 89 and K or Q at position 110; although for the VH domain according to this aspect, the amino acid residue at position 89 will be L, the amino acid residue at position 11 will be V, and the amino acid residues at positions 110 and 112 may be any amino acid residue suitable for these positions). Therefore, such immunoglobulin heavy chain variable domains (VH domains) can have C-terminal extensions as further described herein (including the preferred options for such C-terminal extensions indicated); they can be ISVDs and, in particular, nanobodies, as further described herein.

[0181] Similarly, if the VH domain of this invention has an exposed C-terminal region (e.g., because it forms the C-terminus of the protein, polypeptide or other construct in which it is present), it preferably includes a C-terminal extension (see data shown in Table C below).

[0182] Furthermore, the nanobody of the present invention with L at position 89 and V at position 11 (i.e., with or without C-terminal extension) contains: (i) preferably an amino acid selected from T, I, A, K, or Q (and preferably from T, K, or Q, and particularly may be T) at position 110; (ii) preferably an amino acid selected from S, F, K, or Q (and more preferably S, K, or Q, and particularly may be S) at position 112; and (iii) may, for example, also contain P14A or A14P substitution and / or Q108L substitution. According to a specific embodiment, in the VH domain of this aspect of the invention, the amino acid residue at position 110 is T and the amino acid residue at position 112 is S, and more preferably the C-terminus is VTVSS (SEQ ID NO: 77) or VTVSS(X)n (SEQ ID NO: 78), wherein X and n are as defined herein for the C-terminal extension of other VH domains of the present invention. Furthermore, in the VH domain according to this aspect of the invention: (i) position 41 may, for example, be one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may particularly be (or selected from) proline (P), serine (S), threonine (T), alanine (A) or leucine (L), which are some of the most frequently occurring amino acid residues at this position in humans or camels, and may more particularly be proline (P) or alanine (A); and / or (ii) position 42 may, for example, be one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may particularly be (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may, for example, be WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0183] Therefore, for example, in the VH domain of the present invention where 89 is L and 11 is V, the C-terminus of such VH domain may be (and preferably is) VTVSS (SEQ ID NO:77), VTVSS(X)n (SEQ ID NO:78), VTVKS (SEQ ID NO:1), VTVKS(X)n (SEQ ID NO:21), VTVQS (SEQ ID NO:2), VTVQS(X)n (SEQ ID NO:22), VKVSS (SEQ ID NO:95), VKVSS(X)n (SEQ ID NO:97), VQVSS (SEQ ID NO:96), VQVSS(X)n (SEQ ID NO:98), VZVZS (SEQ ID NO:107, wherein each amino acid residue Z is independently K or Q) or VZVZSX(n) (SEQ ID NO:107). NO:108, wherein each amino acid residue Z is independently one of K or Q (and in particular VTVSS, VKVSS (SEQ ID NO:95), VQVSS (SEQ ID NO:96), VKVSS (X)). n (SEQ ID NO:97) or VQVSS(X) nOne of (SEQ ID NO: 98), and more particularly VTVSS (SEQ ID NO: 77) or VTVSS(X)n (SEQ ID NO: 78), where n and X are as further described herein for the VH domain of the invention where position 112 is Q or K (and where any C-terminal extension is preferably as further described herein for the VH domain of the invention where position 112 is Q or K). Furthermore, similar to the VH domain of the invention where position 112 is Q or K or position 89 is T, when such a VH domain is L at position 89 and V at position 11 is a nanobody, position 14 may be particularly alanine (A) or proline (P) and position 108 may be Q or L (and preferably L in humanized nanobodies); and such nanobodies where position 89 is L and position 11 is V may contain one or more nanobody marker residues and / or may be suitably humanized. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0184] The VH domain of this invention, with L at position 89 and V at position 11, can also be a serum albumin nanobody as further described herein. For example, such a serum albumin-binding nanobody can be one of sequences SEQ ID NO:46 to 75, but with L at position 89 and V at position 11; or another serum albumin-binding nanobody having at least 80%, preferably at least 85%, more preferably at least 90% (e.g., at least 95%) sequence identity (considering both the frame sequence and CDR, but not any C-terminal extension) with L at position 89 and V at position 11, and at least one of Alb-1 (SEQ ID NO:52 of WO 2006 / 122787), Alb-8 (SEQ ID NO:46 herein), and / or Alb-23 (SEQ ID NO:61 herein).

[0185] More generally, the VH domain of serum albumin according to this aspect of the invention can be one of the nanobodies for (human) serum albumin described in WO 2004 / 041865 and, in particular, WO 2006 / 122787 and WO 2012 / 175400 (both applications of the applicant / assignee), wherein the amino acid at position 89 is leucine (L) and the amino acid residue at position 11 is V, and it is optionally provided with a C-terminal extension as described herein (and may also suitably contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 14A, P14A, 14P, A14P, 108L, Q108L, V110K, V110Q, S112K and / or S112Q). Furthermore, it is anticipated that the present invention can also be applied to other heavy chain ISVDs binding to serum albumin, such as those described in WO 03 / 035694, WO 04 / 003019, WO 05 / 118642, WO 06 / 059106, WO08 / 096158, WO 09 / 121804, WO 10 / 108937, or US 2013 / 0129727, i.e., by suitably introducing leucine (L) at position 89 and valine at position 11, and optionally introducing one or more other amino acid residues / substitutions described herein) and optionally (and generally preferred, as described herein) adding C-terminal extensions (as further described herein). In these VH domains targeting serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NO: 95 to 98. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0186] Some preferred, and not limiting, examples of this invention’s nanobody that binds to serum albumin are humanized variants of the amino acid sequence of SEQ ID NO:52 of WO2006 / 122787 (referred to as “Alb-1” in WO 2006 / 122787), wherein the amino acid at position 89 is L and the amino acid at position 11 is V (and it is optionally provided with a C-terminal extension as described herein), such as the humanized variants of Alb-1 given in SEQ ID NO:57 to 64 of WO 2006 / 122787 (in each case having V89L substitution and L11V substitution, and optionally having a C-terminal extension) or the humanized variants of Alb-1 given in SEQ ID NO:3 to 11 of WO 2012 / 175400 (again, in each case having V89L substitution and L11V), wherein SEQ ID NO:3, 4, and 5 may optionally contain a C-terminal extension, and SEQ ID NO:57, 4, and 5 may optionally contain a C-terminal extension. Residues 6 to 11 already contain a C-terminal extension (and similarly, this variant may suitably contain one or more of other specific amino acid residues / substitutions mentioned herein, such as 14A, P14A, 14P, A14P, 108L, Q108L, T110K, T110Q, S112K and / or S112Q). In these VH domains for serum albumin, where L is at position 89 and V is at position 11, the amino acid at position 110 is preferably T and the amino acid residue at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NO: 95 to 98. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0187] Therefore, in another aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), wherein:

[0188] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0189] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0190] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0191] -The amino acid residue at position 89 is L;

[0192] -The amino acid residue at position 11 is V;

[0193] Preferably

[0194] - The amino acid residue at position 110 is one of K, Q, or T, and more preferably T;

[0195] - The amino acid residue at position 112 is one of K, Q or S, and more preferably S;

[0196] And it optionally includes a C-terminal extension (X) at its C-terminus. nWhere 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also suitably contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 14A, P14A, 14P, A14P, 108L, Q108L). Similarly, in the VH domain for serum albumin where L is at position 89 and V is at position 11, the amino acid at position 110 is preferably T and the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus that is one of SEQ ID NO: 95 to 98. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0197] In a particular aspect, the present invention relates to nanobodies (as defined herein) that can bind (and particularly specifically bind) serum albumin (and particularly human serum albumin), said nanobodies being humanized variants of SEQ ID NO:52 of WO 2006 / 122787, wherein:

[0198] -CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41);

[0199] -CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42);

[0200] -CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43);

[0201] -The amino acid residue at position 89 is L;

[0202] -The amino acid residue at position 11 is V;

[0203] And preferably:

[0204] - The amino acid residue at position 110 is one of K, Q, or T, and more preferably T;

[0205] - The amino acid residue at position 112 is one of K, Q or S, and more preferably S;

[0206] And it optionally includes a C-terminal extension (X) at its C-terminus. nWhere 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L and / or Q108L). In the VH domains for serum albumin with L at position 89 and V at position 11, the amino acid residue at position 110 is preferably T and the amino acid residue at position 112 is preferably S, and the VH domains preferably have a C-terminus, which is one of SEQ ID NO: 95 to 98. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0207] In a particular aspect, the present invention relates to nanobodies (as defined herein) capable of binding (and particularly specifically binding) serum albumin (and particularly human serum albumin), said nanobodies having at least 80%, preferably at least 85%, more preferably at least 90% (e.g., at least 95%) sequence identity (considering both the framework sequence and CDR, but not any C-terminal extension), wherein the amino acid residue at position 89 is L and the amino acid residue at position 11 is V, and optionally it includes a C-terminal extension (X) at its C-terminus. nWhere 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (again, this C-terminal extension is preferably as further described herein, and similarly, this nanobody may also suitably contain, for example, one or more of other specific amino acid residues / substitutions mentioned herein, such as 14A, P14A, 14P, A14P, 108L, Q108L, T110K, T110Q, S112K and / or S112Q). The nanobodies of this invention are also preferably humanized variants of Alb-1 (but with V89L and L11V substitutions), and more preferably have at least one, particularly any two, and more particularly all three of CDR1, CDR2, and / or CDR3 respectively given in SEQ ID NOs 41 to 43. In these VH domains targeting serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus, which is one of SEQ ID NOs: 95 to 98. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0208] According to one specific aspect, any of the serum albumin-binding nanobodies of this invention with L at position 89 and V at position 11 can also have the characteristic amino acid residues of Alb-23 and its variants as described in WO 12 / 175400 (i.e., amino acid motif GP at positions 44 and 45, amino acid motif SKN at positions 74 to 76, and preferably G at position 16 and R at position 83, as also optionally). Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0209] Similar to the VH domains of the present invention containing Q or K at position 112 or T at position 89, the VH domains of the present invention containing L at position 89 and V at position 11 show reduced binding to pre-existing antibodies, and particularly reduced binding to pre-existing antibodies (such as those found, for example, in samples obtained from SLE patients) capable of binding to the VH domain and nanobodies in the presence of C-terminal extension. Another noteworthy point in relation to the VH domains of the present invention where position 89 is L and position 11 is V is that these substitutions are known to occur at certain frequencies in human VH domains (see Table A-5 of WO08 / 020079 for position 11, and Table A-7 of WO 08 / 020079 for position 89).

[0210] Some non-limiting examples of the VH domain of the present invention having a V at position 11 and a T at position 89 are shown below. Figure 2The sequences given as SEQ ID NO:109-136 are shown in these VH domains. Among these VH domains, the sequences given as SEQ ID NO:123-136 contain the T110K mutation in addition to the L11V and V89L mutations. These are VH domains that bind human serum albumin and have the CDRs indicated herein for the preferred serum albumin-binding VH domains of the present invention.

[0211] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0212] -The amino acid residue at position 11 is V; and

[0213] - The amino acid residue at position 14 is either A or P; and

[0214] - The amino acid residue at position 41 is either A or P; and

[0215] -The amino acid residue at position 89 is L; and

[0216] - The amino acid residue at position 108 is either Q or L; and

[0217] -The amino acid residue at position 110 is one of T, K, or Q; and

[0218] -The amino acid residue at position 112 is one of S, K, or Q;

[0219] Furthermore, the VH domain optionally includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). Furthermore: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079, and may be particularly (or selected from) threonine (T) or alanine (A). Preferably, in the VH domain according to this paragraph, the amino acid residue at position 110 is T and the amino acid residue at position 112 is S. Likewise, like other VH domains of the present invention, this VH domain can target any suitable target (and particularly therapeutically relevant targets). According to one specific aspect, this VH domain targets serum albumin.

[0220] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0221] - The amino acid residue at position 11 is one of L, V, or K; and

[0222] - The amino acid residue at position 14 is either A or P; and

[0223] - The amino acid residue at position 41 is either A or P; and

[0224] - The amino acid residue at position 89 is one of T, V, or L; and

[0225] - The amino acid residue at position 108 is either Q or L; and

[0226] -The amino acid residue at position 110 is one of T, K, or Q; and

[0227] -The amino acid residue at position 112 is one of S, K, or Q;

[0228] Wherein (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0229] The VH domain optionally includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0230] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0231] - The amino acid residue at position 11 is one of L, V, or K; and

[0232] - The amino acid residue at position 14 is either A or P; and

[0233] - The amino acid residue at position 41 is either A or P; and

[0234] - The amino acid residue at position 89 is one of T, V, or L; and

[0235] - The amino acid residue at position 108 is either Q or L; and

[0236] -The amino acid residue at position 110 is one of T, K, or Q; and

[0237] -The amino acid residue at position 112 is one of S, K, or Q;

[0238] Wherein (i) the amino acid residue at position 112 is one of K or Q, the amino group at position 89 is one of T, V or L (and preferably V), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); and / or (ii) the amino acid residue at position 89 is T, and the amino acid residue at position 112 is one of S, K or Q (and preferably S), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q, and the amino acid residue at position 112 is one of S, K or Q (and preferably S); and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0239] The VH domain optionally includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0240] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0241] - The amino acid residue at position 11 is one of L, V, or K; and

[0242] - The amino acid residue at position 14 is either A or P; and

[0243] - The amino acid residue at position 41 is either A or P; and

[0244] - The amino acid residue at position 89 is one of T, V, or L; and

[0245] - The amino acid residue at position 108 is either Q or L; and

[0246] -The amino acid residue at position 110 is one of T, K, or Q; and

[0247] -The amino acid residue at position 112 is one of S, K, or Q;

[0248] Wherein (i) the amino acid residue at position 112 is one of K or Q; or (ii) the amino acid residue at position 89 is T; or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0249] The VH domain optionally includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0250] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0251] - The amino acid residue at position 11 is one of L, V, or K; and

[0252] - The amino acid residue at position 14 is either A or P; and

[0253] - The amino acid residue at position 41 is either A or P; and

[0254] - The amino acid residue at position 89 is one of T, V, or L; and

[0255] - The amino acid residue at position 108 is either Q or L; and

[0256] -The amino acid residue at position 110 is one of T, K, or Q; and

[0257] -The amino acid residue at position 112 is one of S, K, or Q;

[0258] Wherein (i) the amino acid residue at position 112 is one of K or Q, the amino group at position 89 is one of T, V or L (and preferably V), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); or (ii) the amino acid residue at position 89 is T, and the amino acid residue at position 112 is one of S, K or Q (and preferably S), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); or (iii) the amino acid residue at position 89 is L, and the amino acid residue at position 110 is one of K or Q, and the amino acid residue at position 112 is one of S, K or Q (and preferably S); and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V;

[0259] Furthermore, the VH domain optionally includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0260] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0261] - The amino acid residue at position 11 is one of L, V, or K; and

[0262] - The amino acid residue at position 14 is either A or P; and

[0263] - The amino acid residue at position 41 is either A or P; and

[0264] - The amino acid residue at position 89 is one of T, V, or L; and

[0265] - The amino acid residue at position 108 is either Q or L; and

[0266] -The amino acid residue at position 110 is one of T, K, or Q; and

[0267] -The amino acid residue at position 112 is one of S, K, or Q;

[0268] Wherein (i) the amino acid residue at position 112 is one of K or Q, the amino group at position 89 is V, and the amino acid residue at position 110 is T; or (ii) the amino acid residue at position 89 is T, the amino acid residue at position 112 is S, and the amino acid residue at position 110 is T; or (iii) the amino acid residue at position 89 is L, the amino acid residue at position 110 is one of K or Q, and the amino acid residue at position 112 is S (and preferably S); and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0269] The VH domain optionally includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0270] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0271] - The amino acid residue at position 11 is one of L, V, or K; and

[0272] - The amino acid residue at position 14 is either A or P; and

[0273] - The amino acid residue at position 41 is either A or P; and

[0274] - The amino acid residue at position 89 is one of T, V, or L; and

[0275] - The amino acid residue at position 108 is either Q or L; and

[0276] -The amino acid residue at position 110 is one of T, K, or Q; and

[0277] -The amino acid residue at position 112 is one of S, K, or Q;

[0278] Wherein (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0279] The VH domain includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0280] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0281] - The amino acid residue at position 11 is one of L, V, or K; and

[0282] - The amino acid residue at position 14 is either A or P; and

[0283] - The amino acid residue at position 41 is either A or P; and

[0284] - The amino acid residue at position 89 is one of T, V, or L; and

[0285] - The amino acid residue at position 108 is either Q or L; and

[0286] -The amino acid residue at position 110 is one of T, K, or Q; and

[0287] -The amino acid residue at position 112 is one of S, K, or Q;

[0288] Wherein (i) the amino acid residue at position 112 is one of K or Q, the amino group at position 89 is one of T, V or L (and preferably V), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); and / or (ii) the amino acid residue at position 89 is T, and the amino acid residue at position 112 is one of S, K or Q (and preferably S), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q, and the amino acid residue at position 112 is one of S, K or Q (and preferably S); and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0289] The VH domain includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0290] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0291] - The amino acid residue at position 11 is one of L, V, or K; and

[0292] - The amino acid residue at position 14 is either A or P; and

[0293] - The amino acid residue at position 41 is either A or P; and

[0294] - The amino acid residue at position 89 is one of T, V, or L; and

[0295] - The amino acid residue at position 108 is either Q or L; and

[0296] -The amino acid residue at position 110 is one of T, K, or Q; and

[0297] -The amino acid residue at position 112 is one of S, K, or Q;

[0298] Wherein (i) the amino acid residue at position 112 is one of K or Q; or (ii) the amino acid residue at position 89 is T; or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0299] The VH domain includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0300] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0301] - The amino acid residue at position 11 is one of L, V, or K; and

[0302] - The amino acid residue at position 14 is either A or P; and

[0303] - The amino acid residue at position 41 is either A or P; and

[0304] - The amino acid residue at position 89 is one of T, V, or L; and

[0305] - The amino acid residue at position 108 is either Q or L; and

[0306] -The amino acid residue at position 110 is one of T, K, or Q; and

[0307] -The amino acid residue at position 112 is one of S, K, or Q;

[0308] Wherein (i) the amino acid residue at position 112 is one of K or Q, the amino group at position 89 is one of T, V or L (and preferably V), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); or (ii) the amino acid residue at position 89 is T, and the amino acid residue at position 112 is one of S, K or Q (and preferably S), and the amino acid residue at position 110 is one of T, K or Q (and preferably T); or (iii) the amino acid residue at position 89 is L, and the amino acid residue at position 110 is one of K or Q, and the amino acid residue at position 112 is one of S, K or Q (and preferably S); and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0309] The VH domain includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0310] In another aspect, the present invention relates to a VH domain (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein:

[0311] - The amino acid residue at position 11 is one of L, V, or K; and

[0312] - The amino acid residue at position 14 is either A or P; and

[0313] - The amino acid residue at position 41 is either A or P; and

[0314] - The amino acid residue at position 89 is one of T, V, or L; and

[0315] - The amino acid residue at position 108 is either Q or L; and

[0316] -The amino acid residue at position 110 is one of T, K, or Q; and

[0317] -The amino acid residue at position 112 is one of S, K, or Q;

[0318] Wherein (i) the amino acid residue at position 112 is one of K or Q, the amino group at position 89 is V, and the amino acid residue at position 110 is T; or (ii) the amino acid residue at position 89 is T, the amino acid residue at position 112 is S, and the amino acid residue at position 110 is T; or (iii) the amino acid residue at position 89 is L, the amino acid residue at position 110 is one of K or Q, and the amino acid residue at position 112 is S (and preferably S); and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V; and

[0319] The VH domain includes a C-terminal 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 (preferably naturally occurring) an amino acid residue that is independently selected, and preferably independently selected from the group consisting of: alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) (the C-terminal extension is preferably as further described herein). In addition: (i) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079 and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (ii) position 87 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 87 in Table A-7 of WO 08 / 020079 and may be particularly (or selected from) threonine (T) or alanine (A). Another aspect of the invention relates to a VH domain as described in this paragraph (and particularly as a VH domain of an ISVD, and even more particularly as a VH domain of a nanobody), wherein the amino acid residue at position 11 is V and the amino acid residue at position 110 is K or Q (and wherein the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above emphasis, the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph), and the VH domain may optionally contain a C-terminal extension (X) as described in this paragraph. n ).

[0320] Similarly, in the VH domain of the invention as defined herein, amino acid residues at positions not explicitly defined herein may be any amino acid residue suitable for the VH domain at that position, and particularly suitable for ISVD and even more particularly suitable for nanobodies (including humanized VHH domains). See also prior art referenced herein, such as, for example, Tables A-3 and A-5 to A-8 of WO 08 / 020079. Preferably, in each case, the amino acid residue at position 11 is L or V, and more preferably V. Furthermore: (i) position 41 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 41 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L), which are some of the most frequently occurring amino acid residues at that position in humans or camels, and may be more particularly proline (P) or alanine (A); and / or (ii) position 42 may be, for example, one of the amino acid residues (i.e., human VH3 residues and / or camelid VHH residues) mentioned for position 42 in Table A-6 of WO 08 / 020079, and may be particularly (or selected from) glycine (G) or glutamic acid (E); and / or (iii) position 87 may be, for example, WO One of the amino acid residues mentioned at position 87 in Table A-7 of 08 / 020079 (i.e., human VH3 residues and / or camelid VHH residues) and may be particularly (or selected from) threonine (T) or alanine (A).

[0321] Furthermore, the VH domain of the present invention can target any suitable target, and particularly therapeutic targets. In one aspect, it targets human serum proteins such as human serum albumin.

[0322] The present invention also relates to proteins, peptides, constructs, compounds or other chemical entities (collectively referred to herein as "compounds of the present invention") comprising at least one VH domain of the present invention.

[0323] As further described herein, according to one particular aspect, in the compounds of the present invention, the VH domain of the present invention is present at / forms its C-terminus. In this case, the VH domain of the present invention forming the C-terminus of the compound of the present invention / present at the C-terminus of the compound of the present invention preferably has a C-terminal extension as described herein.

[0324] As further described herein, the compounds of the present invention may be ScFv, biantibodies, or another protein, peptide, or construct (wherein one or more VH domains of the present invention bind to one or more VL domains to form one or more functional antigen-binding sites).

[0325] However, according to a preferred aspect of the invention, the VH domain of the invention is an ISVD and the compounds of the invention are proteins, peptides, constructs, compounds, or other chemical entities comprising at least one ISVD of the invention and optionally one or more additional amino acid sequences, portions, binding domains, or binding units (optionally suitably linked to each other via one or more linkers) or substantially composed thereof. In particular, such compounds of the invention may comprise or be composed of one or more ISVDs, at least one of which is an ISVD of the invention. Such compounds of the invention may particularly have an ISVD of the invention at its C-terminus, in which case the ISVD of the invention preferably also has a C-terminal extension as described herein. Furthermore, if such compounds of the present invention contain two or more ISVDs, the two or more or substantially all of the ISVDs present may be the ISVDs of the present invention (i.e., each having at least one of the following amino acid residues / substitutions: 112K, 112Q, S112K, S112Q, 89T and / or V89T, or a combination of V89L with T110K or T110Q; and optionally one or more other substitutions mentioned herein for the ISVDs of the present invention, such as L11V). Furthermore, in such compounds of the present invention, the ISVDs of the present invention are preferably nanobodies of the present invention, and all or substantially all of the ISVDs present in the compounds of the present invention may be (and preferably are) nanobodies (and particularly the nanobodies of the present invention, i.e. each having at least one of the following amino acid residues / substitutions: 112K, 112Q, S112K, S112Q, 89T and / or V89T, or a combination of V89L with T110K or T110Q; and optionally one or more other substitutions mentioned herein for the nanobodies of the present invention, such as L11V). Based on the further disclosure herein, examples of such compounds of the present invention will be apparent to those skilled in the art.

[0326] Non-limiting examples of proteins, peptides, constructs, compounds, or other chemical entities comprising one or more ISVDs (including at least one ISVD of the present invention) are multivalent, multispecific (e.g., bispecific), or multicomplementary (e.g., bicomplementary) constructs containing two or more ISVDs linked directly or via one or more suitable linkers. Similarly, the ISVD is preferably a nanobody. For some non-limiting examples of such constructs and general teachings on how such constructs can be prepared (particularly based on nanobodies), reference can be made to review articles such as Conrath et al., JBC 276, 10(9), 7346 (2001) and Muyldermans. Reviews in Mol. Biotechnol., 74:27 (2001).

[0327] For example, such compounds of the present invention containing two or more ISVDs (at least one of which is an ISVD of the present invention) can be divalent, trivalent, tetravalent, or pentavalent constructs, and / or can be monospecific, bispecific, or trispecific constructs, and / or can be bicomplementary or tricomplementary constructs. Reference is also made to the prior art concerning IVSD-based and nanobody-based biopharmaceuticals cited herein. Furthermore, such compounds of the present invention may have an increased half-life through functionalization and / or by including in construct portions or binding units that increase the half-life of the construct. Examples of such functionalizations, portions, or binding units will be apparent to those skilled in the art and may include, for example, as described herein, and may include, for example, PEGylation, fusion with serum albumin, or fusion with a peptide or binding unit that can bind serum proteins such as serum albumin. Such serum albumin-binding peptides or binding domains can be any suitable serum albumin-binding peptides or binding domains capable of increasing the half-life of the construct (compared to the same construct without serum albumin-binding peptides or binding domains), and can in particular be serum albumin-binding peptides as described in the applicant's WO 2008 / 068280 (and in particular the applicant's WO 2009 / 127691 and unpublished U.S. application 61 / 301,819), or serum albumin-binding ISVs (such as serum albumin-binding nanobodies; for example, Alb-1 or humanized versions of Alb-1 such as Alb-8, with reference to, for example, WO 06 / 122787), or the ISVDs of the present invention targeting (human) serum proteins such as (human) serum albumin (as further described herein). Generally, any compound of the present invention having an increased half-life will preferably have a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days (as defined herein) in human subjects.

[0328] When the compounds of the present invention comprise at least one (and preferably one) ISVD of the present invention targeting (human) serum proteins, and particularly (human) serum albumin (and especially the nanobodies of the present invention), the compounds of the present invention will generally also contain one or more other therapeutically active amino acid sequences, moieties, binding domains, or binding units (i.e., targeting a therapeutically relevant target, pathway, or mechanism), and the ISVD of the present invention will serve to prolong its (and the compound as a whole) half-life. Similarly, the one or more additional therapeutically active moieties are preferably ISVDs (and more preferably nanobodies), and may also be IVSDs of the present invention (and more preferably nanobodies of the present invention). In such compounds of the present invention, the ISVD of the present invention targeting human serum albumin may also be present at / form the C-terminus of the compound, and in this case may (and preferably) include C-terminal extensions as described herein. When the compounds of the present invention comprise the ISVD of the present invention for (human) serum albumin, the compounds of the present invention preferably have a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days (as defined herein) in human subjects who have been or have been administered the compounds of the present invention. Some non-limiting examples of the ISVDs of the present invention for human serum albumin that can be used for this purpose are further described herein.

[0329] In one aspect, all ISVDs or nanobodies present in the compounds of the present invention are ISVDs of the present invention (meaning they have the characteristic amino acid residues / substitutions of the VH domain of the present invention as defined herein, i.e., at least 112K or Q, or at least 89T, or at least 89L together with 110K or 110Q). When all ISVDs in the compounds of the present invention are ISVDs of the present invention, they may have the same substitutions (e.g., all have S112K or S112Q substitutions) or different substitutions (e.g., one may have S1112K or S112Q substitutions, while another may have a V89L mutation together with T110K or T110Q). Furthermore, generally, only the ISVDs at the C-terminus of the compounds of the present invention have C-terminal extensions (because others will likely be linked at their C-terminus to another ISVD present in the compound).

[0330] Therefore, in another aspect, the present invention relates to proteins, polypeptides or other compounds or molecules (as further described herein) that comprise or are substantially composed of the ISVD of the present invention.

[0331] The present invention also relates to proteins, polypeptides or other compounds or molecules that comprise at least one ISVD of the present invention and at least one other therapeutic portion or entity (directly or via a suitable connector).

[0332] The present invention also relates to proteins, polypeptides or other compounds or molecules comprising at least one ISVD of the present invention targeting (human) serum proteins (and preferably targeting human serum albumin) and at least one other therapeutic portion or entity (directly linked or linked via a suitable connector).

[0333] The present invention also relates to proteins, peptides, or other compounds or molecules comprising at least two (e.g., two, three, or four) immunoglobulin monovariable domains (directly linked or linked via suitable linkers), wherein at least one of them is an ISVD of the present invention. In this respect: (i) the present ISVDs may suitably be the same or different; and when they are different, they may target the same target (e.g., they may have different sequences and / or different epitopes on the same target) or target two or more different targets (i.e., the resulting protein, peptide, or other compound or molecule is a bispecific or multispecific construct); and / or (ii) the ISVD present at the C-terminus of the protein, peptide, or other compound or molecule may or may not be an ISVD of the present invention (but preferably is); and / or (iii) when the ISVD of the present invention is present at the C-terminus of the protein, peptide, or other compound or molecule, it preferably has a C-terminal extension as described herein; and / or (iv) substantially all ISVDs present in the protein, peptide, or other compound or molecule may be ISVDs of the present invention. Furthermore, when the ISVD targets different targets (at least one of which is a therapeutic target), according to another aspect, at least one of the existing ISVDs may target a (human) serum protein such as human serum albumin (and the ISVD may or may not be the ISVD of the present invention; and when it is the ISVD of the present invention, it is preferably a nanobody targeting human serum albumin as further described herein).

[0334] The present invention also relates to proteins, polypeptides or other compounds or molecules that comprise or are substantially composed of two immunoglobulin monovariable domains (directly linked or linked via suitable linkers).

[0335] The present invention also relates to proteins, polypeptides or other compounds or molecules that comprise or are substantially composed of three immunoglobulin monovariable domains (directly linked or linked via suitable linkers).

[0336] The present invention also relates to proteins, polypeptides or other compounds or molecules that comprise or are substantially composed of four immunoglobulin monovariable domains (directly linked or linked via suitable linkers).

[0337] The present invention also relates to proteins, polypeptides, or other compounds or molecules that further comprise at least one portion, binding domain, or binding unit, said at least one portion, binding domain, or binding unit imparting an increased half-life to said protein, polypeptide, or other compound or molecule (i.e., compared to a corresponding protein, polypeptide, or other compound or molecule without said portion, binding domain, or binding unit). According to a more particular aspect, said at least one portion, binding domain, or binding unit imparting the increased half-life to said protein, polypeptide, or other compound or molecule is an immunoglobulin monovariable domain, more particularly for serum proteins (such as serum albumin), and especially for human serum proteins (such as human serum albumin); and as described herein, it can be particularly the ISVD of the present invention. The ISVD for serum proteins may be at the N-terminus, C-terminus, or (if said protein, polypeptide, or other compound or molecule contains more than two ISVDs) of said protein, polypeptide, or other compound or molecule at the middle of the molecule.

[0338] This invention also relates to proteins, polypeptides, or other compounds or molecules that comprise or are substantially composed of the following:

[0339] - Two immunoglobulin monovariable domains (directly linked or linked via a suitable linker), namely, one immunoglobulin monovariable domain (such as a nanobody) that provides an increased half-life and the other immunoglobulin monovariable domain (such as a nanobody) that can specifically target a therapeutic target.

[0340] - Three immunoglobulin monovariable domains (directly linked or linked via suitable linkers), namely, one immunoglobulin monovariable domain (e.g., a nanobody) with an increased half-life and two other immunoglobulin monovariable domains (e.g., two other nanobodies) that can specifically target a therapeutic target (wherein the other two immunoglobulin monovariable domains can target the same target, two different targets, or two different epitopes on the same target); or

[0341] - Four immunoglobulin monovariable domains (directly linked or linked via suitable linkers), namely, one immunoglobulin monovariable domain (e.g., nanobody) that provides an increased half-life and two other immunoglobulin monovariable domains (e.g., two other nanobodies) that can specifically target therapeutic targets (wherein the other three immunoglobulin monovariable domains can target the same target, two or three different targets and / or two or three different epitopes on the same target).

[0342] Similarly, in such proteins, peptides, or other compounds or molecules: (i) the present ISVDs may suitably be the same or different; and when they are different, they may target the same target (e.g., they may have different sequences and / or different epitopes on the same target) or target two or more different targets (i.e., the resulting protein, peptide, or other compound or molecule is a bispecific or multispecific construct); and / or (ii) the ISVD present at the C-terminus of the protein, peptide, or other compound or molecule may or may not be the ISVD of the present invention (but preferably is); and / or (iii) when the ISVD of the present invention is present at the C-terminus of the protein, peptide, or other compound or molecule, it preferably has a C-terminal extension as described herein; and / or (iv) substantially all ISVDs present in the protein, peptide, or other compound or molecule may be the ISVD of the present invention.

[0343] This invention also relates to methods for expressing / producing / preparing the VH domains of the invention and the compounds of the invention (as further described herein). For example, the VH domains of the invention can be expressed / produced by suitably expressing the nucleic acid encoding them in a suitable host organism. Reference is made to, for example, WO 08 / 020079 (and several other applicant / assignee patent applications cited herein), which generally describes methods and techniques suitable for expressing / producing nanobodies, methods which can also be applied to expressing / producing the nanobodies of the invention. Based on this disclosure and the prior art cited herein, methods for expressing the VH domains of the invention, other than nanobodies, will also be apparent to those skilled in the art. The compounds of the invention can be suitably produced / prepared by suitably linking (usually via covalent bonds) one or more VH domains of the invention to one or more additional amino acid residues (and / or other groups or portions) present in the final compound of the invention, optionally via one or more linkers or spacer regions. Alternatively, when the compounds of the invention are proteins or polypeptides, they can be produced / prepared by suitably expressing the nucleic acid encoding them in a suitable host organism. Also refer to, for example, WO 08 / 020079 and other other patent applications of the applicant / assignee cited herein, the general methods described therein.

[0344] This invention also relates to nucleotide sequences and / or nucleic acids encoding the VH domain of the invention or the compounds of the invention. Such nucleic acids may be DNA or RNA; and are preferably DNA and may be in the form of plasmids or vectors. Reference is also made to the general methods described, for example, in WO 08 / 020079 and other applicant / assignee patent applications cited herein.

[0345] The present invention also relates to compositions comprising at least one VH domain of the present invention, a compound of the present invention, or a nucleic acid encoding any of them.

[0346] The present invention also relates to pharmaceutical compositions comprising an ISV (and preferably a therapeutic ISV) or a protein or polypeptide comprising at least one ISV (and preferably at least one therapeutic ISV), wherein the ISV, protein, or polypeptide is as further described herein (i.e., an ISV, protein, or polypeptide according to one or more aspects described herein, and particularly according to one or more aspects described above; and more particularly an ISV, protein, or polypeptide having a C-terminal / sequence according to one or more aspects described herein), and at least one suitable carrier, diluent, or excipient (i.e., suitable for pharmaceutical use), and optionally one or more additional active substances. Such compositions, carriers, diluents, or excipients may be, for example, as described in WO 08 / 020079 for pharmaceutical compositions comprising nanobodies or proteins or polypeptides comprising at least one nanobodies (and as already mentioned, according to the present invention, the ISV is also preferably a nanobodies).

[0347] The present invention also relates to an ISV or a protein or polypeptide comprising at least one ISV for treating diseases in humans (e.g., patients requiring such treatment), wherein the ISV, protein or polypeptide is as further described herein (i.e., an ISV, protein or polypeptide according to one or more aspects described herein, and in particular an ISV, protein or polypeptide according to one or more aspects described above; and more particularly an ISV, protein or polypeptide having a C-terminal / sequence according to one or more aspects described herein).

[0348] The present invention also relates to the use of an ISV or a protein or polypeptide comprising at least one ISV for the preparation of a pharmaceutical composition, wherein the ISV, protein or polypeptide is as further described herein (i.e., an ISV, protein or polypeptide according to one or more aspects described herein, and in particular an ISV, protein or polypeptide according to one or more aspects described above; and more particularly an ISV, protein or polypeptide having a C-terminal / sequence according to one or more aspects described herein).

[0349] The present invention also relates to a treatment method comprising administering to a human subject (e.g., a patient requiring such treatment) an ISV used to prepare a pharmaceutical composition or a protein or polypeptide containing at least one ISV, wherein the ISV, protein, or polypeptide is as further described herein (i.e., an ISV, protein, or polypeptide according to one or more aspects described herein, and particularly according to one or more aspects described above; and more particularly having an ISV, protein, or polypeptide having a C-terminal / sequence according to one or more aspects described herein); or administering a pharmaceutical composition containing at least one such ISV, protein, or polypeptide (as described above).

[0350] Regarding the above, it is clear that the therapeutic use of the ISVs, proteins, and peptides described herein is a very important aspect of the present invention because such therapeutic use (or the clinical development of such ISVs, proteins, and peptides for such therapeutic use) may involve the use of ADA assays to determine whether the ISV, protein, or peptide is immunogenic (i.e., may cause ADA when administered to human subjects). In this regard, it is also clear that the issue of potential immunogenicity will be particularly important to address when the therapeutic agent is used for extended periods (up to weeks, months, or years) and / or in human subjects with a half-life (preferably expressed as t1 / 2-β) of at least 3 days, such as at least one week, and up to 10 days or more.

[0351] Therefore, according to one specific aspect, the present invention relates to ISVs, proteins, peptides, compounds or molecules (or pharmaceutical compositions thereof) of the present invention as described herein, intended for the treatment of chronic diseases in humans, and / or intended for the presence of such ISVs, proteins, peptides as described herein in the circulation (i.e., at pharmacological activity levels) of subjects who have been administered (i.e., at a therapeutically active dose) of the ISV, protein, peptide for at least one week, preferably at least two weeks, or at least several months; and / or such ISVs, proteins, peptides as described herein are such that they have a half-life (preferably expressed as t1 / 2-β) of at least 3 days, or at least one week and up to 10 days or more in human subjects; and / or intended for the administration of such ISVs, proteins, peptides or pharmaceutical compositions as described herein to humans as two or more doses, said two or more doses being administered for at least 3 days, or at least one week, for example at least two weeks or at least one month or even longer (i.e., at least 3 months, at least 6 months or at least one year), or even long-term administration.

[0352] Furthermore, as will be clear to those skilled in the art based on the disclosure herein, the modifications to the VH domain described herein and the resulting modified VH domain will be particularly useful in proteins, peptides, or other compounds or molecules intended to be administered to human subjects (and particularly patients) whose blood / serum contains (or is suspected of containing) a pre-existing antibody of the type found in samples obtained from SLE patients according to the present invention, i.e., a pre-existing antibody that can bind to the C-terminal region of the VH domain even in the presence of the C-terminal extension as described herein. In particular, the modifications to the VH domain described herein and the resulting modified VH domain will be particularly useful in proteins, peptides, or other compounds or molecules intended for the treatment or prevention of diseases or conditions in such patients. This can be any disease or condition, but specifically a disease or condition that causes or is associated with the presence or occurrence of such pre-existing antibodies (an example is SLE, but it is expected that other serious (autoimmune) diseases can also cause such pre-existing antibodies. This can be readily determined by testing samples obtained from the relevant patient population for the presence of such pre-existing antibodies, especially in a manner similar to the testing performed on samples from SLE patients in the experimental section below).

[0353] Therefore, according to one particular aspect, the present invention relates to ISVs, proteins, peptides, compounds or molecules (or pharmaceutical compositions thereof) of the present invention as described herein, intended to be administered to human subjects whose blood contains pre-existing antibodies that can bind to the exposed C-terminal region of the VH domain even when the VH domain includes a C-terminal extension as described herein (or wherein the C-terminus of the VH domain is optionally linked to another protein or peptide, such as another ISV, via a suitable linker).

[0354] In particular, the present invention relates to ISVs, proteins, peptides, compounds, or molecules (or pharmaceutical compositions thereof) of the present invention as described herein, for the treatment of a disease or condition in a human subject whose blood contains a pre-existing antibody that can bind to the exposed C-terminal region of the VH domain even when the VH domain includes a C-terminal extension as described herein (or wherein the C-terminus of the VH domain is optionally linked to another protein or peptide, such as another ISV, via a suitable linker). The disease or condition can be any disease or condition, but can particularly be one that causes or results in the presence of such a pre-existing antibody or a disease or condition associated with it in the blood of such a patient, such as SLE or other (severe) autoimmune diseases.

[0355] Therefore, according to a more particular aspect, the present invention relates to ISVs, proteins, peptides, compounds, or molecules (or pharmaceutical compositions thereof) of the present invention as described herein, for the treatment of a disease or condition in a human subject / patient, wherein said disease or condition is one that causes, results in, the presence of a pre-existing antibody or a disease or condition associated therewith in the blood of said human subject / patient, said pre-existing antibody being able to bind to the exposed C-terminal region of the VH domain even when said VH domain includes a C-terminal extension as described herein (or wherein the C-terminus of the VH domain is optionally linked to another protein or peptide, such as another ISV, via a suitable linker). For example, such ISVs, proteins, peptides, compounds, or molecules (or pharmaceutical compositions thereof) of the present invention as described herein are used to treat SLE or other (severe) autoimmune diseases in a human subject / patient.

[0356] As will be apparent to those skilled in the art, when a protein, polypeptide, compound, or molecule is intended for the prevention or treatment of such a disease or condition, it will contain at least one (e.g., one, two, three, or four) domain, binding unit, or portion or entity that is therapeutically active against the associated disease or condition (e.g., against a target or pathway associated with the treatment of the associated disease or condition). Similarly, such a binding domain or binding unit may be, for example, an ISVD, and according to one aspect, may be particularly the VH domain or ISVD of the present invention. Another general example of such a protein, polypeptide, compound, or molecule is one in which one or more therapeutic domains, binding units, or portions or entities may not be ISVDs (but are derived, for example, from another scaffold), but contain the VH domain of the present invention to prolong its half-life (e.g., serum albumin conjugates, as described herein).

[0357] In another aspect, the VH domain, ISVD, or compound of the present invention (as described herein) is targeted at the ion channel Kv1.3. Some preferred, non-limiting examples of such VH domains, ISVDs, or compounds are given in Example 7, and the VH domains for Kv1.3 described in those examples (and the compounds of the present invention containing them) and the specific compounds of the present invention for Kv1.3 described in those examples form another aspect of the present invention.

[0358] For example, and not as a limitation, compounds of the present invention targeting Kv1.3 may comprise or consist of a single VH domain of the present invention targeting Kv1.3 (and preferably a nanobody), or may comprise or consist of at least two (e.g., two or three) VH domains of the present invention targeting Kv1.3 (and preferably a nanobody). When such a polypeptide comprises more than two VH domains of the present invention targeting Kv1.3, these VH domains may be the same or different, and when they are different, they may target the same epitope or subunit on Kv1.3 or different epitopes or subunits.

[0359] Similarly, as generally described herein with respect to the compounds of the present invention, such compounds may suitably contain one or more linkers, may contain C-terminal extensions (i.e., as further described herein), and may also contain one or more additional binding units or binding domains (or other amino acid sequences or portions), such as additional ISVDs targeting targets other than Kv1.3. For example, and not as a limitation, the compounds of the present invention may (also) contain binding domains or binding units that provide an extended half-life, such as ISVDs targeting serum proteins such as serum albumin (e.g., nanobodies targeting human serum albumin, such as the nanobodies of the present invention targeting human serum albumin).

[0360] In another aspect, the present invention relates to (synthetic) libraries of immunoglobulin variable domain sequences as described herein (i.e., containing amino acid residues / mutations / substitutions as described herein). Such libraries will typically contain at least 100 different sequences, such as at least 1000 different sequences, particularly more than 10 5 10 different sequences, more specifically more than 10 6 Different sequences, such as 10 8 Up to 10 10 One or more different sequences (which in the broadest sense have at least one amino acid difference between the sequences) generally have (substantially) the same framework sequence (which contains the amino acid residues / mutations indicated herein) and different CDRs (meaning that each sequence in the library has at least “one amino acid difference” in at least one CDR compared to other sequences in the library).

[0361] Methods for synthesizing libraries of immunoglobulin single variable domain sequences (e.g., based on human VH sequences or VHH sequences derived from camelids) and for generating / constructing such libraries (including libraries based on predetermined scaffolds and / or containing one or more specific amino acid residues / mutations in the framework region) are known in the art. References include, for example, Tanha et al., J. Biol. Chem., Vol. 276, pp. 24774–24780, 2001; Bond et al., J. Mol. Biol. (2003) 332, 643–655; Mandrup et al., PLOS One, October 2013, Vol. 8, No. 10, e76834; Goldman et al., Anal. Chem., 2006, 78, 8245–8255; Hussak et al., Protein Engineering, Design & Selection vol. 25 no. 6 pp. 313–318, 2012; and Chen et al., Methods Mol. Biol., 2009, 525, 81. The techniques described herein (and similar techniques known per se) can be suitably used or applied to generate immunoglobulin single variable domain libraries according to the invention.

[0362] The ISVDs present in this type of library may appropriately contain any suitable CDR from any suitable source, such as those first used in experiments. "CDRs acquired / produced from mammalian repertoires (such as cameloid species or human sequences), CDRs acquired / produced from animal repertoires (such as cameloid species) that have been appropriately immunized with antigens; fully synthetic CDR libraries; or libraries obtained through techniques such as mutagenesis (e.g., random or site-directed mutagenesis). Such libraries can also be, for example, libraries produced during known affinity maturation processes."

[0363] The framework regions of the ISVDs present in such libraries can be suitably derived from any suitable starting sequence / scaffold, such as scaffolds obtained by starting with a VH sequence (e.g., a human VH sequence) or a nanobody sequence (e.g., a VHH sequence or a humanized VH sequence). It is also possible that the library contains ISVDs derived from more than two different sources or based on more than two different scaffolds (e.g., because the library is obtained by merging two or more libraries obtained from different sources or based on different scaffolds).

[0364] Furthermore, the library (containing the ISVD) can be in the form of a protein or in the form of DNA or RNA encoding the relevant ISVD. For example, the library can be in the form of an expression library suitable for screening and / or selection techniques, and for this purpose, it can be in the form that can be displayed using suitable display techniques, such as a phage display library, a yeast display library, or a ribosome display library.

[0365] Therefore, the present invention also typically relates to libraries (as described herein) containing the VH domains of the present invention (as further described herein). Preferably, according to a specific aspect of such a library, the VH domains present all have the same (or substantially the same) frame sequence, but have different CDR sequences (again, as mentioned, this means that each individual VH domain in the library has at least one amino acid difference in at least one CDR compared to other VH domains in the library).

[0366] In one aspect, such a library of the present invention is a library of the ISVD of the present invention (as further described herein, including a library encoding a suitable nucleic acid of said ISVD), wherein:

[0367] - The amino acid residue at position 11 is one of L, V, or K; and

[0368] - The amino acid residue at position 14 is either A or P; and

[0369] - The amino acid residue at position 41 is either A or P; and

[0370] - The amino acid residue at position 89 is one of T, V, or L; and

[0371] - The amino acid residue at position 108 is either Q or L; and

[0372] -The amino acid residue at position 110 is one of T, K, or Q; and

[0373] -The amino acid residue at position 112 is one of S, K, or Q;

[0374] Wherein (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V. Optionally, the ISVD present in this library may also contain a C-terminal extension (as further described herein with respect to the VH domain of the invention) and / or suitably contain a suitable tag (such as a histidine tag).

[0375] In another aspect, such a library of the present invention is a library of the ISVD of the present invention (as further described herein, including a library encoding a suitable nucleic acid of said ISVD), wherein:

[0376] -The amino acid residue at position 11 is V; and

[0377] - The amino acid residue at position 14 is either A or P; and

[0378] - The amino acid residue at position 41 is either A or P; and

[0379] -The amino acid residue at position 89 is an L; and

[0380] - The amino acid residue at position 108 is either Q or L; and

[0381] -The amino acid residue at position 110 is one of T, K, or Q; and

[0382] -The amino acid residue at position 112 is one of S, K, or Q;

[0383] Optionally, the ISVDs present in such libraries may also contain C-terminal extensions (as further described herein with respect to the VH domain of the present invention) and / or suitably contain suitable tags (such as histidine tags).

[0384] The library of this invention can be used for any suitable / intended purpose known per se. For example, it can be used, for example, for screening and / or selection purposes (or as part of a screening and / or selection method), for / as part of affinity maturation purposes or other methods intended to generate improved VH domains, or, for example, for alanine scanning. In practice, generally, the size, design, and other characteristics of the library will be adapted to its intended use, which will be within the skill of a person skilled in the art.

[0385] The invention will now be further described with reference to the following non-limiting preferred aspects, embodiments, and accompanying drawings, wherein:

[0386] - Figure 1 This is a table that lists 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);

[0387] - Figure 2 The sequences mentioned in this article are listed;

[0388] - Figure 3 The figure shows the results of testing 96 serum samples in Example 4 with a reference nanobody (Reference A, SEQ ID NO:44) that does not have the S112K mutation. Figure 3 The reference (A+S 112K+C-terminal alanine) is compared to a representative nanobody with the S112K mutation (1)). Figure 3 The data points obtained when the middle indicator is (2)) are combined;

[0389] - Figure 4 The figure shows the results of testing 129 serum samples in Example 4 with a reference nanobody (Reference A, SEQ ID NO:44) that does not have the V89T mutation. Figure 4 The middle indicator is (1) compared to the representative nanobody with V89T mutation (reference A+L11V+V89T+C-terminal alanine), Figure 4 The data points obtained when the middle indicator is (2)) are combined;

[0390] - Figure 5 The figure shows the results of Example 5 when 100 serum samples were tested against a reference nanobody (Reference A, SEQ ID NO:44) that did not have any of the following mutations. Figure 5 The middle indication is (1) compared to representative nanobodies with V89L, T110K and / or T110Q mutations (reference A+L11V+V89L+C-terminal Ala, Figure 5 The middle indicator is (2); the reference is A+L11V+V89L+T110K+C-terminal Ala. Figure 5 The indicator in Figure (5) is (3); the reference A+L11V+V89L+T110Q+C-terminal Ala, the indicator in Figure (5) is (4), and the reference A+L11V+T87A+V89L+C-terminal Ala, Figure 5 The data points obtained when the middle indicator is (5)) are combined.

[0391] - Figure 6 The figure shows data points obtained in Example 6 when testing the binding of representative trivalent nanobody constructs to 98 serum samples obtained from healthy subjects. Each point represents a data point collected by testing a specified construct for one of the 98 serum samples. Legend: (1) = Reference X (Nanobody A-35GS-Nanobody A-35GS-Nanobody B); (2) = Reference X + C-terminal Ala; (3) = Reference X + L11V + V89L + C-terminal Ala; (4) = Reference X + L11V + T87A + V89L + C-terminal Ala; (5) = Reference X + L11V + V89L + T110K + C-terminal Ala; (6) = Reference X + L11V + V89L + T110Q + C-terminal Ala.

[0392] - Figure 7The figure shows data points obtained in Example 6 when testing the binding of a representative trivalent nanobody construct to 30 serum samples obtained from healthy subjects (samples were selected such that the pre-existing antibody had a high titer even in the presence of a C-terminal alanine extension or also in the presence of a pre-existing antibody with high binding). Each point represents a data point collected by testing a specified construct for one of the 30 serum samples. Legend: (1) = Reference X + C-terminal Ala; (2) = Reference X + L11V + V89L + C-terminal Ala; (4) = Reference X + L11V + T87A + V89L + C-terminal Ala; (5) = Reference X + L11V + V89L + T110K + C-terminal Ala.

[0393] - Figure 8 This is a graph showing data points obtained in Example 6 when testing the binding of 98 serum samples obtained from healthy subjects to a representative bivalent nanobody construct. Each point represents a data point collected by testing a specified construct for one of 30 serum samples. Legend: (1) = Reference Y (Nanobody A-35GS-Nanobody B); (2) = Reference Y + C-terminal Ala; (3) = Reference Y + L11V + V89L + C-terminal Ala; (4) = Reference Y + L11V + T87A + V89L + C-terminal Ala; (5) = Reference Y + L11V + V89L + T110K + C-terminal Ala; (6) = Reference Y + L11V + V89L + T110Q + C-terminal Ala;

[0394] - Figure 9A and 9B This invention demonstrates a monovalent nanobody targeting ion channel Kv1.3. Figure 9A ) and the trivalent bispecific compound of the present invention with extended half-life ( Figure 9B Preferred, but not limiting, instances of ); and Figure 9C Some preferred CDRs for ISVD for Kv1.3 are listed (categorized according to Kabat and Abm respectively).

[0395] - Figure 10 The figure shows data points obtained in Example 7 when testing the binding of representative trivalent bispecific extended half-life compounds of the present invention against Kv1.3 to 47 serum samples obtained from diabetic subjects. Each point represents a data point collected by testing a specified construct against one of the 47 serum samples. SEQ ID NO indicates the relevant sequence listed in Figure 9;

[0396] - Figure 11The figure shows data points obtained in Example 7 when testing the binding of representative trivalent bispecific extended half-life compounds of the present invention against Kv1.3 to 90 serum samples from healthy volunteers. Each point represents a data point collected by testing a specified construct against one of 47 serum samples. SEQ ID NO indicates the relevant sequence listed in Figure 9;

[0397] - Figure 12A and 12B The CDRs and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting IL-23 based on specified reference sequences are listed. See also Example 8;

[0398] - Figure 13 The CDR and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting OX40-L based on specified reference sequences are listed. See also Example 9;

[0399] - Figure 14 The CDR and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting IgE based on specified reference sequences are listed. See also Example 10;

[0400] - Figure 15A and 15B The CDR and amino acid sequences of some preferred, but not limiting, examples of nanobodies of the present invention targeting CXCR-4 based on specified reference sequences are listed. See also Example 11;

[0401] - Figure 16A and 16B The CDR and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting HER-3 based on specified reference sequences are listed. See also Example 12;

[0402] - Figure 17A and 17B The CDR and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting TNF based on specified reference sequences are listed. See also Examples 13 and 14;

[0403] - Figure 18A and 18B The CDR and amino acid sequences of some preferred, but not limiting, examples of nanobodies of the present invention targeting c-Met based on specified reference sequences are listed. See also Example 15;

[0404] - Figure 19The CDR and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting RANK-L based on specified reference sequences are listed. See also Example 16;

[0405] - Figures 20A to 20C The CDR and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting CXCR-7 based on specified reference sequences are listed. See also Example 17;

[0406] - Figure 21A and 21B The CDR and amino acid sequences of some preferred, non-limiting examples of nanobodies of the present invention targeting A-β based on specified reference sequences are listed. See also Example 18;

[0407] - Figure 22 The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for IL-23 are given;

[0408] - Figure 23 The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for OX40-L are given;

[0409] - Figure 24 The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for IgE are given;

[0410] - Figure 25 The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for CXCR-4 are given;

[0411] - Figure 26 The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for HER-3 are given;

[0412] - Figure 27 The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention targeting TNF are given;

[0413] - Figure 28A and 28B The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for c-Met are given;

[0414] - Figure 29 The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for RANK-L are given;

[0415] - Figure 30The amino acid sequences of some preferred, non-limiting examples of compounds of the present invention for A-β are given;

[0416] - Figure 31A The graph shows data points obtained in Example 19 when testing the binding of representative A-β-targeting trivalent bispecifically extended half-life compounds of the present invention to 92 serum samples obtained from healthy volunteers. Each point represents a data point collected by testing a specified construct against one of the 92 serum samples. The reference numbers are listed in Table CC-1. Similarly, Figure 30 Figure B shows the results of testing 92 serum samples obtained from healthy volunteers in Example 19, indicating the presence of... Figure 31A Data points were obtained when the (monovalent) C-terminal nanobody bound to the construct tested. Each point represents a data point collected by testing a specified construct against one of 92 serum samples. Reference numbers are listed in Table CC-2.

[0417] Experimental Section

[0418] The human samples used in the following experimental sections were obtained from commercial sources or human volunteers (after obtaining all necessary consents and approvals) and used in accordance with applicable laws and regulations (including, but not limited to, those concerning medical secrets and patient privacy).

[0419] In the following examples, ProteOn was used to determine the binding of pre-existing antibodies (i.e., from healthy volunteers, rheumatoid arthritis (RA) patients, and SLE patients) to the tested nanobodies as follows: The binding of pre-existing antibodies to nanobodies captured on human serum albumin (HSA) was evaluated using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween 20) was used as the transport buffer and the experiments were performed at 25°C. The ligand channels of the ProteOn GLC Sensor Chip were activated with EDC / NHS (flow rate 30 μl / min) and injected into HSA at 10 μg / ml in ProteOn acetate buffer pH 4.5 (flow rate 100 μl / min) to provide a fixation level of approximately 3200 RU. After fixation, the surface was passivated with ethanolamine HCl (flow rate 30 μl / min). Nanobodies were injected onto the HSA surface at a rate of 45 μl / min for 2 minutes 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%), followed by injection at 45 μl / min for 2 minutes, and then a dissociation step for 400 seconds. After each cycle (i.e., before new nanobodies capture and blood sample injection steps), the HSA surface was restored by injection of 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 pre-existing antibody binding were obtained after subtracting 1) nanobodies-HSA dissociation and 2) non-specific binding to the reference ligand pathway as a double reference. The binding level of the pre-existing antibody was determined by setting a reporting point at 125 seconds (5 seconds after binding completion). Calculate the percentage reduction in pre-existing antibody binding relative to the reference nanobody at a binding level of 125 seconds.

[0420] Example 1: S112K mutation inhibits the binding of pre-existing antibodies

[0421] The effect of the substitution at position 112 on the binding of pre-existing antibodies to nanobodies in human samples was determined and compared with the effect of C-terminal alanine extension as described in WO 12 / 175741.

[0422] Two reference compounds (reference A without C-terminal alanine extension and reference B with C-terminal alanine extension) and variants of these reference compounds with different mutations at position 112 were tested using serum samples obtained from six different RA patients and eight serum samples obtained from different healthy subjects. Binding of the pre-existing antibody in the samples to the tested nanobody was measured on ProteOn according to the general method described above. The results are shown in Table A below.

[0423] As can be seen, in the mutation at position 112 tested, the S112K mutation resulted in a decrease in binding to pre-existing antibodies present in the tested serum comparable to that of the C-terminal alanine extension (even in the absence of the C-terminal alanine extension in the S112K variant). Similar results were obtained in the case of three human plasma samples (data not shown).

[0424]

[0425] Example 2: Effect of S112K mutation on binding to pre-existing antibodies in human SLE samples

[0426] The same nanobody variant used in Example 1 was tested for binding to pre-existing antibodies from seven serum samples obtained from patients confirmed to be positive for systemic lupus erythematosus (SLE). For comparison, plasma samples from two healthy volunteers were also included.

[0427] The binding of the pre-existing antibody in the sample to the tested nanobody was measured using the general method described above on ProteOn. The results are shown in Table B below.

[0428] As can be seen from the comparison of binding data between Reference A and Reference B and the nanobody of the present invention, samples obtained from some SLE patients showed that they contained certain pre-existing antibodies that could still bind to the nanobody even in the presence of C-terminal alanine residues (C-terminal alanine residues do indeed substantially prevent / remove (partially or substantially completely) all binding of pre-existing antibodies present in plasma samples from healthy subjects).

[0429] It is further evident that the binding of these pre-existing antibodies from SLE samples can be greatly reduced by mutations at positions 11 and 112 (and in the case of position 112, specifically S112K).

[0430]

[0431] Example 3: Combined framework mutation and C-terminal extension against pre-existing antibodies in human SLE samples The effects of combination

[0432] The binding of four different nanobodies (with specific framework mutations and with or without C-terminal alanine extension) to pre-existing antibodies from five serum samples obtained from patients confirmed to be positive for systemic lupus erythematosus (SLE) was tested. For comparison, a plasma sample from a healthy volunteer was included.

[0433] The binding of the pre-existing antibody in the sample to the tested nanobody was measured using the general method described above on ProteOn. The results are shown in Tables C and D below.

[0434] As can be seen from the comparison of binding data between reference A and reference B, the samples obtained from SLE patients showed that they contained certain pre-existing antibodies that could still bind to the nanobodies even in the presence of C-terminal alanine residues (C-terminal alanine residues do indeed essentially prevent / remove all binding of pre-existing antibodies present in plasma samples from healthy volunteers).

[0435] It is also evident that the binding of these pre-existing antibodies from SLE samples can be greatly reduced by mutations at positions 11 and 112 (and in the case of position 112, particularly S112K).

[0436] Example 4: The effect of V89T mutation on the binding of pre-existing antibodies in samples from SLE patients.

[0437] As described in this article, samples obtained from certain SLE patients showed pre-existing antibodies / factors that could still bind to the exposed C-terminus of the VH domain even in the presence of C-terminal extension. The study investigated whether the V89T mutation could reduce or prevent / remove this binding (in the presence or absence of C-terminal extension). The binding is also shown in Tables C and E below.

[0438] As can be seen, the V89T mutation can substantially prevent / remove the binding of pre-existing antibodies present in samples obtained from SLE patients, to a similar extent to the S112K mutation. However, as can be seen from the comparative data given in Tables C and E of nanobodies with the V89T mutation but without C-terminal extension and similar nanobodies with the S112K mutation but without C-terminal extension, the mutation at position 112 in the nanobodies generally reduces the binding of pre-existing antibodies in samples from healthy volunteers to a greater extent compared to the V89T mutation (i.e., 100%, 85%, and 64% for S112K nanobodies, compared to 9%, 11%, and 16% for V89T nanobodies). Therefore, the use of a mutation at position 112 (and particularly S112K) is generally preferred over the use of a mutation at position 89 (such as V89T).

[0439] However, as can also be seen from the data in Tables C and E, adding a C-terminal alanine to the V89T nanobody completely prevents / removes the binding of pre-existing antibodies in samples obtained from healthy volunteers, and therefore, the combination of V89T mutation with C-terminal extension as described herein will generally be preferred (i.e., compared to using V89T without C-terminal extension) if the V89T nanobody or VH domain has, or intends to have, an exposed C-terminal region in the protein or polypeptide therein (e.g., because if its C-terminus is formed).

[0440]

[0441]

[0442]

[0443]

[0444]

[0445] To confirm the broad applicability of the results / findings in the table above, representative nanobodies with S112K and / or V89T mutations were tested in a test group of 96 (S112k) and 129 (V89T) human serum samples. Binding was determined using the methods described above on ProteOn.

[0446] The results are summarized in Figure 3 And Table F (representative nanobodies with S112K mutation) and Figure 4 In Table G (a representative nanobody with the V89T mutation).

[0447] exist Figure 3 In this study, a nanobody with the S112K mutation (reference A + S112K + C-terminal alanine – see Table C above) was compared with a reference nanobody (reference A; SEQ ID NO: 44). Both the nanobody with the S112K mutation and reference A were tested for each serum sample, and the binding level (RU) at 125 seconds was determined. The data were then plotted. Figure 3 In the middle, each point displays reference A ( Figure 3 The middle indicator is (1)) or S112K mutant ( Figure 3 The middle line indicates the binding of one sample (2). The dashed line indicates the measured binding level as 20 RU.

[0448] The same data is also shown in numbers in Table F, which mentions the total number of samples tested for binding levels of more than 20 RU, less than 20 RU (i.e., 0 to 20 RU), and less than 10 RU at 125 seconds for reference A and S112K mutant, respectively.

[0449] As by Figure 3 As can be seen from the plot and the data shown in Table F, for reference A, more than half of the 96 samples tested gave binding levels exceeding 20 RU (in some cases as high as 150-200 RU), indicating that pre-existing antibodies present in the samples bound to reference A. By comparison, for the S112K mutant, no samples gave binding levels exceeding 20 RU (and most were less than 10 RU), indicating that the S122K mutation essentially reduces / prevents the binding of pre-existing antibodies in all 96 samples tested.

[0450] Similar graphs and similar data are shown respectively. Figure 4 In Table G, for representative nanobodies with the V89T mutation (reference A + L11V + V89T + C-terminal alanine; see Table E above), 129 serum samples were tested and compared with reference A ( Figure 4 (1) indicates; in Figure 4 In this context, the V89T mutant is indicated by (2). Similarly, according to Figure 4 The data in Figure 1 and Table G show that, with almost no exceptions (i.e., less than 10% of the tested samples each gave an absolute binding value of less than 100 RU after 125 seconds), the V89T mutation reduced / prevented binding of the pre-existing antibody in most of the 129 tested samples, while in most tested samples, the reference without the V89T mutation was bound by the pre-existing antibody.

[0451] Table F: Representative nanobodies with S112K mutations tested on 96 serum samples.

[0452]

[0453] Table G: Nanobody mutants with V89L and / or T100K / Q mutations were tested on 129 serum samples.

[0454]

[0455] Example 5: The combination of V89L mutation and T110K or T110Q mutation in pre-existing cells in samples from SLE patients The effect of antibody binding.

[0456] As described herein, samples obtained from certain SLE patients exhibited pre-existing antibodies / factors that could still bind to the exposed C-terminus of the VH domain even in the presence of C-terminal extension. The study investigated whether V89L and / or T110Q or T110K mutations (or combinations thereof) could reduce or prevent / remove this binding (with or without C-terminal extension). Results are also shown in Tables H and I, which provide data from two separate experiments (Table D above also shows data on the combination of the S112Q mutation and the V89L mutation of this invention). The nanobodies used in Table I were also used to generate… Figure 5 The data shown in Table J.

[0457]

[0458]

[0459]

[0460] To confirm that the results / findings from the table above are broadly applicable, representative nanobodies with V89L, T110K, and / or T110Q mutations were also tested on a cohort of 99 human serum samples. Binding data were also obtained and presented as in Example 4. Figure 3 and 4 Plot the results and data shown in Tables F and G as indicated.

[0461] The tested nanobodies are (numbered corresponding to...) Figure 5 The reference numbers used are: (1) Reference A; (2) Reference A+L11V+V89L+C-terminal Ala; (3) Reference A+L11V+V89L+T110K+C-terminal Ala; (4) Reference A+L11V+V89L+T110Q+C-terminal Ala; (5) Reference A+L11V+T87A+V89L+C-terminal Ala. The results are shown in Figure 5 And in Table I.

[0462] As can be seen, the mutations introduced into the test also significantly reduce the number of samples in which pre-existing antibodies can bind to the tested nanobodies. It can also be seen that for Figure 5 The nanobody (2) (reference A+L11V+V89L+C-terminal Ala) still showed some samples with pre-existing antibody binding levels exceeding 20 RU (but were much less than 100 RU) after 125 seconds. However, when the V89L mutation was combined with the T110K mutation (nanobody (3)) or the T100Q mutation (nanobody (4)), virtually all 99 tested samples showed binding levels less than 20 RU (and in fact less than 10 RU, see Table J).

[0463] Table J: Representative nanobodies with V89L, T110K and / or T110Q mutations tested on 99 serum samples.

[0464]

[0465] Example 6: Testing the binding of multivalent constructs to pre-existing antibodies

[0466] Multivalent constructs were prepared based on the following nanobodies:

[0467] Nanobody A (targeting therapeutic targets):

[0468] EVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGTLVTVSS(SEQ ID NO: 92)

[0469] Nanobody B (targeting serum albumin):

[0470] EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSSLRSSQGTLVTVSS(SEQ ID NO: 93)

[0471] Nanobody C (targeting therapeutic targets):

[0472] EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS (SEQ ID NO: 94)

[0473] The prepared constructs are listed in Table K below (in each case, the substitutions introduced into the relevant nanobody (if present) are mentioned in parentheses. "HIS6" indicates a his-tag with 6 histidine residues at the N-terminus, and "-Ala" indicates an extension with 1 alanine residue at the C-terminus).

[0474] Table K: Tested multivalent constructs

[0475] HIS6-Nanobody A (E1D, L11V, S112K)-35GS-Nanobody B (L11V, S112K)-Ala HIS6 Nanobody A(E1D)-35GS-Nanobody B HIS6-Nanobody A(E1D)-35GS-Nanobody B-Ala HIS6-Nanobody A (E1D, L11V, S112K)-35GS-Nanobody B (L11V, S112K) HIS6-Nanobody A (E1D, S112K)-35GS-Nanobody B (S112K)-Ala HIS6-Nanobody A (E1D, S112Q)-35GS-Nanobody B (S112Q)-Ala HIS6-Nanobody A (E1D, V89L, S112K)-35GS-Nanobody B (V89L, S112K)-Ala HIS6-Nanobody A (E1D, L11V, S112Q)-35GS-Nanobody B (L11V, S112Q)-Ala HIS6-Nanobody B(E1D)-35GS-Nanobody A HIS6-Nanobody B(E1D)-35GS-Nanobody A-Ala HIS6-Nanobody A (E1D)-35GS-Nanobody B (L11V, S112K)-Ala HIS6-Nanobody B(E1D, L11V, S112K)-35GS-Nanobody A-Ala HIS6-Nanobody B (E1D)-35GS-Nanobody A (L11V, S112K)-Ala HIS6-Nanobody A (E1D, V89L, S112Q)-35GS-Nanobody B (V89L, S112Q)-Ala HIS6-Nanobody B (E1D, L11V, S112K)-35GS-Nanobody A (L11V, S112K)-Ala HIS6-Nanobody B(S112K-Ala Nanobody B(L11V, S112K)-Ala Nanobody B(L11V, S112Q)-Ala Nanobody B(S112Q)-Ala

[0476] Table K (continued)

[0477]

[0478]

[0479] Table K (continued)

[0480]

[0481] Representative multivalent constructs were tested for binding to pre-existing antibodies present in blood or serum samples obtained from patients with SLE and healthy volunteers. Both were determined using ProteOn, essentially as described above.

[0482] Representative constructs tested are listed in Tables L (trivalent constructs) and M (divalent constructs), and the results are shown in... Figure 6-8 In Table NQ, the trivalent constructs tested were derived from the reference construct nanobody A-35GS-nanobody A-35GS-nanobody B (“Reference X”), while the bivalent constructs were derived from the reference construct nanobody A-35GS-nanobody B (“Reference X”). All constructs (except the reference construct) have, as indicated, C-terminal alanine residues, and, in each “nanobody A” and “nanobody B” building block, indicated mutations.

[0483] Table L: Trivalent constructs tested.

[0484]

[0485] Table M: ​​Divalent constructs tested.

[0486]

[0487]

[0488] Table N: Results of testing the trivalent construct against pre-existing antibody binding in 98 serum samples obtained from healthy volunteers. The results were also published in... Figure 6 The graph is shown in the image, where each point represents a data point collected by testing a specified construct from one of 98 serum samples.

[0489]

[0490] Table O: Results of testing the trivalent construct for binding to pre-existing antibodies present in 30 selected serum samples obtained from healthy volunteers. The 30 samples were pre-selected because they had known high titers of pre-existing antibodies or because pre-existing antibodies present in the samples were known to exhibit high binding even in the presence of C-terminal alanine residues. Results are also presented in... Figure 7 The graph is shown in the image, where each point represents a data point collected by testing a specified construct from one of 30 selected serum samples.

[0491]

[0492] Table P: Results of testing the bivalent construct against pre-existing antibody binding in 98 serum samples obtained from healthy volunteers. The results were also presented in... Figure 8 The graph is shown in the image, where each point represents a data point collected by testing a specified construct from one of 98 serum samples.

[0493]

[0494] Three representative trivalent constructs were also tested on serum samples obtained from SLE patients. The results are shown in Table Q.

[0495]

[0496] Example 7: Nanobodies and nanobodies-constructions targeting ion channel Kv1.3.

[0497] In one specific aspect, the VH domain of the present invention (and in particular the ISVD of the present invention and more particularly the nanobodies of the present invention) and the compounds of the present invention can target the ion channel Kv.1.3.

[0498] Meanwhile, the pending U.S. Provisional Application USSN 62 / 014,023 (title: "Kv1.3-binding immunoglobulin"; assignee: Ablynx NV; filing date: June 18, 2014) and the subsequent U.S. Provisional Application with the same title filed on March 16, 2015 (assignee: Ablynx NV) describe immunoglobulin monovariable domains (and particularly nanobodies) targeting the potassium-selective voltage-gated ion channel Kv1.3, as well as proteins, peptides and other nanobodies-based constructs containing at least one such Kv1.3-targeting nanobody.

[0499] The mutations described in this application (optionally and suitably combined with C-terminal extensions as described herein and / or in WO 12 / 175741) may also be suitably applied to nanobodies, proteins, peptides and other nanobodies-based constructs targeting Kv1.3 as described in these two U.S. Provisional Applications.

[0500] Therefore, in one aspect, the present invention relates to a VH domain that is targeted at Kv1.3 and is as further described herein with respect to the ISVD of the invention (i.e., containing amino acid residues / mutations as described herein).

[0501] Similarly, with respect to other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD for Kv1.3 described in this embodiment) or a compound containing it is referred to as “in accordance with the invention” or “as further described herein”, the preferred aspects / implementations and preferred options of the general description of the ISVD or compound of the invention herein also apply particularly to the specific ISVD or compound, unless explicitly stated otherwise or unless required by the specific technical background.

[0502] Therefore, in a particular aspect, the present invention relates to a VH structural domain for Kv1.3, wherein (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in this VH structural domain for Kv1.3:

[0503] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0504] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0505] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0506] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0507] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0508] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0509] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0510] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

[0511] The VH domain for Kv1.3 mentioned in this embodiment may specifically have the CDR (including any preferred aspects / implementations of such CDR) described on pages 5-10 of USSN62 / 014,023 or a sequence-optimized version thereof, as described in the other US provisional applications mentioned above.

[0512] In particular, the VH domain for Kv1.3 mentioned in this embodiment may specifically have a combination of CDR1, CDR2 and CDR3 selected from one of the combinations of CDR1, CDR2 and CDR3 listed as preferred aspects in the list across pages 9 and 10 of USSN62 / 014,023.

[0513] In some preferred, and not limiting, aspects of the invention:

[0514] - In the VH domain of the present invention: (i) CDR1 (according to Kabat) is sequence SED ID NO:166 or an amino acid sequence differing from sequence SEQ ID NO:166 by one or two amino acids; (ii) CDR2 (according to Kabat) is sequence SED ID NO:167 or an amino acid sequence differing from sequence SEQ ID NO:167 by one or two amino acids; and (iii) CDR3 (according to Kabat) is sequence SED ID NO:168 or an amino acid sequence differing from sequence SEQ ID NO:168 by one or two amino acids; and even more preferably: (i) CDR1 (according to Kabat) is sequence SED ID NO:166; (ii) CDR2 (according to Kabat) is sequence SED ID NO:167; and (iii) CDR3 (according to Kabat) is sequence SED ID NO:168; and / or

[0515] - In the VH domain of the present invention: (i) CDR1 (according to Abm) is sequence SED ID NO:169 or an amino acid sequence that differs from sequence SEQ ID NO:169 by one or two amino acids; (ii) CDR2 (according to Abm) is sequence SED ID NO:170 or an amino acid sequence that differs from sequence SEQ ID NO:170 by one or two amino acids; and (iii) CDR3 (according to Abm) is sequence SED ID NO:171 or an amino acid sequence that differs from sequence SEQ ID NO:171 by one or two amino acids; and even more preferably: (i) CDR1 (according to Abm) is sequence SED ID NO:169; (ii) CDR2 (according to Abm) is sequence SED ID NO:170; and (iii) CDR3 (according to Abm) is sequence SED ID NO:178.

[0516] The VH domain of the present invention targeting Kv1.3 can also be, as described herein, and more particularly, an ISVD (and more particularly, a nanobody) targeting Kv1.3 or a protein, peptide, or other compound or construct comprising at least one such ISVD. Such a protein, peptide, or other compound or construct can also be, as further described herein, and can, for example, have an increased half-life (i.e., as described herein, for example, a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days, expressed as t1 / 2β in a human subject), and for this purpose can, for example, comprise a nanobody that binds to serum albumin, which can also be a nanobody of the present invention that binds to serum albumin (again, as described herein).

[0517] Furthermore, such ISVDs may suitably have C-terminal extensions (as further described herein and in WO 12 / 175741), particularly when the ISVD forms the C-terminus of a protein, polypeptide or other compound or construct comprising it (again, as further described herein).

[0518] For example, and not as a limitation, the VH domain of the present invention for Kv1.3 may be one of the sequences listed in Table A-1 of USSN 62 / 014,023 (SEQ ID NO: 1 to 123 in USSN 62 / 014,023) or one of the sequences listed in Table A-1 of the US provisional application entitled “Kv1.3 binding immunoglobulin” (assigned to Ablynx NV; filed March 16, 2015) (SEQ ID NO: 1 to 123, 495, 498 to 513 or 523 to 540 in the aforementioned US provisional application; and particularly the sequence SEQ ID NO: 495), but suitably having the mutated / specific amino acid residues described herein with respect to the ISVD of the present invention, and optionally suitably having a C-terminal extension.

[0519] In one specific aspect, the nanobody of the present invention targeting Kv1.3 is a variant of the nanobody of SEQ ID NO:137 (having at least 90% sequence identity with SEQ ID NO:137), wherein:

[0520] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0521] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0522] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0523] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0524] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0525] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0526] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0527] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

[0528] Some particularly preferred, but not limiting, examples of the nanobodies of the present invention for Kv1.3 and compounds of the present invention comprising therein are listed below. Figure 9A(Monovalent nanobodies: SEQ ID NO: 138 to 155) and Figure 9B (Trivalent bispecific half-life extended constructs: SEQ ID NO: 156 to 164). A further aspect of the invention is formed by comprising at least one (e.g., one, two, or three) anti-Kv1.3 nanobodies selected from SEQ ID NO: 138 to 155, or compounds of the invention composed thereof. Furthermore, each compound of SEQ ID NO: 156 to 164 forms a further aspect of the invention. In one specific aspect, such a compound contains two of the present invention's Kv1.3 nanobodies and one nanobodies targeting human serum albumin (which is also preferably the present invention's nanobodies). Furthermore, such constructs may also contain suitable linkers and C-terminal extensions.

[0529] The monovalent anti-Kv1.3 nanobodies of SEQ ID NO:138 to 155 are generated by introducing the L11V and V89L mutations of the present invention into the starting sequence of SEQ ID NO:137 (reference). Furthermore, different combinations of humanized (or other sequence-optimized) mutations are introduced, such as E1D, A14P, G19R, M53A or M53Q or M53Y, T62S, A74S, K83R, S94G and / or T97E). Specific mutations introduced in each sequence of SEQ ID NO:138 to 155 are described in detail below. Figure 9A The information is provided in the text.

[0530] Figure 9A Some of the monovalent anti-Kv1.3 nanobodies were also formatted into trivalent bispecific constructs, which comprise two nanobodies of the present invention targeting Kv1.3 and one extended-half-life nanobodies of the present invention targeting human serum albumin (SEQ ID NO: 109). Figure 9BAlso referred to as “ALB-82”. A 35GS linker was used, and all constructs had a C-terminal extension (a single C-terminal alanine residue). The sequences of the resulting constructs are given in SEQ ID NO 156 to 164. In these, three constructs (SEQ ID NO: 156, 157, and 160) were tested for binding to pre-existing antibodies obtained from samples from 47 diabetic subjects and 90 healthy subjects, using the general methods described herein. Binding to pre-existing antibodies from samples from both groups was compared to a reference construct SEQ ID NO: 165, which is a corresponding trivalent bispecific construct based on the reference antibody-Kv1.3 structural unit of SEQ ID NO: 137 and serum albumin-binding protein Alb-8 (SEQ ID NO: 46) and also bound with a C-terminal alanine extension. Results showed... Figure 10 (Samples from 47 diabetic patients) and Figure 11 (Samples from 90 healthy volunteers). In each case, the constructs of the present invention with L11V and V89L mutations showed reduced binding to pre-existing antibodies compared to the reference construct.

[0531] Example 8: Targeting the VH domain of IL-23 (and particularly nanobodies) and compounds of the present invention containing therein. things.

[0532] In one specific aspect, the VH domain of the present invention (and in particular the ISVD of the present invention and more particularly the nanobodies of the present invention) and the compounds of the present invention can target IL-23.

[0533] The VH domain of the present invention targeting IL-23 will generally comprise: (i) a suitable framework sequence that suitably comprises the amino acid residues / mutations of the present invention as described herein; and (ii) a CDR sequence that allows the VH domain of the present invention to specifically bind to IL-23. Furthermore, the VH domain of the present invention targeting IL-23 may also suitably have a C-terminal extension as described herein, particularly when the VH domain is monovalent or forms the C-terminus of a compound of the present invention in which the VH domain is present (again, as further described herein). The VH domain of the present invention targeting IL-23 may also be as further described herein, and may particularly be ISVD.

[0534] Similarly, with respect to other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD for IL-23 described in this example) or compound containing it is referred to as “in accordance with the invention” or “as further described herein”, the preferred aspects / implementations and preferred options of the general description of the ISVD or compound of the invention herein also apply particularly to the specific ISVD or compound, unless explicitly stated otherwise or unless required by the specific technical context.

[0535] Therefore, in a particular aspect, the present invention relates to a VH structural domain (and particularly ISVD) for IL-23, wherein (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in this VH structural domain for IL-23:

[0536] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0537] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0538] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0539] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0540] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0541] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0542] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0543] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

[0544] The VH domain of the present invention targeting IL-23 can also be, as described herein, and more particularly, an ISVD (and more particularly, a nanobody) targeting IL-23, or a protein, peptide, or other compound or construct comprising at least one such ISVD. Such a protein, peptide, or other compound or construct can also be, as further described herein, and can, for example, have an increased half-life (i.e., as described herein, for example, a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days, expressed as t1 / 2β in a human subject), and for this purpose can, for example, comprise a nanobody that binds to serum albumin, which can also be a nanobody of the present invention that binds to serum albumin (again, as described herein).

[0545] Furthermore, such ISVDs may suitably have C-terminal extensions (as further described herein and in WO 12 / 175741), particularly when the ISVD forms the C-terminus of a protein, polypeptide or other compound or construct comprising it (again, as further described herein).

[0546] In a preferred aspect, the VH domain of the present invention for IL-23 comprises (i) a CDR1 sequence, which is sequence SEQ ID NO:173 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:173 by only one amino acid; (ii) a CDR2 sequence, which is sequence SEQ ID NO:174 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:174 by only one or two amino acids; and (iii) a CDR3 sequence, which is sequence SEQ ID NO:175 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:175 by only one or two amino acids.

[0547] More preferably, in the VH domain of the present invention for IL-23 according to this aspect: (i) CDR1 is SEQ ID NO:173; (ii) CDR2 is SEQ ID NO:174; and (iii) CDR3 is SEQ ID NO:175.

[0548] In one specific aspect, the nanobody of the present invention targeting IL-23 is a variant of the nanobody of SEQ ID NO:172 (having at least 90% sequence identity with SEQ ID NO:172, such as at least 95% sequence identity), wherein:

[0549] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0550] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0551] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0552] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0553] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0554] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0555] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0556] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR of such ISV is preferably as defined in the preceding two paragraphs.

[0557] In another preferred aspect, the VH domain of the present invention for IL-23 comprises (i) a CDR1 sequence, which is the sequence SEQ ID NO:191 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:191 by only one amino acid; (ii) a CDR2 sequence, which is the sequence SEQ ID NO:192 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:192 by only one or two amino acids; and (iii) a CDR3 sequence, which is the sequence SEQ ID NO:193 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:193 by only one or two amino acids.

[0558] More preferably, in the VH domain of the present invention for IL-23 according to this aspect: (i) CDR1 is SEQ ID NO:191; (ii) CDR2 is SEQ ID NO:192; and (iii) CDR3 is SEQ ID NO:193.

[0559] In one specific aspect, the nanobody of the present invention targeting IL-23 is a variant of the nanobody of SEQ ID NO:172 (having at least 90% sequence identity with SEQ ID NO:190, such as at least 95% sequence identity), wherein:

[0560] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0561] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0562] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0563] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0564] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0565] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0566] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0567] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Similarly, the CDR of such an ISV is preferably as defined in the preceding two paragraphs.

[0568] Some particularly preferred, but not limiting, examples of this invention’s nanobody targeting IL-23 are respectively in Figure 12A In which SEQ ID NO:176 to 189 and in Figure 12B The nanobodies are listed as SEQ ID NO:194 to 207; and each of these nanobodies forms another aspect of the present invention.

[0569] The present invention also relates to compounds of the invention targeting IL-23 comprising at least one (e.g., one, two, or three) of the nanobodies of the invention listed in SEQ ID NO: 176 to 189 and / or 194 to 207. Such compounds of the invention targeting IL-23 may also be as further described herein, and therefore may, for example, contain suitable linkers, may contain C-terminal extensions as described herein, and may have extended half-lives (e.g., because they contain nanobodies targeting human serum albumin, such as (preferably) the nanobodies of the invention targeting human serum albumin). See Table R below.

[0570] As described, for example, in WO 2009 / 068627, WO 2010 / 142534 and WO2011 / 135026, a particularly preferred class of nanobody-based compounds targeting IL-23 are bicomponent site compounds. Therefore, in one aspect of the invention, the compound of the invention targeting IL-23 is a bicomponent site construct comprising an ISV of SEQ ID NO:172 or (preferably) an ISV of the invention derived from SEQ ID NO:172 (as described in Example 8) and an ISV of SEQ ID NO:190 or (preferably) an ISV of the invention derived from SEQ ID NO:190 (as described in Example 8), provided that at least one (and preferably both) of these ISVs is an ISV of the invention. Such bicomponent site constructs can also have an extended half-life (i.e., by means of an ISV that binds to serum albumin). Some specific examples of such bicomponent site constructs are given in SEQ ID NO:514 to 549.

[0571] Some particularly preferred examples of the compounds of the present invention targeting IL-23 are in Figure 22 The compounds are given as SEQ ID NO:514 to 549; and each of these compounds forms another aspect of the invention. Therefore, in another aspect, the invention relates to polypeptides targeting IL-23 and having an amino acid sequence selected from the group consisting of SEQ ID NO:514 to 549. More generally, the compounds of the invention targeting IL-23 can be as described in WO 2009 / 068627, WO 2010 / 142534 and WO2011 / 135026, but contain the ISV of the invention. They can also be used for the purposes described in WO 2009 / 068627, WO2010 / 142534 and WO2011 / 135026.

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579] Example 9: The present invention relates to the VH domain of OX40-L (and particularly to nanobodies) and compounds comprising therein. Compound.

[0580] In one specific aspect, the VH domain of the present invention (and in particular the ISVD of the present invention and more particularly the nanobodies of the present invention) and the compounds of the present invention can target OX40-L.

[0581] Such a VH domain of the present invention targeting OX40-L will generally comprise: (i) a suitable framework sequence that suitably comprises the amino acid residues / mutations of the present invention as described herein; and (ii) a CDR sequence that allows the VH domain of the present invention to specifically bind to OX40-L. Furthermore, such a VH domain of the present invention targeting OX40-L may also suitably have a C-terminal extension as described herein, particularly when the VH domain is monovalent or forms the C-terminus of a compound of the present invention in which the VH domain is present (again, as further described herein). Such a VH domain of the present invention targeting OX40-L may also be as further described herein, and may particularly be ISVD.

[0582] Similarly, with respect to other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD for OX40-L described in this example) or compound containing therein is referred to as “in accordance with the invention” or “as further described herein”, the preferred aspects / implementations and preferred options of the general description of the ISVD or compound of the invention herein also apply particularly to the specific ISVD or compound, unless explicitly stated otherwise or unless required by the specific technical background.

[0583] Therefore, in a particular aspect, the present invention relates to a VH structural domain (and particularly ISVD) for OX40-L, wherein (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in this VH structural domain for OX40-L:

[0584] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0585] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0586] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0587] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0588] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0589] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0590] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0591] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

[0592] The VH domain of the present invention targeting OX40-L can also be, as described herein, and more particularly, an ISVD (and more particularly, a nanobody) targeting OX40-L, or a protein, peptide, or other compound or construct comprising at least one such ISVD. Such a protein, peptide, or other compound or construct can also be, as further described herein, and can, for example, have an increased half-life (i.e., as described herein, for example, a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days, expressed as t1 / 2β in a human subject), and for this purpose can, for example, comprise a nanobody that binds to serum albumin, which can also be a nanobody of the present invention that binds to serum albumin (again, as described herein).

[0593] Furthermore, such ISVDs may suitably have C-terminal extensions (as further described herein and in WO 12 / 175741), particularly when the ISVD forms the C-terminus of a protein, polypeptide or other compound or construct comprising it (again, as further described herein).

[0594] In a preferred aspect, the VH domain of the present invention for OX40-L comprises (i) a CDR1 sequence, which is sequence SEQ ID NO:209 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:209 by only one amino acid; (ii) a CDR2 sequence, which is sequence SEQ ID NO:210 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:210 by only one or two amino acids; and (iii) a CDR3 sequence, which is sequence SEQ ID NO:211 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:211 by only one or two amino acids.

[0595] More preferably, in the VH structural domain of the present invention for OX40-L according to this aspect: (i) CDR1 is SEQ ID NO:209; (ii) CDR2 is SEQ ID NO:210; and (iii) CDR3 is SEQ ID NO:211.

[0596] In one specific aspect, the nanobody of the present invention targeting OX40-L is a variant of the nanobody of SEQ ID NO:208 (having at least 90% sequence identity with SEQ ID NO:208, such as at least 95% sequence identity), wherein:

[0597] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0598] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0599] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0600] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0601] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0602] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0603] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0604] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR of such ISV is preferably as defined in the preceding two paragraphs.

[0605] Some particularly preferred, but not limiting, examples of the nanobodies of the present invention for OX40-L are described below. Figure 13 The nanobodies are listed as SEQ ID NO:212 to 225; and each of these nanobodies forms another aspect of the present invention.

[0606] The present invention also relates to compounds of the invention targeting OX40-L, comprising at least one (e.g., one, two, or three) of the nanobodies of the invention listed in SEQ ID NO: 212 to 225. Such compounds of the invention targeting OX40-L may also be as further described herein, and therefore may, for example, contain suitable linkers, may contain C-terminal extensions as described herein, and may have extended half-lives (e.g., because they contain nanobodies targeting human serum albumin, such as (preferably) the nanobodies of the invention targeting human serum albumin). See Table S below.

[0607] Some particularly preferred examples of the compounds of the present invention for OX40-L are in Figure 23 The compounds are given as SEQ ID NO: 550 to 585; and each of these compounds forms another aspect of the invention. Therefore, in another aspect, the invention relates to polypeptides targeting OX40-L and having an amino acid sequence selected from the group consisting of SEQ ID NO: 550 to 585. More generally, the compounds of the invention targeting OX40-L can be as described in WO 2011 / 073180, but contain the ISV of the invention. They can also be used for the purposes described in WO 2011 / 073180.

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616] Example 10: Targeting the VH domain of IgE (and particularly nanobodies) and compounds of the present invention containing it. things.

[0617] In one specific aspect, the VH domain of the present invention (and in particular the ISVD of the present invention and more particularly the nanobodies of the present invention) and the compounds of the present invention can target IgE.

[0618] Such an IgE-targeting VH domain of the present invention will generally comprise: (i) a suitable framework sequence suitably comprising the amino acid residues / mutations of the present invention as described herein; and (ii) a CDR sequence that allows the VH domain of the present invention to specifically bind to IgE. Furthermore, such an IgE-targeting VH domain of the present invention may also suitably have a C-terminal extension as described herein, particularly when the VH domain is monovalent or forms the C-terminus of a compound of the present invention in which the VH domain is present (again, as further described herein). Such an IgE-targeting VH domain of the present invention may also be as further described herein, and may particularly be ISVD.

[0619] Similarly, with respect to other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD for IgE described in this example) or compound containing it is referred to as “in accordance with the invention” or “as further described herein”, the preferred aspects / implementations and preferred options of the general description of the ISVD or compound of the invention herein also apply particularly to the specific ISVD or compound, unless explicitly stated otherwise or unless required by the specific technical context.

[0620] Therefore, in a particular aspect, the present invention relates to a VH structural domain (and particularly ISVD) for IgE, wherein (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in this VH structural domain for IgE:

[0621] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0622] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0623] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0624] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0625] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0626] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0627] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0628] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

[0629] The VH domain of the present invention targeting IgE can also be an ISVD (and more particularly a nanobody) targeting IgE, as described herein, or a protein, peptide, or other compound or construct comprising at least one such ISVD. Such a protein, peptide, or other compound or construct can also be as further described herein and can, for example, have an increased half-life (i.e., as described herein, for example, a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days, expressed as t1 / 2β in a human subject), and for this purpose can, for example, comprise a nanobody that binds serum albumin, which can also be a nanobody of the present invention that binds serum albumin (again, as described herein).

[0630] Furthermore, such ISVDs may suitably have C-terminal extensions (as further described herein and in WO 12 / 175741), particularly when the ISVD forms the C-terminus of a protein, polypeptide or other compound or construct comprising it (again, as further described herein).

[0631] In a preferred aspect, the VH domain of the present invention for IgE comprises (i) a CDR1 sequence, which is sequence SEQ ID NO:227 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:227 by only one amino acid; (ii) a CDR2 sequence, which is sequence SEQ ID NO:228 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:228 by only one or two amino acids; and (iii) a CDR3 sequence, which is sequence SEQ ID NO:229 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:229 by only one or two amino acids.

[0632] More preferably, in the VH domain of the present invention for IgE according to this aspect: (i) CDR1 is SEQ ID NO:227; (ii) CDR2 is SEQ ID NO:228; and (iii) CDR3 is SEQ ID NO:229.

[0633] In one specific aspect, the nanobody of the present invention targeting IgE is a variant of the nanobody of SEQ ID NO:226 (having at least 90% sequence identity with SEQ ID NO:226, such as at least 95% sequence identity), wherein:

[0634] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0635] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0636] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0637] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0638] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0639] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0640] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0641] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR of such ISV is preferably as defined in the preceding two paragraphs.

[0642] Some particularly preferred, but not limiting, examples of the nanobodies of the present invention targeting IgE are described below. Figure 14 The nanobodies are listed as SEQ ID NO:230 to 243; and each of these nanobodies forms another aspect of the present invention.

[0643] The present invention also relates to compounds of the invention targeting IgE, comprising at least one (e.g., one, two, or three) of the nanobodies of the invention as described herein in SEQ ID NO: 230 to 243. Such compounds of the invention targeting IgE may also be as further described herein, and therefore may, for example, contain suitable linkers, may contain C-terminal extensions as described herein, and may have extended half-lives (e.g., because they contain nanobodies targeting human serum albumin, such as (preferably) the nanobodies of the invention targeting human serum albumin). See Table T below.

[0644] Some particularly preferred examples of the compounds of the present invention targeting IgE are in Figure 24 The compounds are given as SEQ ID NO: 586 to 594; and each of these compounds forms another aspect of the invention. Therefore, in another aspect, the invention relates to polypeptides targeting IgE and having an amino acid sequence selected from the group consisting of SEQ ID NO: 586 to 594.

[0645] More generally, the compounds of the present invention targeting IgE can be as described in the relevant portions of WO 2012 / 175740 and WO2012 / 175400, but contain the ISV of the present invention. They can also be used for the purposes described in WO 2012 / 175740.

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653] Example 11: Targeting the VH domain of CXCR4 (and particularly nanobodies) and the present invention comprising therein. Compound.

[0654] In one specific aspect, the VH domain of the present invention (and in particular the ISVD of the present invention and more particularly the nanobodies of the present invention) and the compounds of the present invention can target CXCR4.

[0655] The VH domain of the present invention targeting CXCR4 will generally comprise: (i) a suitable framework sequence that suitably comprises the amino acid residues / mutations of the present invention as described herein; and (ii) a CDR sequence that allows the VH domain of the present invention to specifically bind to CXCR4. Furthermore, the VH domain of the present invention targeting CXCR4 may also suitably have a C-terminal extension as described herein, particularly when the VH domain is monovalent or forms the C-terminus of a compound of the present invention in which the VH domain is present (again, as further described herein). The VH domain of the present invention targeting CXCR4 may also be as further described herein, and may particularly be ISVD.

[0656] Similarly, with respect to other aspects and embodiments of the invention described herein, when a specific ISVD (such as the ISVD for CXCR4 described in this example) or compound containing it is referred to as “in accordance with the invention” or “as further described herein”, the preferred aspects / implementations and preferred options of the general description of the ISVD or compound of the invention herein also apply particularly to the specific ISVD or compound, unless explicitly stated otherwise or unless the specific technical background requires otherwise.

[0657] Therefore, in a particular aspect, the present invention relates to a VH structural domain (and particularly ISVD) for CXCR4, wherein (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in this VH structural domain for CXCR4:

[0658] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0659] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0660] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0661] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0662] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0663] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0664] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0665] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

[0666] The VH domain of the present invention targeting CXCR4 can also be, as described herein, and more particularly, an ISVD (and more particularly, a nanobody) targeting CXCR4, or a protein, peptide, or other compound or construct comprising at least one such ISVD. Such a protein, peptide, or other compound or construct can also be, as further described herein, and can, for example, have an increased half-life (i.e., as described herein, for example, a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days, expressed as t1 / 2β in a human subject), and for this purpose can, for example, comprise a nanobody that binds to serum albumin, which can also be a nanobody of the present invention that binds to serum albumin (again, as described herein).

[0667] Furthermore, such ISVDs may suitably have C-terminal extensions (as further described herein and in WO 12 / 175741), particularly when the ISVD forms the C-terminus of a protein, polypeptide or other compound or construct comprising it (again, as further described herein).

[0668] In a preferred aspect, the VH domain of the present invention for CXCR4 comprises (i) a CDR1 sequence, which is sequence SEQ ID NO:245 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:245 by only one amino acid; (ii) a CDR2 sequence, which is sequence SEQ ID NO:246 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:246 by only one or two amino acids; and (iii) a CDR3 sequence, which is sequence SEQ ID NO:247 (preferred) or an amino acid sequence differing from sequence SEQ ID NO:247 by only one or two amino acids.

[0669] More preferably, in the VH domain of the present invention for CXCR4 according to this aspect: (i) CDR1 is SEQ ID NO:245; (ii) CDR2 is SEQ ID NO:246; and (iii) CDR3 is SEQ ID NO:247.

[0670] In one specific aspect, the nanobody of the present invention targeting CXCR4 is a variant of the nanobody of SEQ ID NO:244 (having at least 90% sequence identity with SEQ ID NO:244, such as at least 95% sequence identity), wherein:

[0671] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0672] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0673] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0674] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0675] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0676] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0677] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0678] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR of such ISV is preferably as defined in the preceding two paragraphs.

[0679] In another preferred aspect, the VH domain of the present invention for CXCR4 comprises (i) a CDR1 sequence, which is sequence SEQ ID NO:263 (which is preferred) or an amino acid sequence differing from sequence SEQ ID NO:263 by only one amino acid; (ii) a CDR2 sequence, which is sequence SEQ ID NO:264 (which is preferred) or an amino acid sequence differing from sequence SEQ ID NO:264 by only one or two amino acids; and (iii) a CDR3 sequence, which is sequence SEQ ID NO:265 (which is preferred) or an amino acid sequence differing from sequence SEQ ID NO:265 by only one or two amino acids.

[0680] More preferably, in the VH domain of the present invention for CXCR-4 according to this aspect: (i) CDR1 is SEQ ID NO:263; (ii) CDR2 is SEQ ID NO:264; and (iii) CDR3 is SEQ ID NO:265.

[0681] In one specific aspect, the nanobody of the present invention targeting IL-23 is a variant of the nanobody of SEQ ID NO:262 (having at least 90% sequence identity with SEQ ID NO:262, such as at least 95% sequence identity), wherein:

[0682] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0683] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0684] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0685] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0686] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0687] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0688] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0689] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Similarly, the CDR of such an ISV is preferably as defined in the preceding two paragraphs.

[0690] Some particularly preferred, but not limiting, examples of the nanobodies of the present invention targeting CXCR4 are described below. Figure 15A Listed as SEQ ID NO:248 to 261 and in Figure 15B The nanobodies are listed as SEQ ID NO:266 to 279; and each of these nanobodies forms another aspect of the present invention.

[0691] The present invention also relates to compounds of the present invention targeting CXCR4, comprising at least one (e.g., one, two, or three) of the nanobodies of the present invention SEQ ID NO: 248 to 261 and / or 266 to 279. Such compounds of the present invention targeting CXCR4 may also be as further described herein, and therefore may, for example, contain suitable linkers, may contain C-terminal extensions as described herein, and may have extended half-lives (e.g., because they contain nanobodies targeting human serum albumin, such as (preferably) the nanobodies of the present invention targeting human serum albumin). See Table U below.

[0692] As described, for example, in WO 2009 / 138519, WO 2011 / 042398, and WO 2011 / 161266, a particularly preferred class of nanobody-based compounds targeting CXCR4 are bicomponent compounds. Therefore, in one aspect of the invention, the compound of the invention targeting CXCR4 is a bicomponent construct comprising an ISV of SEQ ID NO:244 or (preferably) an ISV of the invention derived from SEQ ID NO:244 (as described in Example 11) and an ISV of SEQ ID NO:262 or (preferably) an ISV of the invention derived from SEQ ID NO:262 (as described in Example 11), provided that at least one (and preferably both) of these ISVs is an ISV of the invention. Such bicomponent constructs can also have an extended half-life (i.e., by means of an ISV that binds to serum albumin). Some specific examples of such bicomponent constructs are listed in SEQ ID NO:595 to 603.

[0693] Some particularly preferred examples of the compounds of the present invention for CXCR-4 are in Figure 25 The compounds are given as SEQ ID NO: 595 to 603; and each of these compounds forms another aspect of the invention. Therefore, in another aspect, the invention relates to polypeptides targeting CXCR-4 and having an amino acid sequence selected from the group consisting of SEQ ID NO: 595 to 603. More generally, the compounds of the invention targeting CXCR-4 can be as described in WO 2009 / 138519, WO 2011 / 042398, and WO 2011 / 161266, but contain the ISV of the invention. They can also be used for the purposes described in WO 2009 / 138519, WO 2011 / 042398, and WO 2011 / 161266.

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703] Example 12: Targeting the VH domain of HER-3 (and particularly nanobodies) and the present invention comprising therein. Compound.

[0704] In one specific aspect, the VH domain of the present invention (and in particular the ISVD of the present invention and more particularly the nanobodies of the present invention) and the compounds of the present invention can target HER-3.

[0705] Such a HER-3-targeting VH domain of the present invention will generally comprise: (i) a suitable framework sequence suitably comprising the amino acid residues / mutations of the present invention as described herein; and (ii) a CDR sequence that allows the VH domain of the present invention to specifically bind to HER-3. Furthermore, such a HER-3-targeting VH domain of the present invention may also suitably have a C-terminal extension as described herein, particularly when the VH domain is monovalent or forms the C-terminus of a compound of the present invention in which the VH domain is present (again, as further described herein). Such a HER-3-targeting VH domain of the present invention may also be as further described herein, and may particularly be ISVD.

[0706] Similarly, with respect to other aspects and embodiments of the invention described herein, when a specific ISVD (such as the HER-3 ISVD described in this embodiment) or compound containing it is referred to as “in accordance with the invention” or “as further described herein,” the preferred aspects / implementations and preferred options of the general description of the ISVD or compound of the invention herein also apply particularly to the specific ISVD or compound, unless explicitly stated otherwise or unless the specific technical context requires otherwise.

[0707] Therefore, in a particular aspect, the present invention relates to a VH structural domain (and particularly ISVD) for HER-3, wherein (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). In particular, in this VH structural domain for HER-3:

[0708] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0709] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0710] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0711] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0712] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0713] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0714] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0715] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v).

[0716] The VH domain of the present invention targeting HER-3 can also be, as described herein, and more particularly, an ISVD (and more particularly, a nanobody) targeting HER-3, or a protein, peptide, or other compound or construct comprising at least one such ISVD. Such a protein, peptide, or other compound or construct can also be, as further described herein, and can, for example, have an increased half-life (i.e., as described herein, for example, a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, such as at least 10 days, expressed as t1 / 2β in a human subject), and for this purpose can, for example, comprise a nanobody that binds to serum albumin, which can also be a nanobody of the present invention that binds to serum albumin (again, as described herein).

[0717] Furthermore, such ISVDs may suitably have C-terminal extensions (as further described herein and in WO 12 / 175741), particularly when the ISVD forms the C-terminus of a protein, polypeptide or other compound or construct comprising it (again, as further described herein).

[0718] In a preferred aspect, the VH domain of the present invention for HER-3 comprises (i) a CDR1 sequence, which is the sequence SEQ ID NO:281 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:281 by only one amino acid; (ii) a CDR2 sequence, which is the sequence SEQ ID NO:282 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:282 by only one or two amino acids; and (iii) a CDR3 sequence, which is the sequence SEQ ID NO:283 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:283 by only one or two amino acids.

[0719] More preferably, in the VH domain of the present invention for HER-3 according to this aspect: (i) CDR1 is SEQ ID NO:281; (ii) CDR2 is SEQ ID NO:282; and (iii) CDR3 is SEQ ID NO:283.

[0720] In one specific aspect, the nanobody of the present invention targeting HER-3 is a variant of the nanobody of SEQ ID NO:280 (having at least 90% sequence identity with SEQ ID NO:280, such as at least 95% sequence identity), wherein:

[0721] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0722] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0723] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0724] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0725] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0726] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0727] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0728] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). The CDR of such ISV is preferably as defined in the preceding two paragraphs.

[0729] In another preferred aspect, the VH domain of the present invention for HER-3 comprises (i) a CDR1 sequence, which is the sequence SEQ ID NO:299 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:299 by only one amino acid; (ii) a CDR2 sequence, which is the sequence SEQ ID NO:300 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:300 by only one or two amino acids; and (iii) a CDR3 sequence, which is the sequence SEQ ID NO:301 (which is preferred) or an amino acid sequence differing from the sequence SEQ ID NO:301 by only one or two amino acids.

[0730] More preferably, in the VH domain of the present invention for HER-3 according to this aspect: (i) CDR1 is SEQ ID NO:299; (ii) CDR2 is SEQ ID NO:300; and (iii) CDR3 is SEQ ID NO:301.

[0731] In one specific aspect, the nanobody of the present invention targeting HER-3 is a variant of the nanobody of SEQ ID NO:298 (having at least 90% sequence identity with SEQ ID NO:298, such as at least 95% sequence identity), wherein:

[0732] - The amino acid residue at position 11 is preferably selected from L, V, or K (and most preferably V); and

[0733] - The amino acid residue at position 14 is preferably suitably selected from A or P; and

[0734] - The amino acid residue at position 41 is preferably suitably selected from A or P; and

[0735] - The amino acid residue at position 89 is preferably suitably selected from T, V, or L; and

[0736] - The amino acid residue at position 108 is preferably suitably selected from Q or L; and

[0737] - The amino acid residue at position 110 is preferably suitably selected from T, K, or Q; and

[0738] - The amino acid residue at position 112 is preferably suitably selected from S, K or Q;

[0739] Such that (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K or Q; or (v) position 11 is V and position 89 is L; or any suitable combination of (i) to (v). Similarly, the CDR of such an ISV is preferably as defined in the preceding two paragraphs.

[0740] Some particularly preferred, but not limiting, examples of the nanobodies of the present invention targeting HER-3 are respectively described in Figure 16A Listed as SEQ ID NO:284 to 297 and in Figure 16B The nanobodies are listed as SEQ ID NO:302 to 315; and each of these nanobodies forms another aspect of the present invention.

[0741] The present invention also relates to compounds of the invention targeting HER-3, comprising at least one (e.g., one, two, or three) of the nanobodies of the invention listed in SEQ ID NO: 284 to 297 and / or 302 to 315. Such compounds of the invention targeting HER-3 may also be as further described herein, and therefore may, for example, contain suitable linkers, may contain C-terminal extensions as described herein, and may have extended half-lives (e.g., because they contain nanobodies targeting human serum albumin, such as (preferably) the nanobodies of the invention targeting human serum albumin). See Table V below.

[0742] As described, for example, in WO 2011 / 144749, a particularly preferred class of HER-3-targeting nanobody-based compounds are bicomponent site compounds. Therefore, in one aspect of the invention, the HER-3-targeting compound of the invention is a bicomponent site construct comprising an ISV of SEQ ID NO:280 or (preferably) an ISV of the invention derived from SEQ ID NO:280 (as described in Example 12) and an ISV of SEQ ID NO:298 or (preferably) an ISV of the invention derived from SEQ ID NO:298 (as described in Example 12), provided that at least one (and preferably both) of these ISVs is an ISV of the invention. Such bicomponent site constructs can also have an extended half-life (i.e., by means of an ISV that binds to serum albumin). Some specific examples of such bicomponent site constructs are given in SEQ ID NO:604 to 639.

[0743] Some particularly preferred examples of the compounds of the present invention targeting HER-3 are in Figure 26The compounds are given as SEQ ID NO: 604 to 639; and each of these compounds forms another aspect of the invention. Therefore, in another aspect, the invention relates to polypeptides targeting HER-3 and having an amino acid sequence selected from the group consisting of SEQ ID NO: 604 to 639. More generally, the compounds of the invention targeting HER-3 can be as described in WO 2011 / 144749, but contain the ISV of the invention. They can also be used for the purposes described in WO 2011 / 144749.

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752] Example 13: VH domains of TNF (and particularly nanobodies) and compounds of the present invention containing them. things.

[0753] In one specific aspect, the VH domain of the present invention (and in particular the ISVD of the present invention and more particularly the nanobodies of the present invention) and the compounds of the present invention can target TNF.

[0754] The VH domain of the present invention targeting TNF will generally comprise: (i) a suitable framework sequence that suitably comprises the amino acid residues / mutations of the present invention as described herein; and (ii) a CDR sequence that allows the VH domain of the present invention to specifically bind to TNF. Furthermore, the VH domain of the present invention targeting TNF may also suitably have a C-terminal extension as described herein, particularly when the VH domain is monovalent or forms the C-terminus of a compound of the present invention in which the VH domain is present (again, as further described herein). The VH domain of the present invention targeting TNF may also be as further described herein, and may particularly be ISVD.

[0755] Similarly, with respect to other aspects and embodiments of the invention described herein, when a specific ISVD (such as the TNF-targeting ISVD described in this example) or compound containing it is referred to as “in accordance with the invention” or “as further described herein”, the preferred aspects / implementations and preferred options of the general description of the ISVD or compound of the invention herein also apply particularly to the specific ISVD or compound, unless explicitly stated otherwise or unless required by the specific technical context.

[0756] Therefore, in a particular aspect, the present invention relates to a VH domain (and particularly ISVD) targeting TNF, wherein (i) position 112 is K or Q; or (ii) position 110 is K or Q and position 11 is V; or (iii) position 89 is T; or (iv) position 89 is L and position 110 is K ...

Claims

1. Heavy chain immunoglobulin single variable domain (ISVD), wherein the amino acid residues / positions are indicated according to Kabat numbering. (1) The frame regions 1-4 of the ISVD are the same as frame regions 1-4 of any one of SEQ ID NOs: 109, 140, 141, 142, 149, 150, 151, 273, 471 and 489, or (2) The frame regions 1-4 of the ISVD have only the following amino acid differences from the frame regions 1-4 of any one of SEQ ID NOs: 109, 140, 141, 142, 149, 150, 151, 273, 471 and 489, said amino acid differences being selected from: The amino acid residue at position 87 is A; The amino acid residue at position 110 is K or Q; The amino acid residue at position 112 is K or Q; and Any combination thereof; Frame region 1 consists of amino acid residues at positions 1-30, frame region 2 consists of amino acids at positions 36-49, frame region 3 consists of amino acid residues at positions 66-94, and frame region 4 consists of amino acid residues at positions 103-113.

2. The heavy chain ISVD according to claim 1, wherein (i) it contains an amino acid selected from K or Q at position 110; and (ii) it contains an amino acid selected from K or Q at position 112.

3. The heavy chain ISVD according to claim 1, wherein an amino acid selected from K or Q is present at position 110.

4. The heavy chain ISVD according to claim 1, wherein an amino acid selected from K or Q is present at position 112.

5. The heavy-chain ISVD according to claim 1, wherein the VH domain includes a C-terminal extension (X). n , where n is 1; and X is alanine (A).

6. The heavy chain ISVD according to claim 1, wherein the C-terminus is VTVSS (SEQ ID NO: 76) or VTVSS(X). n (SEQ ID NO: 77), wherein X and n are as defined in claim 5.

7. The heavy-chain ISVD according to claim 1, wherein the C-terminus of the VH domain is one of the following: VTVSS (SEQ ID NO:76), VTVSS(X)n (SEQ ID NO:77), VTVKS (SEQ ID NO:1), VTVKS(X)n (SEQ ID NO:21), VTVQS (SEQ ID NO:2), VTVQS(X)n (SEQ ID NO:22), VKVSS (SEQ ID NO: 95), VKVSS(X)n (SEQ ID NO:97), VQVSS (SEQ ID NO: 96), VQVSS(X)n (SEQ ID NO: 98), VZVZS (SEQ ID NO: 107) or VZVZSX(n) (SEQ ID NO:108), wherein n is 1; and X is alanine (A), and each amino acid residue Z is independently K or Q.

8. The heavy chain ISVD according to claim 7, wherein the C-terminus of the VH domain is one of the following: VTVSS (SEQ ID NO: 76), VKVSS (SEQ ID NO: 95), VQVSS (SEQ ID NO: 96), VKVSS(X)n (SEQ ID NO: 97) or VQVSS(X)n (SEQ ID NO: 98), wherein X and n are defined as in claim 7.

9. The heavy chain ISVD according to claim 7 or 8, wherein the C-terminus of the VH domain is one of the following: VTVSS (SEQ ID NO: 76) or VTVSS(X)n (SEQ ID NO: 77), wherein X and n are defined as in claim 7.

10. The heavy chain ISVD according to claim 1 or 9, wherein it is a VHH domain or a camel-derived VH domain.

11. The heavy chain ISVD according to claim 10, wherein it is a humanized VHH domain.

12. The heavy chain ISVD according to claim 1, which is capable of specifically binding to human serum albumin.

13. The heavy chain ISVD according to claim 12, wherein the amino acid residue at position 89 is L and the amino acid residue at position 11 is V, the heavy chain ISVD is capable of specifically binding to human serum albumin, and the heavy chain ISVD has at least 80% sequence identity with at least one of Alb-1, Alb-8 (SEQ ID NO: 46) and / or Alb-23 (SEQ ID NO: 61) of SEQ ID NO: 52 of WO2006 / 122787.

14. The heavy chain ISVD according to claim 12, which is capable of specifically binding to human serum albumin, wherein the heavy chain ISVD: - CDR1 is the amino acid sequence SFGMS (SEQ ID NO:41); - CDR2 is the amino acid sequence SIGSGSDTLYADSVKG (SEQ ID NO:42); - CDR3 is the amino acid sequence GGSLSR (SEQ ID NO:43).

15. A library of heavy chain ISVDs according to any one of claims 1 to 14.

16. A library encoding nucleic acid residues of the heavy chain ISVD according to any one of claims 1 to 14.

17. The library according to claim 16, wherein it is an expression library.

18. The library according to any one of claims 15-17, wherein it is a synthesized library.

19. The library according to any one of claims 15-17, comprising at least 100 different sequences.

20. A polypeptide comprising at least one heavy chain immunoglobulin monovariable domain according to any one of claims 1 to 14.

21. A nucleic acid encoding a single variable domain of a heavy chain immunoglobulin according to any one of claims 1 to 14 or a polypeptide according to claim 20.

22. A method for preparing a single variable domain of a heavy chain immunoglobulin according to any one of claims 1 to 14 or a polypeptide according to claim 20, optionally comprising expressing the nucleic acid according to claim 21 in a suitable host organism.

23. A composition comprising: a single variable domain of a heavy chain immunoglobulin according to any one of claims 1 to 14, a polypeptide according to claim 20, or a nucleic acid according to claim 21, wherein the composition is optionally a pharmaceutical composition.

24. The heavy chain immunoglobulin with a single variable domain according to claim 1 or the polypeptide according to claim 20, for pharmaceutical use.

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