Improved immunoglobulin variable domains

Abstract

To provide improved heavy chain immunoglobulin variable domains.SOLUTION: In a VH domain, (i) the amino acid residue at position 112 is 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 K or Q; and (iv) in each of cases (i) to (iii), the amino acid at position 11 is preferably V. The VH domain contains a C-terminal extension (X)n [in the extension, n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5; and each X is a (preferably naturally occurring) amino acid residue that is independently selected from alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I) and preferably independently selected from the group consisting of them].SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] The present invention particularly refers to improved heavy chain immunoglobulin variable domains used (or intended to be used) in applications having either an exposed C-terminal region or end (see also International Publication No. 12 / 175741, as further described herein), or having an exposed C-terminal region or end (again, as further described herein). Some preferred, non-limiting examples of the former are immunoglobulin single variable domains (also referred herein as "ISV" or "ISVD"), e.g., nanobodies (including VHH, humanized VHH, and camelized VH, e.g., camelized human VH), VH domains or antibodies obtained from VH domains (single-domain antibodies), and VH domains or dAbs obtained from VH domains. Some preferred, non-limiting examples of the latter are VH domains used (or intended to be used) in single-stranded FV (ScFv) or diabodies.

[0003] The present invention also relates to proteins, polypeptides, and other constructs, molecules, or chemical components comprising (one or more) the improved heavy immunoglobulin variable domains of the present invention as described herein, or essentially consisting thereof; methods for expressing / producing the improved heavy immunoglobulin variable domains of the present invention, and / or methods for expressing / producing proteins, polypeptides, and other constructs, molecules, or chemical components comprising them; compositions and products (e.g., pharmaceutical compositions and products), and / or proteins, polypeptides, and other constructs, molecules, or chemical components comprising them; nucleotide sequences and nucleic acids encoding and / or proteins or polypeptides comprising the improved heavy immunoglobulin variable domains of the present invention; and the use (and in particular therapeutic, prophylactic, and diagnostic uses) of the improved heavy immunoglobulin variable domains of the present invention and proteins, polypeptides, and other constructs, molecules, or chemical components comprising them.

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

[0005] In this application, amino acid residues / positions in the immunoglobulin heavy chain variable domain will be indicated by numbering according to Kabat. For convenience, Figure 1 provides a table listing some of the amino acid positions that will be specifically referred to herein, along with their numbering according to several alternative numbering systems (e.g., Aho and IMGT; Note: Kabat numbering is definitive for this description and the claims, and other numbering systems are provided for reference only).

[0006] Furthermore, in the present invention, the immunoglobulin variable domain is a constant domain (for example, C H If it is not associated with or linked to a domain, it can be said to have an "exposed C-terminus or region." See the relevant prior art cited herein. [Background technology]

[0007] In particular, as described in International Publication No. 12 / 175741, the C-terminal region (as this term is also used herein) is part of a virtual epitope on the ISV and also includes, among other residues, the amino acid residue at position 14 (and the amino acid residues next to / near the same residue in the amino acid sequence, e.g., positions 11, 13, and 15), the amino acid residue at position 83 (and the amino acid residues next to / near the same residue in the amino acid sequence, e.g., positions 82, 82a, 82b, and 84), and / or the amino acid residue at position 108 (and the amino acid residues next to / near the same residue in the amino acid sequence, e.g., position 107). Similar to International Publication No. 12 / 175741, the putative epitope is also collectively referred to herein as the "C-terminal region." This C-terminal region includes at least the C-terminal sequence VTVSS (i.e., at positions 109, 110, 111, 112, and 113, respectively) and the amino acid residue at position 14, and may also include the amino acid residues at positions 83 and 108, and it is understood that it may also include the amino acid residues at positions 13, 15, 82b, 83, 84, and 107.

[0008] As a result of investigations in single-chain Fv or ScFv (a construct containing an immunoglobulin variable domain not associated with a constant domain, similar to ISVD), it has been described in the art that the C-terminus of the immunoglobulin variable domain is buried at the interface between the variable and constant domains in a conventional full-length antibody, but contains a hydrophobic patch that is solvent-exposed when the variable domain is not associated with 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] Furthermore, it is well known that epitopes that are normally embedded within the structure of proteins (also called "neo-epitopes" or "latent epitopes") can trigger the immune system when exposed to solvents, for example, by causing degradation, misfolding, or aggregation of the involved proteins. For example, in the case of embedded hydrophobic regions of biomolecules (so-called "hyppos"), it has been suggested that when hyppos are exposed to solvents, they form part of a common injury-associated molecular pattern that leads to an innate immune response (see, for example, Seong and Matzinger, Nature Reviews 2004, 469). Various examples of hydrophobic patches whose origins trigger immune responses 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, International Publication No. 11 / 07586, p. 10; and Kolaskar, FEBS 276, 172-174 (1990)). Similarly, it has been reported that hydrophobic patches at the C-terminus of heavy chain variable domains (as described above by Nieba et al. and Harmsen et al.) may generate (or be pre-existing) anti-drug antibodies and / or form B cell epitopes that can interact with such antibodies (International Publication No. 11 / 07586). For this reason, it has been proposed to mutate some of the amino acid residues that form part of the C-terminus of the variable domain to reduce hydrophobicity and / or remove B cell epitopes. For example, Nieba et al. have proposed mutating positions 11, 14, 41, 84, 87, and / or 89 (numbering according to Kabat) of the VH region.On the one hand, International Publication No. 11 / 07586 proposes mutating positions 99, 101, and / or 148 (AHo numbering) of the VL domain, or positions 12, 97, 98, 103, and / or 144 (again, AHo numbering - these positions correspond to positions 11, 83, 84, 85, 89, and 103 according to Kabat) of the VH domain. Similarly, Harmsen et al. proposed compensating for the absence of the C domain by mutating positions 12 and 101 (IMGT numbering, which are positions 11 and 89 according to Kabat). The authors also identified a specific subfamily of VHHs (referred to as "VHH4") containing amino acids that are candidates for substitution at these positions. H 1 domain, which is proposed to mutate positions 12 and 101 (IMGT numbering, which are positions 11 and 89 according to Kabat). The authors also identified a specific subfamily of VHHs (referred to as "VHH4") containing amino acids that are candidates for substitution at these positions.

[0010] In addition, it has also been described in the art that a biological sample obtained from a human subject may contain (existing) proteins or factors capable of binding to the exposed C-terminal region or end of an immunoglobulin variable domain (e.g., the C-terminal region or end of the ISVD or VH or VL domain in a ScFv or diabody) (see, for example, International Publication No. 12 / 175741 and the references cited in the following paragraph).

[0011] For example, International Publication No. 2013 / 024059 states that "serum from some healthy naive human subjects contains both existing anti-VH autoantibodies capable of binding to both VH domain antibodies and VHH molecules, and anti-VL (e.g., V kappa (VK)) autoantibodies capable of binding to VL molecules", and that "existing ADAs that bind to VH dAbs are similar to anti-hinge antibodies that bind to IgG fragments but not to the same sequences found in situ on intact IgG".

[0012] Holland et al., J. Clin. Immunol. 2013, 33(7):1192-203, reported that approximately half of the blood of normal and healthy humans contains fully human V " HIt is described that this includes a novel classification of anti-IgG autoantibodies (also called "HAVH autoantibodies" in Holland et al.) at varying levels that can bind to the framework sequence of domain antibodies. Furthermore, Holland et al. states that these autoantibodies are primarily of the IgG isotype, and V H Relatively high affinity for the sequence (approximately 10 -10 It is thought that this represents M), and the released C-terminus is V of these HAVH autoantibodies. H It is mentioned that this is considered important for joining to the domain.

[0013] Furthermore, issues concerning existing biotherapeutic-responsive antibodies to biotherapeutic molecules and their regulatory effects are discussed comprehensively in Xue et al., AAPS J. 2013; 15(3):852-5.

[0014] Furthermore, the aforementioned prior art has focused on methods for modifying the sequence of immunoglobulin variable domains in order to prevent or reduce the binding of such existing antibodies / factors to the variable domain. In this regard, International Publication No. 2011 / 07586 suggests introducing one or more mutations in the amino acid sequence of the variable domain at several specific locations (these locations are surface-exposed) within the domain. International Publication No. 12 / 175741 describes how the binding of such existing antibodies / factors can be reduced by adding a few amino acid residues (and at least one alanine residue) to the C-terminus of the VH domain, and / or by introducing one or more specific substitutions or deletions within the C-terminal region of the variable domain. This is described in International Publication No. 12 / 175741 as including at least the C-terminal amino acid sequence VTVSS and the amino acid residue at position 14 (for these positions, International Publication No. 12 / 175741 teaches that the presence of an alanine residue provides reduced binding of existing antibodies compared to the presence of proline, which is a “human” amino acid residue), and may also include the amino acid residues at positions 108 and 83, as well as amino acid residues near the aforementioned positions (International Publication No. 2013 / 024059 essentially provides the same teaching as International Publication No. 12 / 175741).

[0015] For example, prior to the filing of International Publication No. 12 / 175741, an applicant / assignee-initiated investigation found that adding a single alanine residue to the C-terminal region or end of an exposed VH domain would typically prevent / remove the binding (essentially all binding) of 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, line 3 of International Publication No. 12 / 175741). These findings have been confirmed by further results obtained by the applicant / assignee after the filing of International Publication No. 12 / 175741, applying the C-terminal alanine substitution of International Publication No. 12 / 175741 to other nanobodies (data not shown).

[0016] Furthermore, in the Applicant / Assignee's International Publication Nos. 12 / 175741 and 12 / 175400, the C-terminal extension described in International Publication No. 12 / 175741 is applied to confirm serum albumin-binding nanobodies (see, for example, the constructs shown in International Publication No. 12 / 175741: Sequence IDs: 37, 51-53, and 55-64, and Sequence IDs: 41, 43, and 44, and International Publication No. 12 / 175400: Sequence IDs: 6-11).

[0017] Furthermore, Figure 9 of International Publication No. 12 / 175741 also describes two albumin-binding sequences that will be used as reference sequences in the following experiments. These are Sequence ID No. 37 (hereinafter also referred to as "Reference B," and its sequence is provided herein as Sequence ID No. 45) from Figure 9 of International Publication No. 12 / 175741 and "Sequence ID No. 37 without the added C-terminal amino acid residue" (hereinafter also referred to as "Reference A," and its sequence is provided as Sequence ID No. 44) from Figure 9 of International Publication No. 12 / 175741. Both Reference A and Reference B are obtained from the sequence of the humanized anti-albumin nanobody "Alb-8," which is provided as Sequence ID No. 62 in International Publication No. 06 / 122787 (and also referred to herein as "Alb-11"). On the other hand, 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. References A, B, and Alb-8 / Alb-11 all contain the CDRs provided for SEQ ID NOs: 41-43, respectively.

[0018] Other examples of nanobodies and other immunoglobulin single variable domains having C-terminal elongation and / or mutations in the C-terminal region can be found, for example, in the prior art described below: International Publication No. 06 / 129843 (see, e.g., Sequence IDs: 4, 6, 8, and 10); International Publication No. 03 / 035695 (see, e.g., some sequences listed on pages 61-64); Vu et al., Molecular Immunology, 1121-1131. 1997 (see, for example, some of the sequences listed in Figure 2); International Publication No. 11 / 003622 (see, for example, sequences provided as sequence numbers 10-27); same publication No. 09 / 058383 (see, for example, the sequence of TAR2h-10-27 mentioned on page 51); same publication No. 10 / 042815 (see, for example, sequences of sequence numbers 15, 17, 27, and 30); and same publication No. 04 / 044204 (see, for example, sequences of sequence numbers 31, 35, 37, 47, and 49).

[0019] Furthermore, some of the references cited herein 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 is substituted with another amino acid, and / or because the serine at position 113 is deleted and a C-terminal amino acid is added (in fact, the final result regarding the C-terminal amino acid sequence would be the same).

[0020] Furthermore, some of the references cited herein also provide examples of nanobodies and other immunoglobulin single variable domains in which the 112th position is an amino acid other than serine. For example, International Publication No. 12 / 175741 describes a nanobody with glycine (G) at the 112th position. Vu et al. (above) describes a nanobody with alanine (A) or isoleucine (I) at the 112th position. International Publication No. 13 / 024059 exemplifies the S112A substitution. International Publication No. 08 / 020079, cited below, exemplifies the S112F substitution and also describes overall that the nanobodies described in that document can contain a limited number of amino acid residues added to the carboxyl terminus of the nanobody's amino acid sequence.

[0021] In investigations conducted up to the present invention, it was established that adding a C-terminal extension (which can simply be a single C-terminal alanine residue; see again International Publication No. 12 / 175741, Example 3) to the C-terminal region or end of a nanobody essentially prevents / removes the binding of existing antibodies / factors in most samples from human subjects / patients. Following this, it was investigated whether samples obtained from human subjects (healthy volunteers and / or subjects with disease or disability) might contain (other) existing antibodies or factors that can bind to the exposed C-terminal region of a nanobody (or other VH domain), even in the presence of a C-terminal extension. By doing so, the inventors have found that such existing antibodies that bind to a C-terminally extended VH domain have not been found in any significant way in the blood or serum of healthy volunteers, or in the blood or serum obtained from human patients suffering from one of many types of diseases (certain inflammatory diseases or autoimmune disorders - data not shown). However, it is believed that some blood or serum samples obtained from certain (but not all) human subjects suffering from certain severe (auto)immune disorders (e.g., systemic lupus erythematosus, also abbreviated herein as "SLE") contain some existing antibodies / factors that can bind to the nanobody, even if the nanobody includes a C-terminal extension.

[0022] Therefore, generally speaking, the object of the present invention is to provide improved heavy-chain immunoglobulin variable domains (and in particular improved heavy-chain ISVDs, and especially improved nanobodies) that, when having an exposed C-terminal region or end, have little tendency to be bound by existing antibodies or factors, such as those found in blood or serum samples obtained from human subjects.

[0023] In particular, an object of the present invention is to provide an improved heavy chain immunoglobulin variable domain that, when having an exposed C-terminal region or end, can also bind to the exposed C-terminal region or end of the heavy chain variable domain, even when the domain includes a C-terminal extension (for example, as described in International Publication Nos. 12 / 175741, 13 / 024059, and further prior art referenced herein), and which has little tendency to be bound by existing antibodies or factors (again, for example, those found in blood or serum samples obtained from human subjects).

[0024] As described herein, existing antibodies capable of binding to a heavy chain variable domain having a C-terminal extension have been found by the inventors to be present in blood or serum samples obtained from human subjects suffering from certain (auto)immune diseases or disorders (e.g., SLE) that severely affect / activate the immune system.

[0025] Therefore, in particular, an object of the present invention is to provide improved heavy chain immunoglobulin variable domains (and in particular improved heavy chain ISVDs, and in particular improved nanobodies) that, when having an exposed C-terminal region or end, have little tendency to be bound by existing antibodies or factors, such as those found in blood or serum samples obtained from human subjects suffering from certain (auto)immune diseases or disorders (e.g., SLE) that severely affect / activate the immune system.

[0026] Furthermore, an object of the present invention is to provide improved heavy-chain immunoglobulin variable domains (and in particular improved heavy-chain ISVDs, and especially improved nanobodies) that, when having an exposed C-terminal region or end, are found in blood or serum samples obtained from human subjects suffering from certain (auto)immune diseases or disorders, and that have little tendency to be bound by existing antibodies or factors that can also bind to the exposed C-terminal region or end of the VH domain, even when the VH domain includes a C-terminal extension.

[0027] Currently, it has been found that the binding of existing antibodies / factors to heavy chain variable domains with exposed C-terminal regions can be (further) reduced by mutations in serine at position 112 (Kabat numbering) to either lysine (K) or glutamine (Q). In particular, it has been found that such S112K or S112Q mutations (S112K or S112Q mutations) to heavy chain variable domains that include C-terminal elongation (but do not contain the S112K or S112Q mutation) are (further) reduced, or their binding is essentially blocked / removed, in existing antibodies / factors found in the blood or serum of human subjects with severe autoimmune disorders, such as SLE.

[0028] Introducing certain mutations disclosed herein (and in particular the L11V mutation described below in combination with V89L, and optionally further in combination with T110K) can improve, or contribute to, the (further) improvement of the solubility of immunoglobulin single variable domains, such as ISVD, as generally and specifically disclosed herein (data not shown).

[0029] Therefore, in a first aspect, the present invention relates to an immunoglobulin heavy chain variable domain (VH domain) in which the amino acid residue at position 112 (Kabat numbering) is either a lysine (K) residue or a glutamine (Q) residue. Such an immunoglobulin heavy chain variable domain is also referred to herein as the "VH domain of the present invention." If the VH domain of the present invention is an immunoglobulin single variable domain (likewise preferred), the immunoglobulin single variable domain will also be referred to herein as the "ISVD domain of the present invention." Similarly, if the VH domain of the present invention is a nanobody (likewise even more preferred), the nanobody will also be referred to herein as the "nanobody of the present invention."

[0030] Generally, the VH domain of the present invention is intended to have either an exposed C-terminus or region, and / or to be present in and / or to have an exposed C-terminus or region in a protein, polypeptide, compound, component, or construct, and / or to have an exposed C-terminus or region in use (e.g., use in a protein, polypeptide, compound, component, or construct intended to form a C-terminus or region).

[0031] In one aspect of the present invention, the VH domain of the present invention is a (heavy chain) immunoglobulin single variable domain, meaning a heavy chain variable domain capable of forming a functional antigen-binding site without interaction with the VL domain. For example, the VH domain of the present invention may be a nanobody (including VHH, humanized VHH, and / or camelized VH, e.g., camelized human VH), a VH domain, or a (single) domain antibody obtained from a VH domain, or a VH domain, or a dAb obtained 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 camelized VH domain, e.g., camelized human VH domain).

[0032] According to another aspect of the present invention, the VH domain of the present invention may be a heavy chain variable domain in which it exists, in a protein, polypeptide, protein, or construct, that requires interaction with a VL domain to form an antigen-binding site and has or forms an exposed C-terminus or region. For example, the VH domain of this aspect of the present invention may be a VH domain present in and / or used in an ScFv and / or diabody.

[0033] According to a more specific embodiment of the present invention, the VH domain of the present invention has a C-terminal sequence (positions 109-113 according to Kabat) which is VTVKS (SEQ ID NO: 1) or VTVQS (SEQ ID NO: 2), or a sequence that has one amino acid difference from either sequence VTVKS and / or VTVQS (i.e., one of positions 109, 110, 111, or 113), and still has either lysine (K) or glutamine (G) at position 112.

[0034] Furthermore, as further described herein, in a particularly preferred embodiment of the present invention, the VH domain of the present invention contains a C-terminal extension (e.g., as described in International Publication Nos. 12 / 175741 and / or 13 / 024059), in particular, a C-terminal extension as further defined herein, at its C-terminus (i.e., linked to a serine residue at the end of a VTVKS-, VTVQS-, or similar motif). However, as further described herein, the VTVKS-, VTVQS-, or similar motif may form the C-terminus of the VH domain (although this would generally be less desirable), or the VH domain of the present invention may be linked at its C-terminus (and optionally via a suitable linker) to another amino acid sequence, sub-domain, or binding unit. For example, if the VH domain of the present invention is an ISVD, the VH domain can be linked at its C-terminus to another ISVD, optionally via a linker (and, for example, the other ISVD can also be the VH domain of the present invention).

[0035] Overall, as is generally known about immunoglobulin variable domains, the VH domain of the present invention will comprise four framework regions (FW1, FW2, FW3, and FW4) and three CDRs (CDR1, CDR2, and CDR3). Generally, as with immunoglobulin variable domains, the sequences of the CDRs will be determined by the antigen / target to which the VH domain of the present invention is intended to arise and / or bind. The framework regions can generally be any suitable framework region of the VH domain (although positions 112 and / or 89 will be as further described herein). If the VH domain of the present invention is an ISVD, the VH domain will have a framework sequence suitable for an ISVD (in some cases in association with one or more CDRs). For example, if the VH domain of the present invention is a nanobody, the framework region will generally contain an appropriate number of VHH hallmark residues (see, for example, International Publication No. 08 / 020079 and some other patent applications of the applicant / assignee cited herein).

[0036] Therefore, for example, if the VH domain of the present invention is a nanobody, the nanobody of the present invention may contain one or more "Hallmark residues" characteristic of the VHH / nanobody (e.g., 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 International Publication No. 08 / 020079); one or more other amino acid residues that may be present in the VHH / nanobody (e.g., one or more humanization substitutions that are themselves known for VHH and nanobody, see, for example, the teachings in International Publication No. 08 / 020079 and again see, Tables A-3 and A-5 to A-8 mentioned above); and / or one or more other suitable amino acid residues or substitutions for the VHH / nanobody; or any suitable combination of such amino acid residues / substitutions.

[0037] The nanobody of the present invention, having K or Q at position 112 (i.e., with or without C-terminal elongation), preferably contains an amino acid at position 11, 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 (V being the most preferred). For example, the nanobody of the present invention may, but is not limited to, contain the L11K or L11V mutation compared to the most frequently occurring leucine residue in VHH. Furthermore, the nanobody of the present invention may, but is not limited to, contain the Q108L mutation (a well-known humanization substitution for VHH / nanobody). Other amino acid residues that may be present (again, not limited to, for example, other native amino acid residues at this position in human VH or VHH) include, for example, alanine (A) at position 14 (a very frequently occurring amino acid residue at this position in native VHH), proline at position 14 (the most common amino acid at this position in the human VH domain), and the mutations suggested by Harmsen et al. (in particular, those suggested by Harmsen et al. based on VHH4-class VHH, e.g., V89M or V89T), and / or one or more of the (other) mutations at the position suggested by Nieba (e.g., at one or more of positions 11, 87, and / or 89, Nieba, p. 437, right column). Another suitable mutation is, for example, T110K or T110Q. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L) (these amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas), and, in particular, may be either proline (P) or alanine (A).(ii) The 42nd position may also be one of the amino acid residues for the 42nd position (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and in particular may be (or selected from) glycine (G) or glutamic acid (E). (iii) The 87th position may also be one of the amino acid residues for the 87th position (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and in particular may be (or selected from) threonine (T) or alanine (A).

[0038] According to a more specific embodiment of the present invention, the VH domain of the present invention (as further described herein) has a framework 4 (FW4 sequence) which is any of the following: a) One of the FW4 sequences with sequence numbers 3-20 mentioned in Table 1 below. [Table 1] Or, b) Sequence number: A sequence having an amino acid difference of less than 3, preferably less than 2, from at least one of the FW4 sequences of sequence number 3 to 20. Herein, (i) the amino acid residue at the position corresponding to position 112 of the Kabat numbering is either K or Q, (ii) the amino acid residue at the position corresponding to position 103 of the Kabat numbering is preferably W or R, (iii) the amino acid residue at the position corresponding to position 104 of the Kabat numbering is preferably G, (iv) the amino acid residue at the position corresponding to position 106 of the Kabat numbering is preferably G, and (v) the amino acid residue at the position corresponding to position 107 of the Kabat numbering is preferably T. (vi) The amino acid residue at the position corresponding to position 108 of the Kabat numbering is preferably Q or L (and preferably L in humanized nanobodies), (vii) The amino acid residue at the position corresponding to position 109 of the Kabat numbering is preferably V, (viii) The amino acid residue at the position corresponding to position 110 of the Kabat numbering is preferably T (or alternatively, K or Q), and (ix) The amino acid residue at the position corresponding to position 111 of the Kabat numbering is preferably V. Table 2 below provides some non-limiting examples of amino acid residues that can be present at various positions (numbered according to Kabat) of such FW4 sequences.

[0039] [Table 2]

[0040] Preferably, the VH domain of the present invention has a framework 4 (FW4 sequence) which is one of the following: a) WGQGTQVTVKS (Sequence ID: 3) or WGQGTQVTVQS (Sequence ID: 12) Or, b) Sequences having less than 3, preferably less than 2 amino acid differences (e.g., only 1 amino acid difference) with respect to sequence number 3 and / or sequence number 12, wherein (i) the amino acid residue at position 112 of the Kabat numbering is either K or Q, where (ii) the amino acid residue at position 103 of the Kabat numbering is preferably W or R, (iii) the amino acid residue at position 104 of the Kabat numbering is preferably G, (iv) the amino acid residue at position 106 of the Kabat numbering is preferably G, and (v) the amino acid residue at position 107 of the Kabat numbering is preferably T. (vi) The amino acid residue at the position corresponding to position 108 of the Kabat numbering is preferably Q or L (and preferably L in humanized nanobodies), (vii) The amino acid residue at the position corresponding to position 109 of the Kabat numbering is preferably V, (viii) The amino acid residue at the position corresponding to position 110 of the Kabat numbering is preferably T (or alternatively, K or Q), and (ix) The amino acid residue at the position corresponding to position 111 of the Kabat numbering is preferably V. Again, Table 2 provides some non-limiting examples of amino acid residues that can be present at various positions (numbered according to Kabat) of such FW4 sequences.

[0041] As further described herein, in preferred embodiments of the present invention, the VH domain of the present invention comprising the FW4 sequence described above preferably also includes a C-terminal extension (as further described herein). However, as further described herein, the FW4 sequence may also form the C-terminus of the VH domain (although this would generally be undesirable), or the VH domain of the present invention may be linked at its C-terminus (optionally via a suitable linker) to another amino acid sequence, portion, domain, or binding unit. For example, if the VH domain of the present invention is an ISVD, the VH domain may be linked at its C-terminus to another ISVD, optionally via a linker (and, for example, the other ISVD may also be a VH domain of the present invention).

[0042] As shown herein, according to preferred and non-limiting embodiments of the present invention, the VH domain of the present invention contains a C-terminal extension, for example, described in International Publication No. 12 / 175741 and / or No. 13 / 024059, and in particular the C-terminal extension described in International Publication No. 12 / 175741.

[0043] Therefore, according to this embodiment, 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 numbering) is not a serine residue, but preferably either a lysine (K) residue or a glutamine (Q) residue, and (ii) at its C-terminus (i.e., at position 113 according to Kabat numbering, or linked to an amino acid residue at a position corresponding to position 113), each is independently selected from appropriate amino acid residues, preferably independently selected from native amino acids, more preferably Each is independently linked to a further amino acid sequence (i.e., a "C-terminal extension") containing 1 to 5 amino acid residues (e.g., 1, 2, 3, 4, or 5, and preferably 1, 2, or 3, and most preferably only 1 or 2, e.g., only 1) selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I) (however, as can be seen from the data presented in International Publication No. 12 / 175741, other amino acid residues, e.g., serine, proline, threonine, and / or lysine, may also be used as part of the C-terminal extension).

[0044] In particular, according to this aspect of the present invention, the VH domain of the present invention is preferably VTVKS(X) n (Sequence ID: 21) or VTVQS(X) nIt has a C-terminal sequence that is (sequence number: 22) (or has one amino acid difference from either of the sequences VTVKS and / or VTVQS at the position of the VTVKS motif or VTVQS motif, and further has either lysine (K) or glutamine (Q) at position 112). Here, (i) the amino acid residues of the VTVKS or VTVQS motif (or VTVKS or VTVQS-like motif) correspond to positions 109 - 113 of the VH domain according to Kabat numbering, (ii) n is 1 - 10, preferably 1 - 5, for example, 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example, 1), and (iii) each X is an (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these.

[0045] In particular, according to this aspect of the present invention, the VH domain of the present invention can have, as its FW4 sequence, one of the FW4 sequences of sequence numbers: 3 - 20 (or an amino acid sequence having less than 3, preferably less than 2 amino acid differences from at least one of the FW4 sequences of sequence numbers: 3 - 20, where the amino acid residue at the position corresponding to position 112 of Kabat numbering is either K or Q). Here, the FW4 sequence has, at its C-terminus, a C-terminal extension (X) n [During the same extension, n is 1 - 10, preferably 1 - 5, for example, 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example, 1), and each X is an (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] linked thereto.

[0046] Therefore, in this aspect of the present invention, the VH domain of the present invention can have, at its C-terminus, any of the following. a) One of the amino acid sequences provided as sequence numbers: 23 - 40 in Table 3 [Table 3] [Here, (i) Sequence ID: (X) in 23-40 n The amino acid residues of the FW4 sequence preceding C-terminal elongation correspond to the amino acid positions of FW4 in the VH domain (i.e., positions 103-113 according to Kabat numbering), (ii)n is 1-10, preferably 1-5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and (iii) each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these. Or, b) SEQ ID NO: An amino acid sequence having a difference of less than 3, preferably less than 2, amino acids from at least one of the amino acid sequences of 23-40 [where the amino acid difference is at the position corresponding to the amino acid position of FW4 in the VH domain, i.e., at positions 103-113 according to Kabat numbering, provided that C-terminal elongation (X) nAny amino acid differences in the middle are ignored. Herein, (i) the amino acid residue at the position corresponding to position 112 of the Kabat numbering is either K or Q, (ii) n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and (iii) each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these. Again, regarding the features (i) to (iii) mentioned in the previous text, in such an amino acid sequence, (iv) the amino acid residue at the position corresponding to position 103 of Kabat numbering is preferably W or R, (v) the amino acid residue at the position corresponding to position 104 of Kabat numbering is preferably G, (vi) the amino acid residue at the position corresponding to position 106 of Kabat numbering is preferably G, (vii) the amino acid residue at the position corresponding to position 107 of Kabat numbering is preferably T, (v iii) The amino acid residue at position 108 of the Kabat numbering is preferably Q or L (and preferably L in humanized nanobodies), (ix) the amino acid residue at position 109 of the Kabat numbering is preferably V, (x) the amino acid residue at position 110 of the Kabat numbering is preferably T (or alternatively, K or Q), and (xi) the amino acid residue at position 111 of the Kabat numbering is preferably V. Again, Table 2 is referenced for the potential amino acid residues that may be present at each position.

[0047] Preferably, according to this aspect of the present invention, the VH domain of the present invention has one of the following at its C-terminus: a)WGQGTQVTVKS(X) n (Sequence ID: 23) or WGQGTQVTVQS(X) n(Sequence ID: 32) [In the same sequence, (ii)n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and (iii) each X is an amino acid residue (preferably a native one), independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these]. or b)WGQGTQVTVKS(X) n (Sequence ID: 23) or WGQGTQVTVQS(X) n An amino acid sequence having a difference of less than 3, preferably less than 2, amino acids from at least one of the amino acid sequences of (SEQ ID NO: 32) (where the amino acid difference is at the position corresponding to the amino acid position of FW4 in the VH domain, i.e., at positions 103-113 according to Kabat numbering, provided that C-terminal elongation (X) n (Any amino acid differences in the middle are ignored.) Here, (i) the amino acid residue at the position corresponding to position 112 of the Kabat numbering is either K or Q, (ii) n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and (iii) each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these. Again, features (vi) to (xi) described in the previous paragraph are also preferably applied to such an amino acid sequence.

[0048] Furthermore, as mentioned herein, the VH domain of the present invention may itself contain, in the relevant positions, other amino acid residues or substitutions known in the art for the VH domain and, in particular, for ISVD (and especially for nanobodies). Some non-limiting examples are as mentioned herein and include, for example, one or more "Hallmark residues" characteristic of VHH / nanobodies (e.g., including leucine (L) at position 11), other amino acid residues that occur spontaneously in VHH (e.g., alanine (A) at position 14), humanization substitutions known for VHH / nanobodies (e.g., Q108L and A14P), one or more mutations suggested by Harmsen (e.g., V89M or V89T), and / or positions suggested by Nieba (e.g., 11, 87, or 89), or any suitable combination thereof, and / or, for example, T110K, T110Q, or V89L mutations.

[0049] The VH domain of the present invention undergoes C-terminal elongation (X). n If it contains, according to a preferred and non-limiting example of such extension, X and n may be as follows: (a) n=1 and X=Ala; (b) n=2 and each X=Ala; (c) n=3 and each X=Ala; (d) n=2 and at least one X=Ala (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (e) n=3 and at least one X=Ala (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (f) n=3 and at least two X=Ala (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (g)n=1 and X=Gly; (h)n=2 and each X=Gly; (i) n=3 and each X=Gly; (j)n=2 and at least one X=Gly (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (k)n=3 and at least one X=Gly (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (l)n=3 and at least two X=Gly (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); (m)n=2 and each X=Ala or Gly; (n)n=3 and each X=Ala or Gly; (o)n=3 and at least one X=Ala or Gly (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile); or (p)n=3 and at least two X=Ala or Gly (where the remaining amino acid residue X is independently selected from any native amino acid, but preferably independently selected from Val, Leu, and / or Ile).

[0050] Here, embodiments (a), (b), (c), (g), (h), (i), (m), and (n) are particularly preferred, embodiments where n=1 or 2 are preferred, and embodiments where n=1 are particularly preferred.

[0051] Furthermore, it should be noted that, preferably, any C-terminal extension present in the VH domain of the present invention does not contain a (free) cysteine ​​residue (unless such cysteine ​​residue is used for further functionalization, e.g., pegylation, or is intended for such functionalization).

[0052] Furthermore, with respect to the C-terminal extension used pursuant to International Publication No. 12 / 175741 (which is also the preferred C-terminal extension used for the VH domain of the present invention), the selections shown on pages 35-41 of International Publication No. 12 / 175741 also apply to the preferred C-terminal extension used for the VH domain of the present invention. These selections pursuant to International Publication No. 12 / 175741 are also incorporated herein by reference.

[0053] Preferably, the VH domain of the present invention undergoes C-terminal elongation (X) n If it contains, n=1, 2, or 3, and each X is either Ala or Gly. More preferably, each X is Ala, and n=1 or 2, preferably 1.

[0054] If the VH domain of the present invention contains a C-terminal extension, the VH domain of the present invention will typically be present at (often forming) the C-terminus of the protein, polypeptide, compound, construct, or other chemical component in which it exists. Again, such a protein, polypeptide, compound, construct, or other chemical component may contain one or more other VH domains of the present invention (i.e., not at the C-terminus). In such cases, the 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, at its C-terminus, it may be linked (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 component, for example, to the VH domain of the present invention having a C-terminal extension present at the C-terminus).

[0055] When the VH domain of the present invention (and the proteins, polypeptides, compounds, constructs, and other chemical components, including those further described herein) is particularly useful (and intended to be used) for pharmaceutical applications (e.g., prevention, treatment, and / or diagnosis of diseases and disorders in human subjects requiring such use), the VH domain of the present invention preferably has a high degree of sequence homology in the framework region of a human VH domain. In particular, the VH domain of the present invention preferably has an overall degree of sequence identity of at least 80%, preferably at least 85%, for example, 90% or more, with respect to at least one human germline sequence (e.g., DP-47, DP-51, or DP-29) (determined as further described herein, considering only the framework region, without considering the CDR, and without considering substitutions at position 112 and any C-terminal extensions, if present). In particular, the VH domain of the present invention preferably has an overall degree of sequence identity of at least 80%, preferably at least 85%, for example, 90% or more, to at least one of the following human germline sequences: DP-47, DP-51, and / or DP-29 (determined as further described herein, considering only the framework region, without considering the CDR, substitution at position 112, and any C-terminal extensions, if present).

[0056] As further described herein, according to one aspect of the present invention, the VH domain of the present invention may be a heavy chain variable domain that interacts with / associates with (or is intended to interact with / associate with) a VL domain to form an antigen-binding site in the protein, polypeptide, protein, or construct in which it exists, where at least the VH domain has an exposed C-terminus or region. For example, the VH domain in this aspect of the present invention may be a VH domain present in and / or used in ScFv and / or diabodies. In this case, the VH domain of the present invention will associate with a VL domain to form an antigen-binding site.

[0057] However, according to a preferred embodiment of the present invention, the VH domain of the present invention is a (heavy chain) immunoglobulin single variable domain or "ISVD," meaning a heavy chain variable domain capable of forming a functional antigen-binding site without interaction with the VL domain. For example, the VH domain of the present invention may be a nanobody (including VHH, humanized VHH, and / or camelized VH, e.g., camelized human VH), a VH domain, or a (single-domain) antibody obtained from a VH domain, or a VH domain, or a dAb obtained from a VH domain. Preferably, the VH domain of the present invention is a nanobody (and more preferably, a VHH domain, a humanized VHH domain, or a camelized VH domain, e.g., a camelized human VH domain).

[0058] In this specification, The term "nanobody" is generally defined as in International Publication No. 08 / 020079 or No. 09 / 138519, and therefore, in specific embodiments, it generally means VHH, humanized VHH, or camelized VH (e.g., camelized human VH domain), or generally sequence-optimized VHH (e.g., optimization of chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression). It should be noted that the terms Nanobody or Nanobodies are registered trademarks of Ablynx NV, and therefore may also be referred to as Nanobody (registered trademark) or Nanobodies (registered trademark).

[0059] In its broadest sense, as used herein, the term “ISVD” (or “ISV”) also includes “ISVD-based biological products,” and, if the ISVD is a nanobody, it also includes “nanobody-based biological products.” “ISVD-based biological products” are defined herein as proteins, polypeptides, or other biological agents comprising at least one (e.g., one, two, or three) ISVDs, or essentially consisting of such ISVDs. Similarly, “nanobody-based biological products” are defined as proteins, polypeptides, or other biological agents comprising at least one (e.g., one, two, or three) nanobodies, or essentially consisting of such nanobodies. As with the term “ISVD,” where the term “ISVD-based biological products” is used, it should be understood that such ISVD-based biological products are preferably nanobody-based biological products. In the context of the present invention, both “ISVD-based biological agents” and “nanobody-based biological agents” can be, for example, monovalent, divalent (or polyvalent), bispecific (or multiplespecific), and biantigen-binding (or multipleantigen-binding) ISVD constructs or nanobody constructs, respectively. Furthermore, any ISVD-based or nanobody-based biological agent may include, for example, one or more (e.g., 1, 2, or 3) ISVDs or nanobodies, in addition to optionally one or more (e.g., 1 or 2) other further therapeutic parts and / or one or more (e.g., 1 or 2) other parts that affect the pharmacological or pharmacodynamic properties (e.g., half-life) of the ISVD-based or nanobody-based biological agent. Suitable examples of such further therapeutic or other parts will be apparent to those skilled in the art and may include, for example, any therapeutically active protein, polypeptide, or other binding domain or unit, and modifications, such as those described on pages 149-152 of International Publication No. 09 / 138159.ISVD-based biological agents or nanobody-based biological agents are preferably therapeutic agents or are intended for use as therapeutic agents (including prophylactic and diagnostic), and for this purpose preferably contain at least one ISVD against a therapeutic-related target (e.g., RANK-L, vWF, IgE, RSV, CXCR-4, IL-23, or other interleukins). For specific, non-limiting examples of some such ISVD-based biological agents or nanobody-based biological agents, see Examples 8 to 18, and also see, for example, various applications by Ablynx NV (e.g., not limited to International Publication Nos. 2004 / 062551, 2006 / 122825, 2008 / 020079, and 2009 / 068627), and for example (and not limited to), applications such as International Publication Nos. 06 / 038027, 06 / 059108, 07 / 063308, 07 / 063311, 07 / 066016, and 07 / 085814. Furthermore, as further described herein, the ISVDs or nanobodies described herein can be bound to (human) serum proteins, such as (human) serum albumin. Such ISVDs or nanobodies can also find therapeutic uses, particularly in the half-life of the therapeutic moiety and compound, and / or for extending the half-life (for example, in and for the ISV-based biological agents described herein). See, for example, International Publication Nos. 2004 / 041865, 2006 / 122787, and 2012 / 175400. These International Publications generally describe the use of serum albumin-binding nanobodies for extending the half-life. Furthermore, unless otherwise specified, all terms referred to herein have the meanings provided in International Publication No. 09 / 138519 (or the prior art cited in International Publication No. 09 / 138519) or International Publication No. 08 / 020079 (or the prior art cited in International Publication No. 08 / 020079).Furthermore, if a method or technique is not specifically described herein, it may be carried out as described in International Publication No. 09 / 138519 (or the prior art cited in the same publication) or International Publication No. 08 / 020079 (or the prior art cited in the same publication).

[0060] Furthermore, as used herein or in the claims, the following terms: “agonist,” “antagonist,” “reverse agonist,” “nonpolar, uncharged amino acid residue,” “polar, uncharged amino acid residue,” “polar, charged amino acid residue,” “sequence identity,” “exactly the same,” and “amino acid difference” (when referring to a sequence comparison of two amino acid sequences), “essentially isolated (in form),” “domain,” “binding domain,” “antigenic determinant,” “epitope,” “against,” “(antigen),” “against,” “specificity,” and “half-life” have the same meanings as provided on pages 62-75 of International Publication No. 09 / 138519, and / or their applicability can be determined in the manner described on those pages. In addition, the terms “modulating,” “for modulating,” “interaction site,” “specific to,” “cross-blocking,” “cross-blocked,” and “cross-blocking,” as well as “essentially independently of pH,” are defined (and / or may be determined as described on the same pages) of the Applicant’s International Publication No. 10 / 130832. Furthermore, when referring to the constructs, compounds, proteins, or polypeptides of the present invention, terms such as “monovalent,” “divalent” (or “polyvalent”), “bispecificity” (or “multispecificity”), and “biantigen-binding” (or “multiantigen-binding”) may have the meanings provided in International Publication Nos. 09 / 138519, 10 / 130832, or 08 / 020079.

[0061] With respect to ISVDs, nanobodies, ISVD-based biological agents, nanobody-based biological agents, or any other amino acid sequences, compounds, or polypeptides referred to herein, the term “half-life” as used herein may generally be defined as described in paragraph o) on page 57 of International Publication No. 08 / 020079 and as referred herein, for example, meaning the time it takes for the serum concentration of an amino acid sequence, compound, or polypeptide to decrease by 50% in vivo due to degradation and / or clearance or sequestration by the intrinsic mechanism of the sequence or compound. The in vivo half-life of the amino acid sequences, compounds, or polypeptides of the present invention may be determined by any method known in itself, for example, by pharmacokinetic analysis. Appropriate techniques will be apparent to those skilled in the art and may generally be described, for example, as described in paragraph o) on page 57 of International Publication No. 08 / 020079. Furthermore, as mentioned in paragraph o) on page 57 of International Publication No. 08 / 020079, the half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta, and area under the curve (AUC). In this regard, as used herein, the term “half-life” means, in particular, t1 / 2-beta or terminal half-life (where t1 / 2-alpha and / or AUC or both may not be considered). For example, the following experimental sections and standard reference books, e.g., Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996), are referred to. Also, “Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982) are referred to.Similarly, the terms “extend half-life” or “extended half-life” are defined in paragraph o) on page 57 of International Publication No. 08 / 020079, and in particular mean the extension of t1 / 2-beta, regardless of the extension of t1 / 2-alpha and / or AUC or both.

[0062] Unless otherwise specifically defined herein, terms shall have their ordinary meanings in the art as would be apparent to the comparator. For example, standard reference books such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd.Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, "Genes II", John Wiley & Sons, New York, NY, (1985);Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd edition, University of California Press, Berkeley, CA (1981);Roitt et al., "Immunology" (6th. Ed.), Mosby / Elsevier, Edinburgh (2001);Roitt et al., Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al. See al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background technologies cited herein.

[0063] Furthermore, as already shown herein, the amino acid residues of the nanobodies are numbered according to the general numbering for VH domains provided by Kabat et al ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). Similarly, the same numbering applied to VHH from camelids in the literature Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195, which is also referred to herein. According to this numbering, FR1 of the nanobodies contains amino acid residues at positions 1-30. CDR1 of the nanobodies contains amino acid residues at positions 31-35. FR2 of the nanobodies contains amino acid residues at positions 36-49. CDR2 of the nanobodies contains amino acid residues at positions 50-65. FR3 of the nanobodies contains amino acid residues at positions 66-94. The CDR3 of the nanobody contains amino acid residues at positions 95-102. The FR4 of the nanobody contains amino acid residues at positions 103-113. [In this regard, it should be noted that, as is well known in the art with respect to the VH domain and VHH domain, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number recognized by Kabat numbering). This generally means that the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.However, generally speaking, according to Kabat numbering, regardless of the number of amino acid residues in the CDR, position 1 according to Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to Kabat numbering corresponds to the start of FR4 and vice versa.

[0064] Furthermore, alternative methods for numbering the amino acid residues of the VH domain can be applied to methods similar to those used for VHH domains and nanobodies from camelids, such as the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition." However, in this description, embodiments, and drawings, numbering will be followed according to Kabat, as applied to VHH by Riechmann and Muyldermans, unless otherwise specified.

[0065] Furthermore, it should be noted that the drawings, any sequence listings, and experimental sections / examples are provided solely to illustrate the present invention and should not be understood or construed as limiting the scope of the present invention and / or the appended claims in any way, unless otherwise specified.

[0066] The VH domain of the present invention can target any suitable or desired antigen or target, including any pharmaceutically and / or therapeutically relevant target described herein.

[0067] In specific embodiments of the present invention, the VH domain of the present invention is for serum proteins, and in particular for human serum proteins. In preferred embodiments, if the VH domain of the present invention is for serum proteins, the VH domain of the present invention is for serum albumin, and in particular for human serum albumin. For this reason, the present invention relates to a VH domain of the present invention (including selections defined for the VH domain of the present invention as defined herein) that can specifically bind to serum proteins, in particular for human serum proteins, and preferably specifically to serum albumin, and more preferably to human serum albumin. Again, such a VH domain is preferably an ISVD (as described in the present invention), and more preferably a nanobody.

[0068] For example, the VH domain of the present invention relative to serum albumin may be one of the nanobodies relative to (human) serum albumin described in International Publication No. 2004 / 041865, and in particular No. 2006 / 122787 and No. 2012 / 175400 (all applications of the applicant / assignee), wherein the amino acid at position 112 is substituted with either K or Q, and the VH domain optionally has the C-terminal extension described herein (and, for example, the present invention It may also appropriately contain one or more other specific amino acid residues / substitutions mentioned in the details, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K, and / or T110K, although the presence of a further substitution at position 110 is often not necessary when position 112 is K or Q. In such cases, position 110 is preferably T). Furthermore, it is assumed that the present invention may also be applied to other serum albumin-binding heavy chain ISVDs, for example, those described in International Publication Nos. 03 / 035694, 04 / 003019, 05 / 118642, 06 / 059106, 08 / 096158, 09 / 121804, 10 / 108937, or U.S. Patent Application Publication No. 2013 / 0129727, by appropriately introducing the substitutions described herein (i.e., at least one of S112K, S112Q, and / or V89T, and optionally one or more other amino acid residues / substitutions described herein, e.g., L11V), and optionally (and usually preferably as outlined herein) by adding a C-terminal extension (further described herein).

[0069] Some preferred, non-limiting examples of such serum albumin-binding nanobodies of the present invention are shown in SEQ ID NO: 52 in International Publication No. 2006 / 122787. (This sequence is sequence number 767 in this specification.)Humanized variants of the amino acid sequence (referred to as "Alb-1" in International Publication No. 2006 / 122787) (in these variants, the amino acid at position 112 is substituted with either K or Q (and these variants may have the C-terminal elongation described herein)), for example, humanized variants of Alb-1 provided in SEQ ID NOs. 57-64 in International Publication No. 2006 / 122787 (each having an S112K or S112Q substitution and, in some cases, a C-terminal elongation), or humanized variants of Alb-1 provided in SEQ ID NOs. 3-11 in International Publication No. 2012 / 175400 (again, each having an S112K or S112Q substitution). Of these variants, SEQ ID NOs: 3, 4, and 5 may optionally contain C-terminal elongation, and SEQ ID NOs: 6-11 already contain C-terminal elongation (and again, such variants may contain one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K, and / or T110K, however, usually, if the 112th position is K or Q, the presence of a further substitution at the 110th position will often not be necessary. In that case, the 110th position is preferably T). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A).and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may in particular be glycine (G) or glutamic acid (E) (or can be selected from these). and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may in particular be threonine (T) or alanine (A) (or can be selected from these).

[0070] Therefore, in a further embodiment, the present invention relates to nanobodies of the present invention (as defined herein) that can bind to (and in particular specifically bind to) serum albumin (and in particular human serum albumin), wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -112 is either K or Q. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the same extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, such nanobody i may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K, and / or T110K, provided that normally, if the 112th position is K or Q, the presence of a further substitution at the 110th position is often not necessarily required (in which case the 110th position is preferably T). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0071] In certain embodiments, the present invention relates to nanobodies of the present invention (as defined herein) that can bind (and in particular specifically bind) to serum albumin (and in particular human serum albumin), which is a humanized variant of Sequence ID No. 52 in International Publication No. 2006 / 122787, wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -112 is either K or Q. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the same extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, such nanobody i may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K, and / or T110K, provided that normally, if the 112th position is K or Q, the presence of a further substitution at the 110th position is often not necessarily required (in which case the 110th position is preferably T). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0072] In certain embodiments, the present invention relates to a nanobody of the present invention (as defined herein) that can bind (and in particular specifically bind) to serum albumin (and in particular human serum albumin) having at least 80%, preferably at least 85%, more preferably at least 90%, for example, at least 95%, sequence identity with at least one of Alb-1 (Sequence ID: 52 in International Publication No. 2006 / 122787), Alb-8 (Sequence ID: 46 in this specification), and / or Alb-23 (Sequence ID: 61 in this specification) (considering both framework sequences and CDRs, but not any C-terminal extensions), wherein the amino acid residue at position 112 is either K or Q, and optionally, at its C-terminus, C-terminal extension (X) n[In the same extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, such nanobody i may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 89T, V89T, 89L, V89L, 108L, Q108L, 110Q, T110Q, 110K, and / or T110K, provided that normally, if the 112th position is K or Q, the presence of a further substitution at the 110th position is often not necessarily required (in which case the 110th position is preferably T). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0073] Such nanobodies of the present invention are again preferably humanized variants of Alb-1 (having S112K or S112Q substitution), more preferably having at least one, particularly any two, and in particular all three of CDR1, CDR2, and / or CDR3 provided in SEQ ID NOs: 41-43, respectively.

[0074] In one specific embodiment, any serum albumin-binding nanobody of the present invention may also have an amino acid residue having the characteristics of Alb-23, and its variants described in International Publication No. 12 / 175400 (i.e., the amino acid motif GP at positions 44 and 45, the amino acid motif SKN at positions 74-76, and preferably G at position 16, and optionally R at position 83).

[0075] Some preferred, non-limiting examples of the nanobodies of the present invention relative to human serum albumin are provided in Table 4 and Example 20. [Table 4] TIFF0007881627000005.tif254168 TIFF0007881627000006.tif255168 TIFF0007881627000007.tif254168

[0076] In another embodiment, the present invention relates to a VH domain, and in particular to an ISVD, and especially to a nanobody, wherein the VH domain has the following: a) One of the following FW4 sequences [Table 5]

[0077] Here, (i) Sequence numbers: (X) in sequences 23-40 n The amino acid residues of the FW4 sequence preceding C-terminal elongation correspond to the amino acid positions of FW4 in the VH domain (i.e., positions 103-113 according to Kabat numbering), (ii)n is 1-10, preferably 1-5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and (iii) each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these, or

[0078] c) SEQ ID NO: An amino acid sequence having a difference of less than 3, preferably less than 2, amino acids from at least one of the amino acid sequences of 99 to 106 (where 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 Kabat numbering, provided that C-terminal elongation (X) n(Any amino acid difference in the middle is ignored.) Here, (i) if the amino acid residue at the position corresponding to position 112 of the Kabat numbering is either K or Q, the amino acid difference is at an amino acid position other than 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), (i) if the amino acid residue at the position corresponding to position 110 of the Kabat numbering is either K or Q, the amino acid difference is at an amino acid position other than 110, and position 89 of the VH domain is L, (iii) n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and (iv) each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these. Again, in relation to the features (i) to (iv) mentioned in the previous text, in such an amino acid sequence, (v) the amino acid residue at the position corresponding to Kabat numbering position 103 is preferably S, (vi) the amino acid residue at the position corresponding to Kabat numbering position 104 is preferably S, (vii) the amino acid residue at the position corresponding to Kabat numbering position 106 is preferably G, (viii) the amino acid residue at the position corresponding to Kabat numbering position 107 is preferably T, (ix) the amino acid residue at the position corresponding to Kabat numbering position 108 is preferably Q or L (and preferably L in humanized nanobodies), and (x) the amino acid residue at the position corresponding to Kabat numbering position 109 is preferably V. Again, refer to Table 2 for the potential amino acid residues that can be present at each position.

[0079] Again, in such a 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 either A or P, and the amino acid residue at position 41 is preferably either A or P. Furthermore, such a VH domain of the present invention is preferably an ISVD, more preferably a nanobody, and can again target human serum albumin (in this case, such a VH domain of the present invention preferably has CDR1, CDR2, and CDR3, respectively, corresponding to SEQ ID NOs: 41, 42, and 43). Furthermore, if the VH domain is an ISVD or nanobody for human serum albumin, the VH domain can further be as described herein for the nanobody of the present invention for human serum albumin.

[0080] The VH domain of the present invention (and in particular the serum albumin-binding ISVD of the present invention) with respect to serum albumin can be used to extend the half-life of therapeutically active compounds, (poly)peptides, proteins, binding domains, binding units, or other therapeutically active components or portions by appropriately linking the serum albumin-binding ISVD to a protein, polypeptide, compound, or other component, optionally via an appropriate linker, for example, pages 12 and 13 of International Publication No. 2012 / 175400, in the manner essentially described in the use of serum albumin-binding nanobodies disclosed in the following references (i.e., by appropriately linking the serum albumin-binding ISVD to a protein, polypeptide, compound, or other component, optionally via an appropriate linker, for example, pages 12 and 13 of International Publication No. 2012 / 175400).

[0081] In another embodiment, the present invention relates to an immunoglobulin heavy chain variable domain (VH domain) in which the amino acid residue at position 89 (Kabat numbering) is threonine (T) and the amino acid residue at position 112 is any of serine (S), lysine (K), or glutamine (Q). Furthermore, such an immunoglobulin heavy chain variable domain (VH domain) having T at position 89 is included in the term “the VH domain of the present invention” when used herein in its broadest sense, and the VH domain of the present invention containing K or Q at position 112 may be as described herein. Thus, such an immunoglobulin heavy chain variable domain (VH domain) may have C-terminal extensions as further described herein (including the selections shown for such C-terminal extensions), and may be ISVDs as further described herein, and in particular, may be nanobodies.

[0082] Again, if such a VH domain of the present invention has an exposed C-terminal region (for example, the VH domain of the present invention is for forming the C-terminus of the protein, polypeptide, or other construct in which it exists), the VH domain of the present invention preferably contains a C-terminal extension (see the data shown in Table C below).

[0083] Furthermore, the nanobodies of the present invention, where the 89th position is T (i.e., with or without C-terminal extension), preferably contain an amino acid at the 11th position 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 (V being the most preferred). For example, the nanobodies of the present invention may contain L11K or L11V substitutions, and, for example, P14A or A14P substitutions, Q108L substitutions, and / or T110K, T110Q, S112K, and / or S112Q substitutions (however, usually, when the 89th position is T, the presence of one or two further substitutions at the 110 and / or 112th positions will often not be necessary. In that case, the 110th position is preferably T, and the 112th position is preferably S).

[0084] In particular, the immunoglobulin heavy chain variable domain (VH domain) of the present invention in this particular embodiment has a T at position 89 (Kabat) and 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) , one of VQVSS (SEQ ID NO: 96), VQVSS(X)n (SEQ ID NO: 98), VZVZS (SEQ ID NO: 107 (in the same sequence, each amino acid residue Z is independently K or Q)), or VZVZSX(n) (SEQ ID NO: 108 (in the same sequence, each amino acid residue Z is independently K or Q)) (and in particular VTVKS (SEQ ID NO: 1), VTVQS (SEQ ID NO: 2), VTVSS (SEQ ID NO: 77), VTVKS(X) n (Sequence ID: 21), VTVQS(X) n (Sequence ID: 22), or VTVSS(X) nIt has one of the (SEQ ID NO: 78) and a C-terminus that is either 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 present invention having Q or K at position 112 (and here, any C-terminal extension is preferably as further described herein for the VH domain of the present invention having Q or K at position 112). Also, as in the case of the VH domain of the present invention having Q or K at position 112, if such a VH domain having T at position 89 is a nanobody, then position 11 is preferably leucine (L), position 14 can be particularly alanine (A) or proline (P), and position 108 can be particularly Q or L (and in humanized nanobodies, preferably L). Such a nanobody having T at position 89 can contain one or more nanobody hallmark residues and / or can be appropriately humanized. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0085] Such VH domains of the present invention having a T at position 89 can again be serum albumin nanobodies as further described herein. For example, such serum albumin-binding nanobodies can be one of the sequences of SEQ ID NOs: 46-75 (but having a T at position 89), or another serum albumin-binding nanobodies having a T at position 89 and having at least 80%, preferably at least 85%, more preferably at least 90%, for example at least 95%, sequence identity with at least 95%, to one of Alb-1 (SEQ ID NO: 52 in International Publication No. 2006 / 122787), Alb-8 (SEQ ID NO: 46 as specified herein), and / or Alb-23 (SEQ ID NO: 61 as specified herein) (considering both the framework sequence and CDR, but not any C-terminal extension). Some other examples of such nanobodies having T at position 89 are provided in SEQ ID NOs: 78-91 (these are further variants of Alb-1 / Alb-8 or Alb-23 having T at position 89 and S at position 112).

[0086] More generally, the VH domain for serum albumin in this embodiment of the present invention may be one of the nanobodies for (human) serum albumin described in International Publication No. 2004 / 041865, and in particular No. 2006 / 122787 and No. 2012 / 175400 (all applications of the applicant / assignee), wherein the amino acid at position 89 is threonine (T), and which optionally comprises the C-terminal extension described herein (and, for example, the one mentioned herein). It may also appropriately contain one or more other specific amino acid residues / substitutions, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, S112K, and / or S112Q, although usually, if T is present at position 89, the presence of further substitutions at positions 110 and / or 112 will often not be necessary. In that case, position 110 is preferably T, and position 112 is preferably S). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A). Furthermore, it is assumed that the present invention may also be applied to other serum albumin-binding heavy chain ISVDs, for example, those described in International Publication Nos. 03 / 035694, 04 / 003019, 05 / 118642, 06 / 059106, 08 / 096158, 09 / 121804, 10 / 108937, or U.S. Patent Application Publication No. 2013 / 0129727, by appropriately introducing threonine (T) at position 89 and optionally one or more other amino acid residues / substitutions as described herein, and optionally (and usually preferred as outlined herein) (further described herein) by adding a C-terminal extension.

[0087] Some preferred, non-limiting examples of such serum albumin-binding nanobodies of the present invention are humanized variants of the amino acid sequence of SEQ ID NO: 52 in International Publication No. 2006 / 122787 (referred to as "Alb-1" in International Publication No. 2006 / 122787) (in which the amino acid at position 89 is T (and the variant optionally has the C-terminal elongation described herein)), for example, humanized variants of Alb-1 provided in SEQ ID NOs: 57-64 in International Publication No. 2006 / 122787 (each having a V89T substitution and optionally a C-terminal elongation), or humanized variants of Alb-1 provided in SEQ ID NOs: 3-11 in International Publication No. 2012 / 175400 (again, each having a V89T substitution). Of these variants, SEQ ID NOs: 3, 4, and 5 may optionally contain C-terminal elongation, and SEQ ID NOs: 6-11 already contain C-terminal elongation (and again, such variants may contain one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q, and / or S112K, however, if the 89th position is T, the presence of one or two further substitutions at the 110th and / or 112th positions will often not be necessary. In such cases, the 110th position is preferably T, and the 112th position is preferably S). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A).and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may in particular be glycine (G) or glutamic acid (E) (or can be selected from these). and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may in particular be threonine (T) or alanine (A) (or can be selected from these).

[0088] Therefore, in a further embodiment, the present invention relates to nanobodies of the present invention (as defined herein) that can bind to (and in particular specifically bind to) serum albumin (and in particular human serum albumin), wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -89 is T. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, such nanobodies are, for example, as referred to herein It may contain one or more other specific amino acid residues / substitutions, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q, and / or S112K, however, if the 89th position is T, the presence of one or two further substitutions at the 110th and / or 112th positions will often not be necessary. In that case, the 110th position is preferably T and the 112th position is preferably S). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0089] In certain embodiments, the present invention relates to a nanobody (as defined herein) that can bind to (and in particular specifically bind to) serum albumin (and in particular to human serum albumin), which is a humanized variant of Sequence ID No. 52 in International Publication No. 2006 / 122787, wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -89 is T. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, such nanobodies are, for example, as referred to herein It may contain one or more other specific amino acid residues / substitutions, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q, and / or S112K, however, if the 89th position is T, the presence of one or two further substitutions at the 110th and / or 112th positions will often not be necessary. In that case, the 110th position is preferably T and the 112th position is preferably S). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0090] In certain embodiments, the present invention relates to a nanobody of the present invention (as defined herein) that can bind (and in particular specifically bind) to serum albumin (and in particular human serum albumin) having at least 80%, preferably at least 85%, more preferably at least 90%, for example, at least 95%, sequence identity with at least one of Alb-1 (Sequence ID: 52 in International Publication No. 2006 / 122787), Alb-8 (Sequence ID: 46 in this specification), and / or Alb-23 (Sequence ID: 61 in this specification) (considering both framework sequences and CDRs, but not any C-terminal extensions), wherein the amino acid residue at position 89 is T, and optionally, at its C-terminus, C-terminal extension (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, such nanobodies are, for example, as referred to herein It may contain one or more other specific amino acid residues / substitutions, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, Q108L, 110K, T110K, 110Q, T110Q, 112Q, 112K, S112Q, and / or S112K, however, if the 89th position is T, the presence of one or two further substitutions at the 110th and / or 112th positions will often not be necessary. In that case, the 110th position is preferably T and the 112th position is preferably S). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0091] In one specific embodiment, any serum albumin-binding nanobody of the present invention having T at position 89 may also have amino acid residues that have the characteristics of Alb-23 and its variants as described in International Publication No. 12 / 175400 (i.e., amino acid motifs GP at positions 44 and 45, amino acid motifs SKN at positions 74-76, and preferably G at position 16, and optionally R at position 83).

[0092] 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 (and optionally Q or K at position 112, and / or C-terminal extension) exhibits reduced binding by existing antibodies, and in particular by such existing antibodies capable of binding to the VH domain and nanobody in the presence of C-terminal extension (e.g., found in samples obtained from SLE patients).

[0093] In yet another aspect, the present invention relates to an immunoglobulin heavy chain variable domain (VH domain) in which the amino acid residue at position 89 (Kabat numbering) is leucine (L) and the amino acid residue at position 110 is either a lysine (K) residue or a glutamine (Q) residue. Furthermore, such an immunoglobulin heavy chain variable domain (VH domain) having L at position 89 and K or Q at position 110 is included in the term “VH domain of the present invention” when used herein in its broadest sense, and further, other VH domains of the present invention (i.e., those containing K or Q at position 112, or those containing T at position 89) may be as described herein. Thus, such an immunoglobulin heavy chain variable domain (VH domain) may have C-terminal extensions as further described herein (including the selections shown for such C-terminal extensions), and may be ISVDs as further described herein, and in particular, may be nanobodies.

[0094] Again, if such a VH domain of the present invention has an exposed C-terminal region (for example, the VH domain of the present invention is for forming the C-terminus of the protein, polypeptide, or other construct in which it exists), the VH domain of the present invention preferably contains a C-terminal extension (see the data shown in Table C below).

[0095] Furthermore, the nanobodies of the present invention, where position 89 is L and position 110 is K or Q (i.e., with or without C-terminal extension), preferably contain an amino acid at position 11, 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 (V is most preferred). For example, the nanobodies of the present invention may contain L11K or L11V substitutions, and, for example, P14A or A14P substitutions and / or Q108L substitutions (the nanobodies of the present invention may also appropriately contain S112K and / or S112Q substitutions; however, usually, when position 89 is L and position 110 is K or Q, the presence of further substitutions at position 112 is often not necessarily required. In that case, position 112 is preferably S). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E). and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0096] In one embodiment, in such a VH domain of the present invention, where 89 is L and 110 is K or Q, the amino acid residue at position 112 is serine (S). In particular, the C-terminus of such a VH domain is VKVSS (SEQ ID NO: 95), VQVSS (SEQ ID NO: 96), VKVSS(X) n (Sequence ID: 97), or VQVSS(X) nIt can be one of (and preferably one of these) (SEQ ID NO: 98), where n and X are as further described herein for the VH domain of the present invention, having Q or K at position 112 (and here any C-terminal extension is as further described herein for the VH domain of the present invention, having Q or K at position 112). Also, as in the case of the VH domain of the present invention, having Q or K at position 112 or T at position 89, if such a VH domain having L at position 89 and K or Q at position 110 is a nanobody, then position 11 is preferably leucine (L), position 14 can be alanine (A) or proline (P) in particular, and position 108 can be Q or L in particular (and preferably L in humanized nanobodies). Such a nanobody having L at position 89 and K or Q at position 110 can contain one or more nanobody hallmark residues and / or can be appropriately humanized. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0097] Again, such VH domains of the present invention having L at position 89 and K or Q at position 110 can also be serum albumin nanobodies as further described herein. For example, such serum albumin-binding nanobodies can be other serum albumin-binding nanobodies having L at position 89 and K or Q at position 110, having at least 80%, preferably at least 85%, more preferably at least 90%, for example at least 95%, sequence identity with one of the sequences of SEQ ID NOs: 46-75 (provided to have L at position 89 and K or Q at position 110), or at least one of Alb-1 (SEQ ID NO: 52 in International Publication No. 2006 / 122787), Alb-8 (SEQ ID NO: 46 as specified herein), and / or Alb-23 (SEQ ID NO: 61 as specified herein) (considering both framework sequences and CDRs, but not any C-terminal extensions).

[0098] More generally, the VH domain for serum albumin in this embodiment of the present invention may be one of the nanobodies for (human) serum albumin described in International Publication No. 2004 / 041865, and in particular Nos. 2006 / 122787 and 2012 / 175400 (all 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, wherein C as described herein may be used. It may also contain terminal elongation (and may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, and / or Q108L, as well as S112K or S112Q, although the presence of a further substitution at position 112 is often not necessarily required when position 89 is L and position 110 is K or Q, in which case position 112 is preferably S). Furthermore, it is assumed that the present invention may also be applied to other serum albumin-binding heavy chain ISVDs, for example, those described in International Publication Nos. 03 / 035694, 04 / 003019, 05 / 118642, 06 / 059106, 08 / 096158, 09 / 121804, 10 / 108937, or U.S. Patent Application Publication No. 2013 / 0129727, by appropriately introducing leucine (L) at position 89 and either K or Q at position 110, and optionally one or more other amino acid residues / substitutions as described herein, and optionally (and usually, preferably as outlined herein) (further described herein) by adding a C-terminal extension. In these VH domains for serum albumin, the amino acid at position 112 is preferably S. The VH domain preferably has a C-terminus that is one of sequence numbers 95 to 98.Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E). and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0099] Some preferred, non-limiting examples of such serum albumin-binding nanobodies of the present invention include a humanized variant of the amino acid sequence of SEQ ID NO: 52 in International Publication No. 2006 / 122787 (referred to as "Alb-1" in International Publication No. 2006 / 122787) (in which the amino acid at position 89 is L, and the amino acid at position 110 is K or Q (and the variant optionally comprises the C-terminal extension described herein) For example, humanized variants of Alb-1 provided in SEQ ID NO: 57-64 in International Publication No. 2006 / 122787 (each having a V89L substitution and a T110Q or T110K substitution, and possibly a C-terminal elongation), or humanized variants of Alb-1 provided in SEQ ID NO: 3-11 in International Publication No. 2012 / 175400 (again, each having a V89L substitution and a T110Q or T110K substitution). Of these variants, SEQ ID NOs: 3, 4, and 5 may optionally contain C-terminal elongation, and SEQ ID NOs: 6-11 already contain C-terminal elongation (and again, such variants may also contain one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, and / or Q108L, as well as S112K or S112Q, although the presence of a further substitution at position 112 is often not necessarily required when position 89 is L and position 110 is K or Q. In such cases, position 112 is preferably S). In these VH domains for serum albumin, the amino acid at position 112 is preferably S. The VH domain preferably has a C-terminus which is one of SEQ ID NOs: 95-98. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L).These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (iii) position 87 can be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-7 of International Publication No. 08 / 020079, and can be (or selected from) threonine (T) or alanine (A).

[0100] Therefore, in a further embodiment, the present invention relates to nanobodies of the present invention (as defined herein) that can bind to (and in particular specifically bind to) serum albumin (and in particular human serum albumin), wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -89 is L. The amino acid residue at position -110 is either K or Q. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the same extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, this Such nanobodies may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, and / or Q108L; as well as S112K or S112Q, although the presence of a further substitution at position 112 is often not necessary, as is usually the case when position 89 is L and position 110 is K or Q. In these VH domains for serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus that is one of sequence numbers 95-98. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0101] In certain embodiments, the present invention relates to nanobodies of the present invention (as defined herein) that can bind (and in particular specifically bind) to serum albumin (and in particular human serum albumin), which is a humanized variant of Sequence ID No. 52 in International Publication No. 2006 / 122787, wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -89 is L. The amino acid residue at position -110 is either K or Q. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the same extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, this Such nanobodies may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, and / or Q108L; as well as S112K or S112Q, although the presence of a further substitution at position 112 is often not necessary, as is usually the case when position 89 is L and position 110 is K or Q. In these VH domains for serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus that is one of sequence numbers 95-98. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0102] In certain embodiments, the present invention relates to a nanobody of the present invention (as defined herein) that can bind (and in particular specifically bind) to serum albumin (and in particular human serum albumin) having at least 80%, preferably at least 85%, more preferably at least 90%, for example, at least 95%, sequence identity with at least one of Alb-1 (Sequence ID: 52 in International Publication No. 2006 / 122787), Alb-8 (Sequence ID: 46 in this specification), and / or Alb-23 (Sequence ID: 61 in this specification) (considering both framework sequences and CDRs, but not any C-terminal extensions), wherein the amino acid residue at position 89 is L, the amino acid residue at position 110 is K or Q, and optionally, at its C-terminus, C-terminal extension (X) n[In the same extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] containing (again, such C-terminal extensions are preferably as further described herein, and again, this Such nanobodies may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, and / or Q108L; as well as S112K or S112Q, although the presence of a further substitution at position 112 is often not necessarily required, as is usually the case when position 89 is L and position 110 is K or Q. In that case, position 112 is preferably S). Again, such nanobodies of the present invention are preferably humanized variants of Alb-1 (but having the V89L and T110K or T110Q substitutions), and more preferably have at least one, particularly any two, and especially all three, CDR1, CDR2, and / or CDR3 provided in SEQ ID NOs: 41-43, respectively. In these VH domains for serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus that is one of sequence numbers 95-98. Also, (i) position 41 can be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be either proline (P) or alanine (A), among others.and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may in particular be glycine (G) or glutamic acid (E) (or can be selected from these). and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may in particular be threonine (T) or alanine (A) (or can be selected from these).

[0103] In one specific embodiment, any such serum albumin-binding nanobody of the present invention having L at position 89 and K or Q at position 110 may also have amino acid residues and their variants having the characteristics of Alb-23 as described in International Publication No. 12 / 175400 (i.e., amino acid motifs GP at positions 44 and 45, amino acid motifs SKN at positions 74-76, and preferably G at position 16, and optionally R at position 83). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as mentioned in Table A-6 of International Publication No. 08 / 020079, in particular proline (P), serine (S), threonine (T), alanine (A), or leucine (L) (or can be selected from these). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (iii) position 87 can be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-7 of International Publication No. 08 / 020079, and can be (or selected from) threonine (T) or alanine (A).

[0104] Similar to the VH domains of the present invention that contain Q or K at position 112 or have T at position 89, the VH domains of the present invention that contain L at position 89 and K or Q at position 110 exhibit reduced binding by existing antibodies, and in particular by such existing antibodies capable of binding to the VH domain and nanobody in the presence of C-terminal extension (e.g., found in samples obtained from SLE patients).

[0105] Some non-limiting examples of the VH domains of the present invention having L at position 89 and K at position 110 (and in addition, V at position 11) are provided in Figure 2 as Sequence IDs: 123-136. These are VH domains that bind to human serum albumin and have the CDRs shown herein for preferred serum albumin-binding VH domains of the present invention.

[0106] In yet another embodiment, the present invention relates to an immunoglobulin heavy chain variable domain (VH domain) in which the amino acid residue at position 89 (Kabat numbering) is leucine (L) and the amino acid residue at position 11 is valine (V). Furthermore, such an immunoglobulin heavy chain variable domain (VH domain) having L at position 89 and V at position 11 is included in the term “VH domain of the present invention” when used herein in its broadest sense, and furthermore, other VH domains of the present invention described herein (i.e., those containing K or Q at position 112, those containing T at position 89, or those containing L at position 89 and K or Q at position 110) may be as described herein. Notwithstanding the VH domains of this embodiment, the amino acid residue at position 89 may be L, the amino acid residue at position 11 may be V, and the amino acid residues at positions 110 and 112 may each be any amino acid residue appropriate to those positions. Therefore, such immunoglobulin heavy chain variable domains (VH domains) may have C-terminal extensions as further described herein (including the selections shown for such C-terminal extensions), ISVDs as further described herein, and in particular, nanobodies.

[0107] Again, if such a VH domain of the present invention has an exposed C-terminal region (for example, the VH domain of the present invention is for forming the C-terminus of the protein, polypeptide, or other construct in which it exists), the VH domain of the present invention preferably contains a C-terminal extension (see the data shown in Table C below).

[0108] Furthermore, the nanobody of the present invention, having L at position 89 and V at position 11 (i.e., with or without C-terminal extension), (i) preferably contains an amino acid at position 110 selected from T, I, A, K, or Q (and preferably selected from T, K, or Q, and in particular can be T), (ii) preferably contains an amino acid at position 112 selected from S, F, K, or Q (and more preferably selected from S, K, or Q, and in particular can be S), and (iii) may also contain, for example, P14A or A14P substitutions and / or Q108L substitutions. According to one specific embodiment, in the VH domain of this aspect of the present invention, the amino acid residue at position 110 is T, the amino acid residue at position 112 is S, and more preferably the C-terminus is either VTVSS (SEQ ID NO: 77) or VTVSS(X)n (SEQ ID NO: 78). Here, X and n are as defined herein for the C-terminal extension of other VH domains of the present invention. Also, in the VH domain of this embodiment of the present invention, (i) position 41 can be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0109] Therefore, for example, in such a VH domain of the present invention where 89 is L and 11 is V, the C-terminus of such a VH domain is VTVSS (sequence number: 77), VTVSS(X)n (sequence number: 78), VTVKS (sequence number: 1), VTVKS(X)n (sequence number: 21), VTVQS (sequence number: 2), VTVQS(X)n (sequence number: 22), VKVSS (sequence number: 95), VKVSS(X)n (sequence number: 97), VQVSS (sequence number: 77) It can be one of the following (and preferably one of these) (and especially VTVSS, VKVSS (SEQ ID NO: 95), VQVSS (SEQ ID NO: 96), VKVSS(X)n (SEQ ID NO: 98), VZVZS (SEQ ID NO: 107, in the same sequence, each amino acid residue Z is independently K or Q), or VZVZSX(n) (SEQ ID NO: 108, in the same sequence, each amino acid residue Z is independently K or Q) (and preferably one of these) (and especially VTVSS, VKVSS (SEQ ID NO: 95), VQVSS (SEQ ID NO: 96), VKVSS(X) n (Sequence ID: 97), or VQVSS(X) n(SEQ ID NO: 98) is one of the following, and in particular can be either VTVSS (SEQ ID NO: 77) or VTVSS(X)n (SEQ ID NO: 78). Here, n and X are as further described herein for the VH domain of the present invention, having Q or K at position 112 (and here, any C-terminal extension is preferably as further described herein for the VH domain of the present invention, having Q or K at position 112). Also, as in the case of the VH domain of the present invention, having Q or K at position 112 or L at position 89, if such a VH domain having L at position 89 and V at position 11 is a nanobody, then position 14 can be alanine (A) or proline (P), in particular, and position 108 can be Q or L (and preferably L in humanized nanobodies). Such a nanobody having L at position 89 and V at position 11 can contain one or more nanobody hallmark residues and / or can be appropriately humanized. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0110] Such VH domains of the present invention having L at position 89 and V at position 11 can again be serum albumin nanobodies as further described herein. For example, such serum albumin-binding nanobodies can be one of the sequences of SEQ ID NOs: 46-75 (with L at position 89 and V at position 11), or another serum albumin-binding nanobodies having L at position 89 and V at position 11, having at least 80%, preferably at least 85%, more preferably at least 90%, for example at least 95%, sequence identity with at least 95%, for example at least 95%, to Alb-1 (SEQ ID NO: 52 in International Publication No. 2006 / 122787), Alb-8 (SEQ ID NO: 46 as specified herein), and / or Alb-23 (SEQ ID NO: 61 as specified herein) (considering both framework sequences and CDRs, but not any C-terminal extensions).

[0111] More generally, the VH domain for serum albumin in this embodiment of the present invention may be one of the nanobodies for (human) serum albumin described in International Publication No. 2004 / 041865, and in particular Nos. 2006 / 122787 and 2012 / 175400 (all applications of the applicant / assignee), wherein the amino acid at position 89 is leucine (L), the amino acid residue at position 11 is V, and optionally comprises the C-terminal extension described herein (and may appropriately contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 14A, P14A, 14P, A14P, 108L, Q108L, V110K, V110Q, S112K, and / or S112Q). Furthermore, it is assumed that the present invention may also be applied to other serum albumin-binding heavy chain ISVDs, for example, those described in International Publication Nos. 03 / 035694, 04 / 003019, 05 / 118642, 06 / 059106, 08 / 096158, 09 / 121804, 10 / 108937, or U.S. Patent Application Publication No. 2013 / 0129727, by appropriately introducing leucine (L) at position 89 and valine at position 11, and optionally one or more other amino acid residues / substitutions as described herein, and optionally (and usually, preferably as outlined herein) (further described herein) by adding a C-terminal extension. In these VH domains for serum albumin, the amino acid at position 112 is preferably S. The VH domain preferably has a C-terminus that is one of sequence numbers 95-98. Also, (i) position 41 can be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) mentioned in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L).These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (iii) position 87 can be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-7 of International Publication No. 08 / 020079, and can be (or selected from) threonine (T) or alanine (A).

[0112] Some preferred, non-limiting examples of such serum albumin-binding nanobodies of the present invention are humanized variants of the amino acid sequence of SEQ ID NO: 52 in International Publication No. 2006 / 122787 (referred to as "Alb-1" in International Publication No. 2006 / 122787) (in which the amino acid at position 89 is L and the amino acid at position 11 is V (and the variant optionally has the C-terminal elongation described herein)), for example, humanized variants of Alb-1 provided in SEQ ID NOs: 57-64 in International Publication No. 2006 / 122787 (each having a V89L substitution and an L11V substitution, and optionally having a C-terminal elongation), or humanized variants of Alb-1 provided in SEQ ID NOs: 3-11 in International Publication No. 2012 / 175400 (again, each having a V89L substitution and an L11V). Of these variants, SEQ ID NOs: 3, 4, and 5 may optionally contain C-terminal elongation, and SEQ ID NOs: 6-11 pre-contain C-terminal elongation (and again, such variants may appropriately contain one or more other specific amino acid residues / substitutions mentioned herein, e.g., 14A, P14A, 14P, A14P, 108L, Q108L, T110K, T110Q, S112K, and / or S112Q). In these VH domains for serum albumin having L at position 89 and V at position 11, the amino acid at position 110 is preferably T, and the amino acid residue at position 112 is preferably S. The VH domain preferably has a C-terminus which is one of SEQ ID NOs: 95-98. Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and may be, in particular, either proline (P) or alanine (A).and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may in particular be glycine (G) or glutamic acid (E) (or can be selected from these). and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may in particular be threonine (T) or alanine (A) (or can be selected from these).

[0113] Therefore, in a further embodiment, the present invention relates to nanobodies of the present invention (as defined herein) that can bind to (and in particular specifically bind to) serum albumin (and in particular human serum albumin), wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -89 is L. The amino acid residue at position -11 is V. Here, preferably, The amino acid residue at position -110 is one of K, Q, or T, and more preferably T. The amino acid residue at position -112 is one of K, Q, or S, and more preferably S. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the extension, n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (again, such C-terminal extensions are preferably as further described herein, and again, such nanobodies can appropriately contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 14A, P14A, 14P, A14P, 108L, Q108L). Again, in these VH domains for serum albumin having L at position 89 and V at position 11, the amino acid at position 110 is preferably T, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus which is one of sequence numbers 95-98. Also, (i) position 41 can be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be either proline (P) or alanine (A) in particular. and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0114] In certain embodiments, the present invention relates to nanobodies of the present invention (as defined herein) that can bind (and in particular specifically bind) to serum albumin (and in particular human serum albumin), which is a humanized variant of Sequence ID No. 52 in International Publication No. 2006 / 122787, wherein, -CDR1 is the amino acid sequence SFGMS (SEQ ID NO: 41); -CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (Sequence ID: 42); -CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43); The amino acid residue at position -89 is L. The amino acid residue at position -11 is V. And preferably, The amino acid residue at position -110 is one of K, Q, or T, and more preferably T. The amino acid residue at position -112 is one of K, Q, or S, and more preferably S. In some cases, C-terminal extension (X) occurs at the C-terminus. n[In the extension, n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (again, such C-terminal extensions are preferably as further described herein, and again, such nanobodies may contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 11L, L11V, L11K, 14A, P14A, 14P, A14P, 108L, and / or Q108L). In these VH domains for serum albumin having L at position 89 and V at position 11, the amino acid residue at position 110 is preferably T, the amino acid residue at position 112 is preferably S, and the VH domain preferably has a C-terminus that is one of sequence numbers 95-98. Also, (i) position 41 can be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be either proline (P) or alanine (A), among others. and / or, (ii) position 42 may be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and may be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0115] In certain embodiments, the present invention relates to a nanobody of the present invention (as defined herein) that can bind (and in particular specifically bind) to serum albumin (and in particular human serum albumin) having at least 80%, preferably at least 85%, more preferably at least 90%, for example, at least 95%, sequence identity with at least one of Alb-1 (Sequence ID: 52 in International Publication No. 2006 / 122787), Alb-8 (Sequence ID: 46 in this specification), and / or Alb-23 (Sequence ID: 61 in this specification) (considering both framework sequences and CDRs, but not any C-terminal extensions), wherein the amino acid residue at position 89 is L, the amino acid residue at position 11 is V, and optionally, at its C-terminus, C-terminal extension (X) n[In the extension, n is 1 to 10, preferably 1 to 5, e.g., 1, 2, 3, 4, or 5 (and preferably 1 or 2, e.g., 1), and each X is a (preferably native) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (again, such C-terminal extensions are preferably as further described herein, and again, such nanobodies may appropriately contain, for example, one or more other specific amino acid residues / substitutions mentioned herein, e.g., 14A, P14A, 14P, A14P, 108L, Q108L, T110K, T110Q, S112K, and / or S112Q). Again, such nanobodies of the present invention are preferably humanized variants of Alb-1 (having V89L and L11V substitutions), and more preferably have at least one, particularly any two, and especially all three, CDR1, CDR2, and / or CDR3 provided in SEQ ID NOs: 41-43, respectively. In these VH domains relative to serum albumin, the amino acid at position 112 is preferably S, and the VH domain preferably has a C-terminus that is one of SEQ ID NOs: 95-98. Also, (i) position 41 can be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E).and / or, (iii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or selected from) threonine (T) or alanine (A).

[0116] In one specific embodiment, any such serum albumin-binding nanobody of the present invention having L at position 89 and V at position 11 may also have amino acid residues and their variants having the characteristics of Alb-23 as described in International Publication No. 12 / 175400 (i.e., amino acid motifs GP at positions 44 and 45, amino acid motifs SKN at positions 74-76, and preferably G at position 16, and optionally R at position 83). Furthermore, (i) position 41 may be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) as mentioned in Table A-6 of International Publication No. 08 / 020079, in particular proline (P), serine (S), threonine (T), alanine (A), or leucine (L) (or can be selected from these). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (iii) position 87 can be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-7 of International Publication No. 08 / 020079, and can be (or selected from) threonine (T) or alanine (A).

[0117] 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 exhibit reduced binding by existing antibodies, and in particular, by such existing antibodies capable of binding to the VH domain and nanobodies in the presence of C-terminal extension (e.g., found in samples obtained from SLE patients). One further point to note regarding the VH domains of the present invention having L at position 89 and V at position 11 is that these substitutions are known to occur with some frequency in human VH domains (see Table A-5 of International Publication No. 08 / 020079 for position 11, and Table A-7 of International Publication No. 08 / 020079 for position 89).

[0118] Some non-limiting examples of the VH domains of the present invention having V at position 11 and T at position 89 are provided in Figure 2 as SEQ ID NOs: 109-136. Of these VH domains, the sequences provided as SEQ ID NOs: 123-136 also contain the T110K mutation in addition to the L11V and V89L mutations. These are VH domains that bind to human serum albumin and have the CDRs shown herein for the preferred serum albumin-binding VH domains of the present invention.

[0119] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is V, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is L, and, The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, the VH domain may be C-terminally elongated (X) n [In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A). Preferably, in the VH domain of this paragraph, the amino acid residue at position 110 is T, and the amino acid residue at position 112 is S. Again, as with other VH domains of the present invention, such a VH domain may target any suitable target (and in particular a therapeutic target). According to one specific embodiment, such a VH domain may target serum albumin.

[0120] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either 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, the amino acid residue at position 110 is either K or Q, and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain may be C-terminally elongated (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0121] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either K or Q, the amino acid at position 89 is one of T, V, or L (and preferably V), 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, the amino acid residue at position 112 is one of S, K, or Q (and preferably S), 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, the amino acid residue at position 110 is either K or Q, the amino acid residue at position 112 is one of S, K, or Q (and preferably S), and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain may be C-terminally elongated (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0122] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either 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 either K or Q, and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain may be C-terminally elongated (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0123] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either K or Q, the amino acid 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, 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, the amino acid residue at position 110 is either 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 (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain may be C-terminally elongated (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0124] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either K or Q, the amino acid residue 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 either K or Q, and the amino acid residue at position 112 is S (and preferably S); and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain may be C-terminally elongated (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0125] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either 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, the amino acid residue at position 110 is either K or Q, and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain undergoes C-terminal elongation (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0126] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either K or Q, the amino acid at position 89 is one of T, V, or L (and preferably V), 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, the amino acid residue at position 112 is one of S, K, or Q (and preferably S), 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, the amino acid residue at position 110 is either K or Q, the amino acid residue at position 112 is one of S, K, or Q (and preferably S), and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain undergoes C-terminal elongation (X) n[During the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and in particular can be glycine (G) or glutamic acid (E) (or can be selected from these). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0127] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either 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 either K or Q, and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain undergoes C-terminal elongation (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0128] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, The following are all possible: (i) the amino acid residue at position 112 is either K or Q, the amino acid 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, 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, the amino acid residue at position 110 is either 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 (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain undergoes C-terminal elongation (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0129] In a further embodiment, the present invention relates to VH domains (and in particular VH domains that are ISVDs, and especially VH domains that are nanobodies), wherein, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Here, one of the following applies: (i) the amino acid residue at position 112 is either K or Q, the amino acid 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 either K or Q, and the amino acid residue at position 112 is S (and preferably S); and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. Here, the VH domain undergoes C-terminal elongation (X) n[In the extension, n is 1 to 10, preferably 1 to 5, for example 1, 2, 3, 4, or 5 (and preferably 1 or 2, for example 1), and each X is a (preferably natural) amino acid residue, independently selected from alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I), preferably independently selected from the group consisting of these] (this C-terminal extension is preferably as further described herein). Also, (i) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (ii) position 87 may be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) as referred to, for example, in Table A-7 of International Publication No. 08 / 020079, and may be (or can be selected from) threonine (T) or alanine (A) in particular. Another aspect of the present invention relates to the VH domain described in this paragraph (and in particular the VH domain which is an ISVD, and especially the VH domain which is a nanobody), where the amino acid residue at position 11 is V, and the amino acid residue at position 110 is either K or Q (and here the amino acid residues at positions 14, 41, 89, 108, and 112 may be as listed in the above bullet, and the amino acid residues at positions 42 and 87 may be, for example, as described in this paragraph, and this VH domain may optionally have the C-terminal extension (X) described in this paragraph n (May contain).

[0130] Again, in the VH domain of the present invention as defined herein, amino acid residues at positions not explicitly defined herein can be any amino acid residue suitable for such positions in the VH domain, and in particular the ISVD, and especially in the nanobody (including the humanized VHH domain). Again, refer to the prior art cited herein, e.g., Tables A-3 and A-5 to A-8 of International Publication No. 08 / 020079. Preferably, in each case, the amino acid residue at position 11 is L or V, and more preferably V. Also, (i) position 41 can be one of the amino acid residues for position 41 (i.e., human VH3 residues and / or camelid VHH residues) mentioned in, for example, Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) proline (P), serine (S), threonine (T), alanine (A), or leucine (L). These amino acid residues are some of the amino acid residues that most frequently occur at this position in either humans or llamas, and can be, in particular, either proline (P) or alanine (A). and / or, (ii) position 42 can be one of the amino acid residues for position 42 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-6 of International Publication No. 08 / 020079, and can be (or selected from) glycine (G) or glutamic acid (E). and / or, (iii) position 87 can be one of the amino acid residues for position 87 (i.e., human VH3 residues and / or camelid VHH residues) mentioned, for example, in Table A-7 of International Publication No. 08 / 020079, and can be (or selected from) threonine (T) or alanine (A).

[0131] Furthermore, the VH domain of the present invention can target any suitable target, and in particular, a therapeutic target. In one embodiment, the VH domain of the present invention is targeted at human serum proteins, for example, human serum albumin.

[0132] Furthermore, the present invention relates to proteins, polypeptides, constructs, compounds, or other chemical components (collectively referred to herein as "compounds of the present invention") comprising at least one VH domain of the present invention.

[0133] As further described herein, in one specific embodiment, in the compound of the present invention, the VH domain of the present invention is located at / forms the C-terminus of the compound of the present invention. In such cases, the VH domain of the present invention that forms / forms the C-terminus of the compound of the present invention preferably has the C-terminal extension described herein.

[0134] Furthermore, as further described herein, the compounds of the present invention may be ScFv, diabodies, or other proteins, polypeptides, or constructs in which one or more VH domains of the present invention associate with one or more VL domains to form one or more functional antigen-binding sites.

[0135] However, according to a preferred embodiment of the present invention, the VH domain of the present invention is an ISVD, and the compound of the present invention is a protein, polypeptide, construct, compound, or other chemical component comprising, or essentially comprising, at least one ISVD of the present invention and optionally one or more further amino acid sequences, portions, binding domains, or binding units (optionally appropriately linked to one or more linkers). In particular, such a compound of the present invention may comprise or essentially consist of one or more ISVDs, at least one of which is an ISVD of the present invention. Such a compound of the present invention may, in particular, have an ISVD of the present invention at its C-terminus. In this case, the ISVD of the present invention preferably has the C-terminal extension described herein. Furthermore, if such a compound of the present invention contains two or more ISVDs, two or more or essentially all of the ISVDs present may be 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 and T110K or T110Q, and optionally one or more further substitutions referred herein for ISVDs of the present invention, e.g., L11V). Furthermore, in such compounds of the present invention, the ISVD of the present invention is preferably a nanobody of the present invention, and all or essentially all ISVDs present in the compounds of the present invention may be nanobodies (and in particular, nanobodies of the present invention, i.e., each having one of the following amino acid residues / substitutions: 112K, 112Q, S112K, S112Q, 89T, and / or V89T, or a combination of V89L and T110K or T110Q, and optionally one or more further substitutions referred herein with respect to the nanobodies of the present invention, e.g., L11V) (preferably the same nanobodies). Examples of such compounds of the present invention will be apparent to those skilled in the art based on further disclosures herein.

[0136] Some non-limiting examples of proteins, polypeptides, constructs, compounds, or other chemical components comprising one or more ISVDs (including at least one ISVD of the present invention) are polyvalent, multispecific (e.g., bispecific), or multiantigen-binding (e.g., biantigen-binding) constructs containing two or more ISVDs linked directly or via one or more suitable linkers. Again, the ISVDs are preferably nanobodies. For some non-limiting examples of general teachings in which such constructs and methods for preparing such constructs (particularly based on nanobodies) are, for example, Conrath et al., JBC 276, 10(9), 7346 (2001) and Muyldermans' review, Review in Mol. Biotechnol., 74: 27 (2001).

[0137] 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) may be divalent, trivalent, tetravalent, or pentavalent constructs, and / or monospecific, bispecific, or triplicate constructs, and / or biantigen-binding or triplicate constructs. Again, the prior art in ISVD-based and nanobody-based biological formulations cited herein is referenced. Furthermore, such compounds of the present invention may have an extended half-life by functionalization and / or by including a moiety or binding unit in the construct that extends the half-life of the construct. Examples of such functionalizations, moieties, or binding units will be apparent to those skilled in the art and may, for example, be as described herein, and may include, for example, pegylation, fusion to serum albumin, or fusion to a serum protein, such as a peptide or binding unit that can bind to serum albumin. Such serum albumin-binding peptides or binding domains can be any suitable serum albumin-binding peptide or binding domain that can extend the half-life of the construct (compared to the same construct without the serum albumin-binding peptide or binding domain), and can be, in particular, serum albumin-binding peptides described in the Applicant's International Publication No. 2008 / 068280 (and in particular, both the Applicant's International Publication No. 2009 / 127691 and the unpublished U.S. Provisional Application No. 61 / 301,819), or serum albumin-binding ISVs (e.g., serum albumin-binding nanobodies, e.g., Alb-8 or the humanized version of Akb-1, e.g., Alb-8, for which see, for example, International Publication No. 06 / 122787), or (human) serum proteins, e.g., the ISVD of the present invention for (human) serum albumin (as further described herein). Generally, any compound of the present invention having an extended half-life will preferably have a half-life of at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example, at least 10 days (as defined herein) in a human subject.

[0138] If the compounds of the present invention contain at least one (and preferably one) ISVD (and especially one nanobody) of the present invention for (human) serum proteins, and in particular for (human) serum albumin, the compounds of the present invention will typically further contain one or more therapeutically active amino acid sequences, moieties, binding domains, or binding units (i.e., for therapeutically relevant targets, pathways, or mechanisms), and the ISVDs of the present invention will function to extend the half-lives of them (and the compound as a whole). Again, the one or more further therapeutically active moieties are preferably ISVDs (and more preferably nanobody), and may also be ISVDs (and more preferably nanobody) of the present invention. In such compounds of the present invention, the ISVD of the present invention for human serum albumin may again be present at / form the C-terminus of the compound, and in that case may include (and preferably include) the C-terminal extension described herein. If the compound of the present invention contains the ISVD of the present invention against (human) serum albumin, the compound of the present invention preferably has a half-life (as defined herein) of at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example, at least 10 days, in the human subject to which it is administered / administered. Some non-limiting examples of the ISVD of the present invention against human serum albumin that can be used for this purpose are further described herein.

[0139] In one embodiment, all ISVDs or nanobodies present in the compound of the present invention are ISVDs of the present invention (meaning they have amino acid residues / substitutions that characterize the VH domain of the present invention as defined herein, i.e., at least 112K or Q in combination with 110K or 110Q, or at least 89T or at least 89L). If all ISVDs in the compound of the present invention are ISVDs of the present invention, they can have the same substitution (e.g., all having S112K or S112Q substitutions) or different substitutions (e.g., one may have an S112K or S112Q substitution, and the others may have a V89L mutation in combination with T110K or T110Q). Also, typically only the C-terminal ISVD of the compound of the present invention will have C-terminal elongation (the others will probably be ligated at their C-terminus to another ISVD present in the compound).

[0140] Therefore, in further embodiments, the present invention relates to proteins, polypeptides, or other compounds or molecules comprising, or essentially consisting of, the ISVD of the present invention (as further described herein).

[0141] Furthermore, the present invention relates to a protein, polypeptide, or other compound or molecule comprising at least one ISVD of the present invention and at least one other therapeutic portion or component (linked to each other directly or via a suitable linker).

[0142] Furthermore, the present invention relates to a protein, polypeptide, or other compound or molecule comprising (linked directly to each other or via an appropriate linker) at least one ISVD of the present invention for (human) serum protein (and preferably for human serum albumin) and at least one other therapeutic moiety or component.

[0143] Furthermore, the present invention relates to a protein, polypeptide, or other compound or molecule comprising at least two (e.g., two, three, or four) immunoglobulin single variable domains (linked to each other directly or via appropriate linkers). At least one of the immunoglobulin single variable domains is an ISVD of the present invention. In this embodiment, (i) the ISVDs present may appropriately be the same or different, and if they are different, they may be directed to the same target (e.g., they may have different sequences and / or to different epitopes on the same target) or to two or more different targets (i.e., such that the resulting protein, polypeptide, or other compound or molecule is a bispecific or multispecific construct). and / or, (ii) the ISVD present at the C-terminus of the protein, polypeptide, or other compound or molecule may or may not be an ISVD of the present invention (but preferably it is an ISVD). and / or, (iii) if the ISVD of the present invention is present at the C-terminus of a protein, polypeptide, or other compound or molecule, the ISVD of the present invention preferably has the C-terminal extension described herein. and / or, (iv) essentially all ISVDs present in a protein, polypeptide, or other compound or molecule can be the ISVD of the present invention. Also, if the ISVDs are for different targets (at least one of which is a therapeutic target), according to one further embodiment, at least one of the ISVDs present may be for a (human) serum protein, for example, human serum albumin (this ISVD may or may not be the ISVD of the present invention, and if this ISVD is the ISVD of the present invention, the nanobody for human serum albumin described herein further is preferred).

[0144] Furthermore, the present invention relates to such proteins, polypeptides, or other compounds or molecules comprising, or essentially consisting of, two immunoglobulin single variable domains (linked to each other directly or via appropriate linkers).

[0145] Furthermore, the present invention relates to such proteins, polypeptides, or other compounds or molecules comprising, or essentially consisting of, three immunoglobulin single variable domains (linked to each other directly or via appropriate linkers).

[0146] Furthermore, the present invention relates to such proteins, polypeptides, or other compounds or molecules comprising, or essentially consisting of, four immunoglobulin single variable domains (linked to each other directly or via appropriate linkers).

[0147] Furthermore, the present invention relates to such proteins, polypeptides, or other compounds or molecules, further comprising at least one moiety, binding domain, or binding unit that confers an extended half-life to the protein, polypeptide, or other compound or molecule (i.e., compared to the corresponding protein, polypeptide, or other compound or molecule that does not include the moiety, binding domain, or binding unit). In a more specific embodiment, the at least one moiety, binding domain, or binding unit that confers an extended half-life to the protein, polypeptide, or other compound or molecule is an immunoglobulin single variable domain, in particular an immunoglobulin single variable domain for serum proteins (e.g., serum albumin), especially for human serum proteins (e.g., human serum albumin), and may be, in particular, an ISVD of the present invention, as described herein. The ISVD for serum proteins may be located at the N-terminus, C-terminus, or (if the protein, polypeptide, or other compound or molecule contains three or more ISVDs) in the center of the molecule.

[0148] Furthermore, the present invention relates to such proteins, polypeptides, or other compounds or molecules comprising any of the following, or essentially consisting of any of the following. -Two immunoglobulin single variable domains (linked directly to each other or via appropriate linkers), namely, one immunoglobulin single variable domain (e.g., a nanobody) that confers an extended half-life, and the other immunoglobulin single variable domain (e.g., a nanobody) that can specifically target therapeutic targets; - Three immunoglobulin single variable domains (linked to each other directly or via appropriate linkers), i.e., one immunoglobulin single variable domain (e.g., a nanobody) that confers an extended half-life, and two other immunoglobulin single variable domains (e.g., two other nanobodies) that can specifically target a therapeutic target (where the two other immunoglobulin single variable domains can target the same target, two different targets, or two different epitopes on the same target); or - Four immunoglobulin single variable domains (linked to each other directly or via appropriate linkers), i.e., one immunoglobulin single variable domain (e.g., a nanobody) conferring an extended half-life, and two other immunoglobulin single variable domains (e.g., two other nanobodies) that can specifically target a therapeutic target (where the three other immunoglobulin single variable domains can target the same target, two or three different targets, and / or two or three different epitopes on the same target).

[0149] Again, in such proteins, polypeptides, or other compounds or molecules, (i) the ISVD present may appropriately be the same or different, and if the ISVDs are different, the ISVDs may be for the same target (e.g., the ISVDs may be different sequences and / or for different epitopes on the same target) or for two or more different targets (i.e., the resulting protein, polypeptide, or other compound or molecule may be a dual or multispecific construct). and / or, (ii) the ISVD present at the C-terminus of a protein, polypeptide, or other compound or molecule may be the ISVD of the present invention or not (but preferably the ISVD of the present invention). and / or, (iii) if the ISVD of the present invention is present at the C-terminus of a protein, polypeptide, or other compound or molecule, the ISVD of the present invention preferably has the C-terminal extension described herein. and / or, (iv) essentially all ISVDs present in a protein, polypeptide, or other compound or molecule may be the ISVD of the present invention.

[0150] Furthermore, the present invention relates to methods for expressing / generating / producing the VH domains of the present invention and the compounds of the present invention (as further described herein). For example, the VH domains of the present invention can be expressed / generated by appropriately expressing the nucleic acid encoding the VH domains of the present invention in a suitable host organism. See, for example, International Publication No. 08 / 020079 (and several other patent applications of the applicant / assignee cited herein). This document generally describes methods and techniques suitable for expressing / generating nanobodies. This method can also be appropriately used to express / generate the nanobodies of the present invention. Methods for expressing the VH domains of the present invention other than nanobodies will be apparent to those skilled in the art based on the prior art cited herein and herein. The compounds of the present invention can be appropriately produced / generated by appropriately linking one or more VH domains of the present invention to one or more further amino acid residues (and / or other groups or parts) to be present in the final compound of the present invention, optionally via one or more linkers or spacers (usually via covalent bonds). Alternatively, if the compound of the present invention is a protein or polypeptide, the compound of the present invention can be produced / generated by appropriately expressing the nucleic acid encoding the compound of the present invention in a suitable host organism. For example, see again the general methods described in International Publication No. 08 / 020079 and several other patent applications of the applicant / assignee cited herein.

[0151] Furthermore, the present invention relates to nucleotide sequences and / or nucleic acids encoding the VH domain of the present invention or the compounds of the present invention. Such nucleic acids may be DNA or RNA, and preferably DNA, and may be in the form of plasmids or vectors. See, for example, again, International Publication No. 08 / 020079 and several other patent applications of the applicant / assignee cited herein.

[0152] Furthermore, the present invention relates to a composition comprising at least one VH domain of the present invention, a compound of the present invention, or a nucleic acid encoding any of the above.

[0153] Furthermore, the present invention relates to a pharmaceutical composition comprising an ISV (and preferably a therapeutic ISV) or a protein or polypeptide containing at least one ISV (and preferably at least one therapeutic ISV) [wherein the ISV, protein, or polypeptide is as further described herein (i.e., one or more embodiments described herein, and in particular, one or more embodiments of the ISV, protein, or polypeptide described on the preceding pages, and especially an ISV, protein, or polypeptide having a C-terminus / sequence as one or more embodiments described herein)], at least one suitable (i.e., suitable for pharmaceutical use) carrier, diluent, or excipient, and optionally one or more further active substances. Such compositions, carriers, diluents, or excipients are, for example, as described in International Publication No. 08 / 020079 for a pharmaceutical composition containing a nanobody or a protein or polypeptide containing at least one nanobody (as already mentioned in the present invention, and the ISV is preferably also a nanobody).

[0154] Furthermore, the present invention relates to an ISV or a protein or polypeptide comprising at least one ISV for use in the treatment of a disease in humans (for example, a patient requiring such treatment), wherein the ISV, protein, or polypeptide is as further described herein (i.e., one or more embodiments described herein, and in particular, one or more embodiments of the ISV, protein, or polypeptide described on the preceding pages, and especially an ISV, protein, or polypeptide having a C-terminus / sequence as one or more embodiments described herein).

[0155] Furthermore, the present invention relates to the use of an ISV or a protein or polypeptide comprising at least one ISV in the manufacture of a pharmaceutical composition, wherein the ISV, protein, or polypeptide is as further described herein (i.e., one or more embodiments described herein, and in particular, one or more embodiments of the ISV, protein, or polypeptide described on the preceding pages, and especially an ISV, protein, or polypeptide having a C-terminus / sequence as one or more embodiments described herein).

[0156] Furthermore, the present invention relates to a method of treatment to a human subject (for example, a patient requiring the following treatment), comprising administering an ISV or a protein or polypeptide comprising at least one ISV, wherein the ISV, protein, or polypeptide is as further described herein (i.e., one or more embodiments described herein, and in particular, one or more embodiments of the ISV, protein, or polypeptide described on the preceding pages, and especially an ISV, protein, or polypeptide having a C-terminus / sequence as one or more embodiments described herein), or to a pharmaceutical composition (as described above) comprising at least one such ISV, protein, or polypeptide.

[0157] In relation to the above, it will be apparent that the therapeutic use of ISVs, proteins, or polypeptides described herein is a very important aspect of the present invention. Accordingly, the therapeutic use (or the clinical development of such ISVs, proteins, and polypeptides for such therapeutic use) may include the use of ADA assay methods to determine whether the ISVs, proteins, or polypeptides are immunogenic (i.e., may produce ADA when administered to human subjects). In this regard, it will also be apparent that concerns about potential immunogenicity must be addressed in particular if the therapeutic agent is used for a long period of time (weeks, months, or years) and / or has a half-life in human subjects of at least 3 days, e.g., at least 1 week, and up to 10 days or more (preferably expressed as t1 / 2 beta).

[0158] Therefore, according to one specific embodiment, the present invention relates to ISVs, proteins, polypeptides, compounds, or molecules (or pharmaceutical compositions comprising them) of the present invention as described herein, intended for the treatment of chronic diseases in humans. and / or, such ISVs, proteins, polypeptides as described herein are intended to be present in the circulation (i.e., at pharmacological activity levels) of a subject administered at least one week, preferably at least two weeks, for example, at least one month (i.e., at a therapeutically active dose). and / or, such ISVs, proteins, polypeptides as described herein are intended to have a half-life in a human subject of at least three days, for example, at least one week, and up to 10 days or more (preferably expressed as t1 / 2 beta). And / or, such ISVs, proteins, polypeptides, or pharmaceutical compositions described herein are intended to be administered to humans over a period of at least three days, for example, at least one week, for example, at least two weeks or at least one month, or longer (i.e., at least three months, at least six months, or at least one year), or as two or more chronically administered doses.

[0159] Furthermore, as will be apparent to those skilled in the art based on the disclosure herein, improvements to the VH domain described herein and the resulting improved VH domains will find specific uses in proteins, polypeptides, or other compounds or molecules intended for administration to human subjects (and in particular patients) whose whole blood / serum contains (or is suspected to contain) existing antibodies of the type found in samples obtained from SLE patients, i.e., existing antibodies that can bind to the C-terminal region of the VH domain even in the presence of the C-terminal extension described herein. In particular, improvements to the VH domain described herein and the resulting improved VH domains will find specific uses in proteins, polypeptides, or other compounds or molecules intended for treating or preventing disease or impairment in such patients. This can be any disease or disorder, but in particular, a disease or disorder that results in the presence or development of such pre-existing antibodies, and / or is associated with the presence or development of such pre-existing antibodies (one example being SLE, but other serious (auto)immune disorders are also expected to result in such pre-existing antibodies. Pre-existing antibodies can be readily confirmed by testing samples obtained from the relevant patient population for the presence of such pre-existing antibodies in a manner essentially similar to the tests performed on samples from SLE patients in the laboratory described below).

[0160] Therefore, according to one specific embodiment, the present invention relates to an ISV, protein, polypeptide, compound, or molecule (or a pharmaceutical composition comprising the same) of the present invention as described herein, intended for administration to a human subject in the blood of which an existing antibody can bind even when the exposed C-terminal region of the VH domain contains the C-terminal extension described herein (or when the C-terminus of the VH domain is linked to another protein or polypeptide, e.g., another ISV, optionally via a suitable linker).

[0161] In particular, the present invention relates to the ISVs, proteins, polypeptides, compounds, or molecules (or pharmaceutical compositions comprising them) of the present invention described herein for use in treating diseases or disorders in human subjects in which an existing antibody is present in the blood and can bind even when the exposed C-terminal region of the VH domain contains the C-terminal extension described herein (or when the C-terminus of the VH domain is linked to another protein or polypeptide, e.g., another ISV, optionally via a suitable linker). The disease or disorder may be any disease or disorder, but in particular may be a disease or disorder caused, brought about, or otherwise associated by the presence of such an existing antibody in the blood of such a patient, e.g., SLE or another (serious) autoimmune disease.

[0162] Therefore, in a more specific embodiment, the present invention relates to an ISV, protein, polypeptide, compound, or molecule (or a pharmaceutical composition containing the same) of the present invention as described herein, for use in the treatment of a disease or disorder in a human subject / patient, wherein the disease or disorder is caused, brought about, or otherwise associated by the presence of a pre-existing antibody in the blood of the human subject / patient, which can also bind to the exposed C-terminal region of the VH domain, where the VH domain contains the C-terminal extension described herein (or where the C-terminus of the VH domain is linked to another protein or polypeptide, e.g., another ISV, optionally via an appropriate linker). For example, such an ISV, protein, polypeptide, compound, or molecule (or a pharmaceutical composition containing the same) of the present invention as described herein is for use in the treatment of SLE or another (severe) autoimmune disease in a human subject / patient.

[0163] 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 disease or disorder, it will contain at least one (e.g., one, two, three, or four) domains, binding units, or parts or components that are therapeutic activators for the relevant disease or disorder (e.g., for therapeutically relevant targets or pathways to the relevant disease or disorder). Again, such binding domains or binding units may be, for example, (other) ISVDs, and in one embodiment, in particular, the VH domains or ISVDs of the present invention. Another common example of such a protein, polypeptide, compound, or molecule is a protein, polypeptide, compound, or molecule (e.g., a serum albumin binder described herein) in which the one or more therapeutic domains, binding units, or parts or components are not ISVDs (but are, for example, obtained from another scaffold), but contain the VH domains of the present invention to extend the half-life of the protein, polypeptide, compound, or molecule.

[0164] In further embodiments, the VH domain, ISVD, or compound of the present invention (as described herein) is for the ion channel Kv1.3. Some preferred, non-limiting examples of such VH domains, ISVDs, or compounds are provided in Example 7. The VH domains for Kv1.3 (and compounds of the present invention containing them) described in the above examples and the specific compounds of the present invention for Kv1.3 described in the above examples constitute further embodiments of the present invention.

[0165] For example, but not limited to, compounds of the present invention relative to Kv1.3 may contain or essentially consist of a single VH domain of the present invention relative to Kv1.3—and preferably a nanobody—or may contain or essentially consist of at least two (e.g., two or three) VH domains of the present invention relative to Kv1.3—and preferably a nanobody. If such polypeptide contains two or more VH domains of the present invention relative to Kv1.3, these VH domains may be the same or different. If they are different, they may be relative to the same epitope or subunit on Kv1.3, or to different epitopes or subunits.

[0166] Again, as generally described herein with respect to the compounds of the present invention, such compounds may appropriately contain one or more linkers, may contain a C-terminal extension (i.e., as described herein), and may also contain one or more further binding units or binding domains (or other amino acid sequences or portions), for example, further ISVDs to targets other than Kv1.3. For example, but not limited to, the compounds of the present invention may also contain binding domains or binding units that provide an extended half-life, for example, ISVDs to serum proteins, for example, human serum albumin (e.g., nanobodies to human serum albumin, for example, the nanobodies of the present invention to human serum albumin).

[0167] In a further embodiment, the present invention relates to a (synthetic) library of immunoglobulin variable domain sequences described herein (i.e., containing the amino acid residues / mutations / substitutions described herein). Such a library typically comprises at least 100 sequences, for example, at least 1000 sequences, particularly 1000 sequences, with a variety of CDRs (meaning that each sequence in the library has at least "one amino acid difference" in at least one of its CDRs compared to other sequences in the library) and all (essentially) the same framework sequences (the framework sequences containing the amino acid residues / mutations shown herein). 5 Sequences exceeding the number of types, especially 10 6 Arrays exceeding the number of types, for example, 10 8 ~10 10 It will likely contain more than one type of sequence (in its broadest sense, this means that there is at least one amino acid difference between the sequences).

[0168] Synthetic libraries of immunoglobulin single variable domain sequences (e.g., based on human VH sequences or camel derivatized VHH sequences) and methods for generating / constructing them (including libraries based on a predetermined scaffold and / or containing one or more specific amino acid residues / mutations in a framework region) are well known in the art. 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, Volume 8, Issue 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 in these documents (and similar techniques known in themselves) can be appropriately used or adapted to generate the immunoglobulin single variable domain library of the present invention.

[0169] Such libraries can appropriately contain any suitable CDRs from any suitable source, such as CDRs obtained / generated starting from an immune repertoire from "naive" mammals (e.g., camel species or human sequences), CDRs obtained / generated starting from an immune repertoire from animals appropriately immunized with antigens (e.g., camel species), a completely synthetic CDR repertoire, or a repertoire obtained by techniques such as mutagenesis (e.g., random mutagenesis or site-directed mutagenesis). Such libraries can also be libraries generated, for example, in the process of affinity maturation methods known in themselves.

[0170] The framework region of ISVDs present in such libraries can be appropriately obtained from any suitable starting sequence / scaffold, for example, based on 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). Furthermore, libraries can be obtained from two or more sources, or contain ISVDs based on two or more scaffolds (for example, libraries can be obtained from different sources or by combining two or more libraries based on different scaffolds).

[0171] Furthermore, the library (containing the ISVD) may be in the form of a protein, or in the form of DNA or RNA encoding the associated ISVD. For example, the library may be in the form of an expression library suitable for screening and / or selection techniques, and for this purpose may be in a form that can be displayed using a suitable display technique, such as a phage display library, a yeast display library, or a ribosome display library.

[0172] Therefore, and more generally, the present invention relates to a library (as described herein) containing the VH domains of the present invention (as further described herein). Preferably, according to certain embodiments of such a library, all present VH domains have the same (or essentially the same) framework sequence but different CDR sequences (as again 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).

[0173] In one embodiment, the library of the present invention is a library of ISVDs of the present invention (including a library of nucleic acids suitable for encoding the ISVDs, as further described herein), where, The amino acid residue at position -11 is one of L, V, or K, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is one of T, V, or L, and The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q. Herein, one of the following applies: (i) the amino acid residue at position 112 is either 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, the amino acid residue at position 110 is either K or Q, and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V. In addition, the ISVD present in such a library may contain a C-terminal extension (as further described herein with respect to the VH domain of the present invention) and / or may appropriately contain a suitable tag (e.g., a histidine tag).

[0174] In another embodiment, such a library of the present invention is a library of ISVDs of the present invention (including a library of nucleic acids suitable for encoding the ISVDs, as further described herein), where, The amino acid residue at position -11 is V, and The amino acid residue at position -14 is either A or P, and The amino acid residue at position -41 is either A or P, and The amino acid residue at position -89 is L, and, The amino acid residue at position -108 is either Q or L, and The amino acid residue at position -110 is one of T, K, or Q, and The amino acid residue at position -112 is one of S, K, or Q.

[0175] In addition, depending on the circumstances, the ISVD present in such a library may contain a C-terminal extension (as further described herein with respect to the VH domain of the present invention) and / or may appropriately contain a suitable tag (e.g., a histidine tag).

[0176] The library of the present invention is known in itself and can be used for any suitable / intended purpose. For example, the library of the present invention can be used for screening and / or selection purposes (or as part of a screening and / or selection process), and can be used for affinity maturation purposes or as part of other processes intended for the generation of improved VH domains, for example, in alanine scanning. In practice, the size, design, and other features of the library will usually be adapted to its intended use, which will be within the scope of the art.

[0177] From here, the present invention will be further described by the following non-limiting preferred embodiments, examples, and drawings. Here, [Brief explanation of the drawing]

[0178] [Figure 1] Figure 1 is a table listing some of the amino acid positions that will be specifically referred to herein and their numbering according to several alternative numbering systems (e.g., Aho and IMGT). [Figure 2] Figure 2 lists the sequences mentioned herein. [Figure 3] Figure 3 shows the results of testing 96 serum samples for binding to a representative nanobody with the S112K mutation (reference A + S112K + C-terminal alanine, shown as (2) in Figure 3) compared to a reference nanobody without the S112K mutation (reference A, SEQ ID NO: 44, shown as (1) in Figure 3). [Figure 4]Figure 4 is a plot showing the data points obtained in Example 4 when 129 serum samples were tested for binding to a representative nanobody with the V89T mutation (reference A + L11V + V89T + C-terminal alanine, shown as (2) in Figure 4) compared to a reference nanobody without the V89T mutation (reference A, sequence number: 44, shown as (1) in Figure 4). [Figure 5] Figure 5 is a plot showing the data points obtained in Example 5 when 100 serum samples were tested for binding to representative nanobodies having the V89L, T110K, and / or T110Q mutations (reference A+L11V+V89L+C-terminal Ala, shown as (2) in Figure 5; reference A+L11V+V89L+T110K+C-terminal Ala, shown as (3) in Figure 5; reference A+L11V+V89L+T110Q+C-terminal Ala, shown as (4) in Figure 5; and reference A+L11V+T87A+V89L+C-terminal Ala, shown as (5) in Figure 5) compared to a reference nanobody not having any of these mutations (reference A, SEQ ID NO: 44, shown as (1) in Figure 5). [Figure 6] Figure 6 is a plot showing the data points obtained in Example 6 when 98 serum samples obtained from healthy volunteers were tested for binding to a representative trivalent nanobody construct. Each dot represents a data point collected by testing the indicated construct on 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. [Figure 7]Figure 7 is a plot showing data points obtained in Example 6 when 30 serum samples obtained from healthy volunteers (samples selected for the presence of high-titer existing antibodies or existing antibodies with high binding even if C-terminal alanine elongation is present) were tested for binding to a representative trivalent nanobody construct. Each dot represents a data point collected by testing the indicated construct on one of the 30 serum samples. Legend: (1) = reference X + C-terminal Ala; (2) = reference X + L11V + V89L + C-terminal Ala; (3) = reference X + L11V + T87A + V89L + C-terminal Ala; (4) = reference X + L11V + V89L + T110K + C-terminal Ala. [Figure 8] Figure 8 is a plot showing data points obtained in Example 6 when 98 serum samples obtained from healthy volunteers were tested for binding to a representative divalent nanobody construct. Each dot represents a data point collected by testing the indicated construct on 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. [Figure 9A] Figures 9A and 9B show preferred, non-limiting examples of the monovalent nanobody of the present invention (Figure 9A) and the trivalent bispecific half-life extension compound of the present invention (Figure 9B) for the ion channel Kv1.3. Figure 9C lists some preferred CDRs (classified according to Kabat and Abm, respectively) for ISVDs for Kv1.3. [Figure 9B] Figures 9A and 9B show preferred, non-limiting examples of the monovalent nanobody of the present invention (Figure 9A) and the trivalent bispecific half-life extension compound of the present invention (Figure 9B) for the ion channel Kv1.3. Figure 9C lists some preferred CDRs (classified according to Kabat and Abm, respectively) for ISVDs for Kv1.3. [Figure 9C] Figures 9A and 9B show preferred, non-limiting examples of the monovalent nanobody of the present invention (Figure 9A) and the trivalent bispecific half-life extension compound of the present invention (Figure 9B) for the ion channel Kv1.3. Figure 9C lists some preferred CDRs (classified according to Kabat and Abm, respectively) for ISVDs for Kv1.3. [Figure 10] Figure 10 is a plot showing data points obtained in Example 7 when 47 serum samples obtained from human subjects with diabetes were tested for binding to a representative trivalent bispecific half-life extension compound of the present invention against Kv1.3. Each dot represents a data point collected by testing the indicated construct on one of the 47 serum samples. The sequence numbers refer to the relevant sequences listed in Figure 9. [Figure 11] Figure 11 is a plot showing data points obtained in Example 7 when 90 serum samples obtained from healthy volunteers were tested for binding to representative trivalent bispecific half-life extension compounds of the present invention against Kv1.3. Each dot represents a data point collected by testing the indicated construct against one of 47 serum samples. Sequence numbers refer to the relevant sequences listed in Figure 9. [Figure 12A] Figures 12A and 12B list the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to IL-23, based on the indicated reference sequence. See also Example 8. [Figure 12B] Figures 12A and 12B list the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to IL-23, based on the indicated reference sequence. See also Example 8. [Figure 13] Figure 13 lists the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to OX40-L, based on the shown reference sequence. See also Example 9. [Figure 14]Figure 14 lists the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to IgE, based on the shown reference sequence. See also Example 10. [Figure 15A] Figures 15A and 15B list the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to CXCR-4, based on the indicated reference sequence. See also Example 11. [Figure 15B] Figures 15A and 15B list the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to CXCR-4, based on the indicated reference sequence. See also Example 11. [Figure 16A] Figures 16A and 16B list the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to HER-3, based on the indicated reference sequence. See also Example 12. [Figure 16B] Figures 16A and 16B list the CDR and amino sequences of some preferred, non-limiting examples of the nanobodies of the present invention relative to HER-3, based on the indicated reference sequence. See also Example 12. [Figure 17A] Figures 17A and 17B list preferred, non-limiting examples of CDR and amino sequences of some of the nanobodies of the present invention relative to TNF, based on the indicated reference sequences. See also Examples 13 and 14. [Figure 17B] Figures 17A and 17B list preferred, non-limiting examples of CDR and amino sequences of some of the nanobodies of the present invention relative to TNF, based on the indicated reference sequences. See also Examples 13 and 14. [Figure 18A] Figures 18A and 18B list preferred, non-limiting examples of CDR and amino sequences of some of the nanobodies of the present invention relative to c-Met, based on the indicated reference sequences. See also Example 15. [Figure 18B] Figures 18A and 18B list preferred, non-limiting examples of CDR and amino sequences of some of the nanobodies of the present invention relative to c-Met, based on the indicated reference sequences. See also Example 15. [Figure 19] Figure 19 lists the CDR and amino acid sequences of some preferred, non-limiting examples of the nanobody of the present invention against RANK-L based on the reference sequences shown. See also Example 16. [Figure 20A] Figures 20A - 20C list the CDR and amino acid sequences of some preferred, non-limiting examples of the nanobody of the present invention against CXCR-7 based on the reference sequences shown. See also Example 17. [Figure 20B] Figures 20A - 20C list the CDR and amino acid sequences of some preferred, non-limiting examples of the nanobody of the present invention against CXCR-7 based on the reference sequences shown. See also Example 17. [Figure 20C] Figures 20A - 20C list the CDR and amino acid sequences of some preferred, non-limiting examples of the nanobody of the present invention against CXCR-7 based on the reference sequences shown. See also Example 17. [Figure 21A] Figures 21A and 21B list the CDR and amino acid sequences of some preferred, non-limiting examples of the nanobody of the present invention against A-beta based on the reference sequences shown. See also Example 18. [Figure 21B] Figures 21A and 21B list the CDR and amino acid sequences of some preferred, non-limiting examples of the nanobody of the present invention against A-beta based on the reference sequences shown. See also Example 18. [Figure 22] Figure 22 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against IL-23. [Figure 23] Figure 23 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against OX40-L. [Figure 24] Figure 24 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against IgE. [Figure 25] Figure 25 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against CXCR-4. [Figure 26]Figure 26 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against HER-3. [Figure 27] Figure 27 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against TNF. [Figure 28A] Figures 28A and 28B provide the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against c-Met. [Figure 28B] Figures 28A and 28B provide the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against c-Met. [Figure 29] Figure 29 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against RANK-L. [Figure 30] Figure 30 provides the amino acid sequences of some preferred, non-limiting examples of the compounds of the present invention against A-beta. [Figure 31A] Figure 31A is a plot showing the data points obtained in Example 19 when 92 serum samples obtained from healthy volunteers were tested for binding to a representative trivalent bispecific half-life extended compound of the present invention against A-beta. Each dot represents a data point collected by testing the indicated construct against one of the 92 serum samples. The reference numbers are as listed in Table CC-1. Similarly, Figure 31B is a plot showing the data points obtained in Example 19 when 92 serum samples obtained from healthy volunteers were tested for binding to the (monovalent) C-terminal nanobody present in the construct tested in Figure 31A. Each dot represents a data point collected by testing the indicated construct against one of the 92 serum samples. The reference numbers are as listed in Table CC-2. [Figure 31B]Figure 31A is a plot showing data points obtained in Example 19 when 92 serum samples from healthy volunteers were tested for binding to a representative trivalent bispecific half-life extension compound of the present invention for A-beta. Each dot represents a data point collected by testing the indicated construct against one of the 92 serum samples. Reference numbers are listed in Table CC-1. Similarly, Figure 31B is a plot showing data points obtained in Example 19 when 92 serum samples from healthy volunteers were tested for binding to the (monovalent) C-terminal nanobody present in the construct tested in Figure 31A. Each dot represents a data point collected by testing the indicated construct against one of the 92 serum samples. Reference numbers are listed in Table CC-2. [Examples]

[0179] Experiment Department The human samples used in the following experiments were obtained from either commercial suppliers or human volunteers (after obtaining all necessary consent and approvals) and used in accordance with the requirements of applicable laws and regulations, including but not limited to those relating to medical confidentiality and patient privacy.

[0180] In the following examples, the binding of existing antibodies present in the samples used (i.e., samples from healthy volunteers, rheumatoid arthritis (RA) patients, and SLE patients) to the tested nanobodies was determined using ProteOn as follows: The binding of existing antibodies to nanobodies captured by 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 running buffer. Experiments were performed at 25°C. The ligand lane of the ProteOn GLC sensor chip was activated with EDC / NHS (flow rate 30 μL / min). HSA was injected at 10 μg / mL in ProteOn acetate buffer pH 4.5 (flow rate 100 μL / min) to an immobilization level of approximately 3200 RU. After immobilization, the surface was inactivated with ethanolamine HCl (flow rate 30 μL / min). Nanobodies were injected onto the HSA surface at 45 μL / min for 2 minutes to achieve a nanobody capture level of approximately 200 RU. Samples containing existing antibodies were centrifuged at 14,000 rpm for 2 minutes. The supernatant was diluted 1:10 in PBS-Tween20 (0.005%), then injected at 45 μL / min for 2 minutes, followed by a 400-second dissociation step. After each cycle (i.e., before new nanobody capture and blood sample injection), the HSA surface was regenerated by injecting HCl (100 mM) at 45 μL / min for 2 minutes. Sensational gram processing and data analysis were performed using ProteOn Manager 3.1.0 (Bio-Rad Laboratories, Inc.). Sensorgrams showing the binding of existing antibodies were obtained after double referencing by subtracting 1) nanobody-HSA dissociation and 2) nonspecific binding to the reference ligand lane. The binding level of existing antibodies was determined by setting a reporting point at 125 seconds (5 seconds after the end of association). The percentage decrease in binding of existing antibodies was calculated relative to the binding level of the reference nanobody at 125 seconds.

[0181] Example 1: The S112K mutation inhibits the binding of existing antibodies. The effect of substitution at position 112 on the binding of existing antibodies to nanobodies in human samples was determined and compared with the effect of C-terminal alanine elongation described in International Publication No. 12 / 175741.

[0182] Two reference compounds (reference A without C-terminal alanine elongation and reference B with C-terminal alanine elongation) and variants of these reference compounds with various mutations at position 112 were tested on serum from six different RA patients and eight serum from different healthy human subjects. Binding of existing antibodies in the samples to the tested nanobodies was measured in ProteOn according to the overall protocol outlined above. The results are shown in Table A below.

[0183] As can be seen, among the mutations at position 112 tested, the S112K mutation provided a reduction in binding by existing antibodies present in the tested serum, comparable to a reduction in C-terminal alanine elongation (even when C-terminal alanine elongation was not present in the S112K variant). Similar results were obtained for three human plasma samples (data not shown).

[0184] [Table 6]

[0185] Example 2: Effect of the S112K mutation on the binding of existing antibodies present in human SLE samples. The same nanobody variant used in Example 1 was tested for binding with existing antibodies from seven serum samples obtained from patients confirmed to be positive for systemic lupus erythematosus (SLE). Plasma samples from two healthy human volunteers were included for comparison.

[0186] The binding of existing antibodies in the sample to the tested nanobodies was measured in ProteOn according to the overall protocol outlined above. The results are shown in Table B below.

[0187] As can be seen from the comparison of the binding data for reference A and reference B, as well as for the nanobodies of the present invention, samples obtained from some SLE patients are thought to contain certain existing antibodies that can bind to the nanobody even when a C-terminal alanine residue is present (the C-terminal alanine residue essentially (partially or essentially completely) prevents / removes all binding of existing antibodies present in plasma samples from healthy volunteers).

[0188] It was further found that the binding of these existing antibodies from the SLE samples could be significantly reduced by mutations at positions 11 and 112 (and, in the case of position 112, particularly S112K).

[0189] [Table 7]

[0190] Example 3: Effect of combined framework mutations and C-terminal extension on the binding of existing antibodies present in human SLE samples (With specific framework mutations and with or without a C-terminal alanine extension) Four nanobodies were tested for the binding of existing antibodies from five serum samples obtained from patients confirmed to be positive for systemic lupus erythematosus (SLE). For comparison, one plasma sample from a healthy human volunteer was included.

[0191] The binding of existing antibodies in the samples to the tested nanobodies was measured on a ProteOn according to the overall protocol outlined above. The results are shown in Tables C and D below.

[0192] As can be seen from the comparison of the binding data for reference A and reference B, samples obtained from SLE patients are thought to contain certain existing antibodies that can bind to the nanobody even when a C-terminal alanine residue is present (the C-terminal alanine residue essentially (partially or essentially completely) prevents / removes all binding of existing antibodies present in plasma samples from healthy volunteers).

[0193] It is further evident that the binding of these existing antibodies from SLE samples could be significantly reduced by mutations at positions 11 and 112 (and, in the case of position 112, particularly S112K).

[0194] Example 4: Effect of the V89T mutation on the binding of existing antibodies in samples from SLE patients. As described herein, samples obtained from certain SLE patients are thought to contain pre-existing antibodies / factors capable of binding to the exposed C-terminus of the VH domain, even in the presence of C-terminal elongation. We investigated whether the V89T mutation could reduce, prevent, or eliminate such binding, regardless of the presence of C-terminal elongation. The results are also shown in Tables C and E below.

[0195] As can be seen, the V89T mutation was able to essentially prevent / remove the binding of pre-existing antibodies present in samples obtained from SLE patients to the same extent as the S112K mutation. However, as can be seen from the comparison of data provided in Tables C and E for nanobodies with the V89T mutation and no C-terminal extension and similar nanobodies with the S112K mutation and no C-terminal extension, having a mutation at position 112 in nanobodies without C-terminal extension reduces the overall binding of pre-existing antibodies in samples from healthy volunteers to a greater extent than the V89T mutation (i.e., 100%, 85%, and 64% S112K nanobodies vs. 9%, 11%, and 16% V89T nanobodies, respectively). For this reason, the use of the mutation at position 112 (and in particular S112K or S112K) will often be preferred over the use of the mutation at position 89 (e.g., V89T).

[0196] However, as can be seen from the data in Tables C and E, adding C-terminal alanine to the V89T nanoantibody completely prevents / removes the binding of existing antibodies in samples obtained from healthy volunteers. For this reason, if the V89T nanobody or VH domain has, or is intended to have, a C-terminal region exposed in the protein or polypeptide in which it would be located (for example, if the V89T nanobody or VH domain forms the C-terminus of the protein or polypeptide), the combination of V89T mutation and C-terminal extension described herein would generally be preferred (i.e., over the use of V89T without C-terminal extension).

[0197] [Table 8]

[0198] [Table 9]

[0199] [Table 10]

[0200] To confirm the broad applicability of the results / findings from the table above, representative nanobodies with S112K and / or V89T mutations were tested against a test panel of 96 (S112K) and 129 (V89T) human serum samples. Binding was determined in ProteOn using the protocol described above.

[0201] The results are summarized in Figure 3 and Table F (representative nanobodies with the S112K mutation) and in Figure 4 and Table G (representative nanobodies with the V89T mutation).

[0202] In Figure 3, a nanobody with the S112K mutation (reference A + S112K + C-terminal alanine - see Table C above) was compared to a reference nanobody (reference A; SEQ ID NO: 44). Both the nanobody with the S112K mutation and reference A were tested against their respective serum samples. The binding level (RU) at 125 seconds was determined. The data were then plotted in Figure 3. Here, each point represents the binding measured in a single sample for either reference A (indicated as (1) in Figure 3) or the S112K mutation (indicated as (2) in Figure 3). The dashed line indicates a measured binding level of 20 RU.

[0203] The same data is also represented numerically in Table F. Table F refers to the total number of samples tested that provide binding levels greater than 20 RU, less than 20 RU (i.e., 0-20 RU), and less than 10 RU at 125 seconds (for each of the Reference A and S112K variants).

[0204] For reference A, as plotted in Figure 3 and as can be seen from the data shown in Table F, more than half of the 96 samples tested provided binding levels above 20 RU (in some cases, as high as 150–200 RU). This indicates that the existing antibodies present in the samples bound to reference A. In comparison, for the S112K mutation, no binding levels above 20 RU were provided from the samples (and most were below 10 RU). This indicates that the S112K mutation was able to essentially prevent / remove the binding of existing antibodies in all 96 samples tested.

[0205] For a representative nanobody with the V89T mutation (reference A + L11V + V89T + C-terminal alanine, see Table E above) tested against 129 serum samples, similar plots and data are shown in Figure 4 and Table G, respectively, and compared again with reference A (shown as (1) in Figure 4, the V89T mutation, shown as (2) in Figure 4). Again, from the plots in Figure 4 and the data in Table G, it can be seen that, with few exceptions (i.e., less than 10% of the tested samples, for example, they each provided absolute binding values ​​of approximately 100 RU or less after 125 seconds), the V89T mutation was able to reduce / prevent the binding of existing antibodies in most of the 129 tested samples. On the other hand, the reference without the V89T mutation bound to existing antibodies in most of the tested samples.

[0206] [Table 11]

[0207] [Table 12]

[0208] Example 5: Effect of V89L mutation combined with T110K or T110Q on the binding of existing antibodies in samples from SLE patients. As described herein, samples obtained from specific SLE patients are thought to contain pre-existing antibodies / factors capable of binding to the exposed C-terminus of the VH domain, even in the presence of C-terminal extension. We investigated whether the V89L and / or T110Q or T110K mutations (or combinations thereof) could reduce, prevent, or eliminate such binding, regardless of the presence of C-terminal extension. The results are also shown in Tables H and I below. These tables represent data from two separate experiments (Table D above also shows data for the S112Q mutation of the present invention in combination with the V89L mutation). The nanobodies used in Table I were also used to generate the data shown in Figure 5 and Table J.

[0209] [Table 13]

[0210] [Table 14]

[0211] To confirm that the results / findings from the above table are broadly applicable, representative nanobodies with V89L, T110K, and / or T110Q mutations were again tested against a panel of 99 human serum samples. Binding data were again obtained, and the results and data shown in Figures 3 and 4 and Tables F and G were plotted in the same manner as shown in Example 4.

[0212] The nanobodies tested were numbered according to the numbering used in Figure 5: (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, and (5) Reference A + L11V + T87A + V89L + C-terminal Ala. The results are shown in Figure 5 and Table I.

[0213] As can be seen, reintroducing the tested mutations significantly reduced the number of samples in which the existing antibody was able to bind to the tested nanobodies. For nanobodies (2) in Figure 5 (reference A+L11V+V89L+C-terminal Ala), some samples showed binding of the existing antibody at levels exceeding 20 RU (but less than 100 RU) after 125 seconds. However, when the V89L mutation was combined with the T110K mutation (nanobodies (3)) or the T100Q mutation (nanobodies (4)), all 99 tested samples essentially showed binding levels of less than 20 RU (and actually less than 10 RU; see Table J).

[0214] [Table 15]

[0215] Example 6: Testing of a multivalent construct for binding to an existing antibody. The multivalent constructs are prepared based on the following nanobody. Nanobody A (against therapeutic targets): [Table 16] Nanobody B (against serum albumin): [Table 17] Nanobody C (against therapeutic targets): [Table 18]

[0216] The prepared constructs are listed in Table K below (in each case, substitutions introduced (if any) into the relevant nanobody are indicated in parentheses. "HIS6" means an N-terminal his- tag of six histidine residues. "-Ala" means a C-terminal extension of one alanine residue.

[0217] [Table 19] TIFF0007881627000023.tif240168 TIFF0007881627000024.tif190168

[0218] Representative polyvalent constructs were tested for binding by pre-existing antibodies present in blood or serum samples obtained from patients with SLE and healthy volunteers. Both were determined essentially using the ProteOn described above.

[0219] Representative constructs tested are listed in Table L (trivalent constructs) and Table M (divalent constructs). The results are shown in Figures 6-8 and Tables N-Q. The tested trivalent constructs were obtained from the reference construct nanobody A-35GS-nanobody A-35GS-nanobody B ("reference X"). The divalent constructs were obtained from the reference construct nanobody A-35GS-nanobody B ("reference Y"). All constructs (except the reference constructs) had and exhibited the mutations shown in the C-terminal alanine residue and in the respective "nanobody A" and "nanobody B" construct blocks.

[0220] [Table 20]

[0221] [Table 21]

[0222] [Table 22]

[0223] [Table 23]

[0224] [Table 24]

[0225] Furthermore, three representative trivalent constructs were tested on serum samples obtained from SLE patients. The results are shown in Table Q. [Table 25]

[0226] Example 7: Nanobodies and nanobodies for ion channel Kv1.3 In one specific embodiment, the VH domain of the present invention (and in particular the ISVD of the present invention, and especially the nanobody of the present invention) and the compounds of the present invention can target the ion channel Kv1.3.

[0227] U.S. Provisional Application No. 62 / 014,023 (title of invention: "Kv1.3-binding immunoglobulin"; assignee: Ablynx NV; filing date: June 18, 2014), which is concurrently pending, and a subsequent U.S. Provisional Application for the same invention (filing date: March 16, 2015; assignee: Ablynx NV), describe in particular immunoglobulin single variable domains (and in particular nanobodies) for potassium-selective electroglomerate ion channels Kv1.3, as well as proteins, polypeptides, and other nanobody system constructs comprising such nanobodies for at least one Kv1.3.

[0228] Furthermore, the mutations described herein (in part, appropriately combined with the C-terminal elongation described herein and / or International Publication No. 12 / 175741) can be appropriately applied to nanobodies, proteins, polypeptides, and other nanobody system constructs for Kv1.3 as described in those two U.S. Provisional Applications.

[0229] Therefore, in one embodiment, the present invention relates to the VH domain of the ISVD of the present invention with respect to Kv1.3, as further described herein (i.e., including the amino acid residues / mutations described herein).

[0230] Again, as with other aspects and embodiments of the Invention described herein, where a specific ISVD (e.g., the ISVD for Kv1.3 described in this example) or compound containing it is referred to as "the Invention" or "further described herein," the preferred aspects / embodiments and selections of the ISVD or compound of the Invention described herein as a whole also apply specifically to each of the aforementioned specific ISVDs or compounds, unless otherwise explicitly indicated or otherwise required by specific technical context.

[0231] Therefore, in certain embodiments, the present invention relates to a VH domain for Kv1.3, where (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 such a VH domain for Kv1.3, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, one of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v).

[0232] The VH domain for Kv1.3 referred to in this embodiment may, in particular, have a CDR (including any preferred embodiment / part of such CDR) described on pages 5-10 of U.S. Provisional Application No. 62 / 014,023 or a sequence-optimized version thereof described in other U.S. Provisional Applications referenced above.

[0233] In particular, the VH domain for Kv1.3 as referred to in this embodiment may have a combination of CDR1, CDR2, and CDR3, selected from one of the combinations of CDR1, CDR2, and CDR3 listed as preferred embodiments in the list spanning pages 9 and 10 of U.S. Provisional Application No. 62 / 014,023.

[0234] In some preferred and non-limiting embodiments of the present invention, -In the VH domain of the present invention, (i) CDR1 (by Kabat) is the sequence of SEQ ID NO: 166 or an amino acid sequence having one or two amino acid differences from the sequence of SEQ ID NO: 166, (ii) CDR2 (by Kabat) is the sequence of SEQ ID NO: 167 or an amino acid sequence having one or two amino acid differences from the sequence of SEQ ID NO: 167, and (iii) CDR3 (by Kabat) is the sequence of SEQ ID NO: 168 or an amino acid sequence having one or two amino acid differences from the sequence of SEQ ID NO: 168; and more preferably, (i) CDR1 (by Kabat) is the sequence of SEQ ID NO: 166, (ii) CDR2 (by Kabat) is the sequence of SEQ ID NO: 167, and (iii) CDR3 (by Kabat) is the sequence of SEQ ID NO: 168, and / or -In the VH domain of the present invention, (i) CDR1 (according to Abm) is the sequence of SEQ ID NO: 169 or an amino acid sequence having one or two amino acid differences from the sequence of SEQ ID NO: 169, (ii) CDR2 (according to Abm) is the sequence of SEQ ID NO: 170 or an amino acid sequence having one or two amino acid differences from the sequence of SEQ ID NO: 170, and (iii) CDR3 (according to Abm) is the sequence of SEQ ID NO: 171 or an amino acid sequence having one or two amino acid differences from the sequence of SEQ ID NO: 171; and more preferably, (i) CDR1 (according to Abm) is the sequence of SEQ ID NO: 169, (ii) CDR2 (according to Abm) is the sequence of SEQ ID NO: 170, and (iii) CDR3 (according to Abm) is the sequence of SEQ ID NO: 178.

[0235] Furthermore, the VH domain of the present invention relative to Kv1.3 may be as described herein, and again in particular, an ISVD (and especially a nanobody) relative to Kv1.3, or a protein, polypeptide, or other compound or construct comprising at least one such ISVD. Such a protein, polypeptide, or other compound or construct may also be as described herein, and for example, may have an extended half-life (i.e., as described herein, for example, at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example, a half-life of at least 10 days in human subjects - expressed as t1 / 2 beta), and for this purpose may include, for example, a serum albumin-binding nanobody. The nanobody may also be the serum albumin-binding nanobody of the present invention (again, as described herein).

[0236] Furthermore, such ISVDs can appropriately have C-terminal elongation (as further described herein and in International Publication No. 12 / 175741) in particular when the ISVD forms the C-terminus of a protein, polypeptide, or other compound or construct containing it (as further described herein).

[0237] For example, but not limited to, the VH domain of the present invention for Kv1.3 may be one of the sequences listed in Table A-1 of U.S. Provisional Application No. 62 / 014,023 (Sequence IDs 1-123 in U.S. Provisional Application No. 62 / 014,023), or one of the sequences listed in Table A-1 of U.S. Provisional Application, Title of Invention "Kv1.3-conjugated immunoglobulin" (Assignee: Ablynx NV; Filing Date: March 16, 2015) (Sequence IDs 1-123, 495, 498-513, or 523-540, and in particular, Sequence ID 495 in the aforementioned U.S. Provisional Application), and may appropriately have the mutations / specific amino acid residues described herein for the ISVD of the present invention, and optionally appropriately have a C-terminal extension.

[0238] In one specific embodiment, the nanobody of the present invention relative to Kv1.3 is a variant of the nanobody of SEQ ID NO: 137 (having at least 90% sequence identity with respect to SEQ ID NO: 137), where, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, one of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v).

[0239] Some specific preferred and non-limiting examples of the nanobodies of the present invention against Kv1.3 and some compounds of the present invention containing them are listed in Figure 9A (monovalent nanobodies: SEQ ID NOs: 138-155) and Figure 9B (trivalent bispecific half-life extension constructs: SEQ ID NOs: 156-164). Compounds of the present invention containing, or essentially consisting of, at least one (e.g., one, two, or three) anti-Kv1.3 nanobodies selected from the anti-Kv1.3 nanobodies of SEQ ID NOs: 138-155 constitute further embodiments of the present invention. The compounds of SEQ ID NOs: 156-164 each constitute further embodiments of the present invention. In one specific embodiment, such a compound contains two such nanobodies of the present invention against Kv1.3 and one nanobody against human serum albumin (preferably also a nanobody of the present invention). Again, such a construct may also contain a suitable linker and C-terminal extension.

[0240] Monovalent anti-Kv1.3 nanobodies of sequence numbers 138-155 were generated by introducing the L11V and V89L mutations of the present invention into the start sequence of sequence number 137 (reference). In addition, various combinations of humanization (or other sequence optimization) mutations were introduced (e.g., E1D, A14P, G19R, M53A or M53Q or M53Y, T62S, A74S, K83R, S94G, and / or T97E). Specific mutations introduced into each of the sequences of sequence numbers 138-155 are shown in Figure 9A.

[0241] Furthermore, a portion of the monovalent anti-Kv1.3 nanobodies from Figure 9A were also formatted as trivalent bispecific constructs containing the present invention's nanobodies for two Kv1.3 molecules and the present invention's half-life extension nanobodies for one human serum albumin molecule (SEQ ID NO: 109, also referred to as "ALB-82" in Figure 9B). A 35GS linker was used. All constructs have a C-terminal extension (one C-terminal alanine residue). Sequences of the obtained constructs are provided as SEQ ID NOs: 156-164. Three of these constructs (SEQ ID NOs: 156, 157, and 160) were tested for binding with existing antibodies in samples obtained from 47 human subjects with diabetes and 90 healthy human subjects using the overall protocol described herein. Binding with existing antibodies from samples from these two sets was compared to the reference construct of SEQ ID NO: 165. The construct is a corresponding trivalent, bispecific construct based on the reference anti-Kv1.3 construct block of SEQ ID NO: 137 and serum albumin binder Alb-8 (SEQ ID NO: 46), again combined with C-terminal alanine elongation. The results are shown in Figure 10 (samples from 47 patients with diabetes) and Figure 11 (samples from 90 healthy volunteers). In each case, the constructs with the L11V and V89L mutations of the present invention showed reduced binding with existing antibodies compared to the reference construct.

[0242] Example 8: VH domain (and in particular nanobody) relative to IL-23 and compounds of the present invention containing the same In one specific embodiment, the VH domain of the present invention (and in particular the ISVD of the present invention, and especially the nanobody of the present invention) and the compounds of the present invention can be used with IL-23.

[0243] Such a VH domain of the present invention for IL-23 would generally comprise (i) a suitable framework sequence appropriately comprising the amino acid residues / mutations of the present invention as described herein, and (ii) a CDR sequence that enables the VH domain of the present invention to specifically bind to IL-23. In addition, such a VH domain of the present invention for IL-23 may also appropriately have the C-terminal extension described herein if the VH domain is monovalent or if it forms the C-terminus of the compound of the present invention in which the VH domain is present (again, as further described herein). Furthermore, such a VH domain of the present invention for IL-23 may be as further described herein, and in particular may be ISVD.

[0244] Again, as with other aspects and embodiments of the Invention described herein, where a specific ISVD (e.g., the ISVD for IL-23 described in this example) or compound containing the same is referred to as "the Invention" or "further described herein," the preferred aspects / embodiments and selections of the ISVD or compound of the Invention described herein as a whole also apply specifically to each of the aforementioned specific ISVDs or compounds, unless otherwise explicitly indicated or otherwise required by specific technical context.

[0245] Therefore, in certain embodiments, the present invention relates to a VH domain (and in particular, ISVD) for IL-23, where (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 such a VH domain for IL-23, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, one of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v).

[0246] Furthermore, the VH domain of the present invention relative to IL-23 may be as described herein, and again in particular, an ISVD (and especially a nanobody) relative to IL-23, or a protein, polypeptide, or other compound or construct comprising at least one such ISVD. Such a protein, polypeptide, or other compound or construct may also be as described herein, and for example, may have an extended half-life (i.e., as described herein, for example, at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example, a half-life of at least 10 days in human subjects - expressed as t1 / 2 beta), and for this purpose may include, for example, a serum albumin-binding nanobody. The nanobody may also be the serum albumin-binding nanobody of the present invention (again, as described herein).

[0247] Furthermore, such ISVDs can appropriately have C-terminal elongation (as further described herein and in International Publication No. 12 / 175741) in particular when the ISVD forms the C-terminus of a protein, polypeptide, or other compound or construct containing it (as further described herein).

[0248] In one preferred embodiment, the VH domain of the present invention relative to IL-23 includes (i) a CDR1 sequence which is the sequence of SEQ ID NO: 173 or an amino acid sequence having only one amino acid difference from the sequence of SEQ ID NO: 173 (the sequence of SEQ ID NO: 173 is preferred), (ii) a CDR2 sequence which is the sequence of SEQ ID NO: 174 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 174 (the sequence of SEQ ID NO: 174 is preferred), and (iii) a CDR3 sequence which is the sequence of SEQ ID NO: 175 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 175 (the sequence of SEQ ID NO: 175 is preferred).

[0249] More preferably, in the VH domain of the present invention for IL-23 in this embodiment, (i) CDR1 is sequence number: 173, (ii) CDR2 is sequence number: 174, and (iii) CDR3 is sequence number: 175.

[0250] In one specific embodiment, the nanobody of the present invention relative to IL-23 is a variant of the nanobody of SEQ ID NO: 172 (having at least 90% sequence identity, e.g., at least 95% sequence identity, relative to SEQ ID NO: 172), where, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, any of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v). The CDR of such an ISV is preferably as defined in the previous two paragraphs.

[0251] In another preferred embodiment, the VH domain of the present invention relative to IL-23 includes (i) a CDR1 sequence which is the sequence of SEQ ID NO: 191 or an amino acid sequence having only one amino acid difference from the sequence of SEQ ID NO: 191 (the sequence of SEQ ID NO: 191 is preferred), (ii) a CDR2 sequence which is the sequence of SEQ ID NO: 192 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 192 (the sequence of SEQ ID NO: 192 is preferred), and (iii) a CDR3 sequence which is the sequence of SEQ ID NO: 193 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 193 (the sequence of SEQ ID NO: 193 is preferred).

[0252] More preferably, in the VH domain of the present invention with respect to IL-23 in this embodiment, (i) CDR1 is sequence number: 191, (ii) CDR2 is sequence number: 192, and (iii) CDR3 is sequence number: 193.

[0253] In one specific embodiment, the nanobody of the present invention relative to IL-23 is a variant of the nanobody of SEQ ID NO: 172 (having at least 90% sequence identity, e.g., at least 95% sequence identity, relative to SEQ ID NO: 190), where, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, any of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v). Again, the CDR of such an ISV is preferably as defined in the previous two paragraphs.

[0254] Some specific, preferred, and non-limiting examples of such nanobodies of the present invention relative to IL-23 are listed in Figure 12A as Sequence IDs 176-189 and in Figure 12B as Sequence IDs 194-207, respectively. Each of these nanobodies constitutes a further aspect of the present invention.

[0255] Furthermore, the present invention relates to a compound for IL-23 comprising at least one (e.g., one, two, or three) nanobodies of the present invention of sequence numbers 176-189 and / or 194-207. Again, such a compound for IL-23 of the present invention may be as further described herein and for this purpose may, for example, include a suitable linker, may include the C-terminal extension described herein, and may extend the half-life (for example, since the compound of the present invention comprises a nanobody for human serum albumin, for example (preferably), the nanobody of the present invention for human serum albumin). See Table R below.

[0256] For example, as described in International Publication Nos. 2009 / 068627, 2010 / 142534, and 2011 / 135026, a particularly preferred class of nanobody compounds for IL-23 are biantigen-binding compounds. Therefore, in one aspect of the present invention, the compound for IL-23 is a biantigen-binding construct comprising one ISV of SEQ ID NO: 172 or (preferably) an ISV of the present invention obtained from SEQ ID NO: 172 (as described in Example 8), and one ISV of SEQ ID NO: 190 or (preferably) an ISV of the present invention obtained from SEQ ID NO: 190 (as described in Example 8), provided that at least one (and preferably both) of these ISVs are ISVs of the present invention. Furthermore, such a biantigen-binding construct can also have its half-life extended (i.e., by a serum albumin-binding ISV). Some specific examples of such biantigen-binding constructs are provided in Sequence IDs: 514-549.

[0257] Some specifically preferred examples of the compounds of the present invention for IL-23 are provided in Figure 22 as SEQ ID NOs: 514-549, each of which constitutes a further aspect of the present invention. Thus, in another aspect, the present invention relates to polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 514-549 for IL-23. More generally, the compounds of the present invention for IL-23 can be as described in International Publication Nos. 2009 / 068627, 2010 / 142534, and 2011 / 135026, and include the ISVs of the present invention. The compounds of the present invention can also be used for the purposes described in International Publication Nos. 2009 / 068627, 2010 / 142534, and 2011 / 135026.

[0258] [Table 26] TIFF0007881627000032.tif243168 TIFF0007881627000033.tif244168 TIFF0007881627000034.tif243168 TIFF0007881627000035.tif244170

[0259] Example 9: VH domain (and in particular nanobody) relative to OX40-L and the present invention compound containing the same In one specific embodiment, the VH domain of the present invention (and in particular the ISVD of the present invention, and especially the nanobody of the present invention) and the compound of the present invention can be used with OX40-L.

[0260] Such a VH domain of the present invention for OX40-L would generally comprise (i) a suitable framework sequence appropriately comprising the amino acid residues / mutations of the present invention as described herein, and (ii) a CDR sequence that enables the VH domain of the present invention to bind specifically to OX40-L. In addition, such a VH domain of the present invention for OX40-L may also appropriately have the C-terminal elongation described herein if the VH domain is monovalent or if it forms the C-terminus of the compound of the present invention (as again further described herein). Furthermore, such a VH domain of the present invention for OX40-L may be as further described herein, and in particular may be ISVD.

[0261] Again, as with other aspects and embodiments of the Invention described herein, where a specific ISVD (e.g., the ISVD for OX40-L described in this example) or compound containing the same is referred to as “the Invention” or “further described herein,” the preferred aspects / embodiments and selections of the ISVD or compound of the Invention described herein as a whole also apply specifically to each of the aforementioned specific ISVDs or compounds, unless otherwise explicitly indicated or otherwise required by specific technical context.

[0262] Therefore, in certain embodiments, the present invention relates to a VH domain (and in particular, ISVD) for OX40-L, where (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 such a VH domain for OX40-L, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, one of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v).

[0263] Furthermore, the VH domain of the present invention relative to OX40-L may be as described herein, and again in particular, an ISVD (and especially a nanobody) relative to OX40-L, or a protein, polypeptide, or other compound or construct comprising at least one such ISVD. Such a protein, polypeptide, or other compound or construct may also be as described herein, and for example, may have an extended half-life (i.e., as described herein, for example, at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example, a half-life of at least 10 days in human subjects - expressed as t1 / 2 beta), and for this purpose may include, for example, a serum albumin-binding nanobody. The nanobody may also be the serum albumin-binding nanobody of the present invention (again, as described herein).

[0264] Furthermore, such ISVDs can appropriately have C-terminal elongation (as further described herein and in International Publication No. 12 / 175741) in particular when the ISVD forms the C-terminus of a protein, polypeptide, or other compound or construct containing it (as further described herein).

[0265] In one preferred embodiment, the VH domain of the present invention relative to OX40-L includes (i) a CDR1 sequence which is the sequence of SEQ ID NO: 209 or an amino acid sequence having only one amino acid difference from the sequence of SEQ ID NO: 209 (the sequence of SEQ ID NO: 209 is preferred), (ii) a CDR2 sequence which is the sequence of SEQ ID NO: 210 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 210 (the sequence of SEQ ID NO: 210 is preferred), and (iii) a CDR3 sequence which is the sequence of SEQ ID NO: 211 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 211 (the sequence of SEQ ID NO: 211 is preferred).

[0266] More preferably, in the VH domain of the present invention with respect to OX40-L in this embodiment, (i) CDR1 is sequence number: 209, (ii) CDR2 is sequence number: 210, and (iii) CDR3 is sequence number: 211.

[0267] In one specific embodiment, the nanobody of the present invention relative to OX40-L is a variant of the nanobody of SEQ ID NO: 208 (having at least 90% sequence identity, e.g., at least 95% sequence identity, relative to SEQ ID NO: 208), where, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, any of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v). The CDR of such an ISV is preferably as defined in the previous two paragraphs.

[0268] Some specific, preferred, and non-limiting examples of the nanobodies of the present invention relative to OX40-L are listed in Figure 13 as Sequence IDs: 212-225. Each of these nanobodies constitutes a further embodiment of the present invention.

[0269] Furthermore, the present invention relates to a compound of the present invention for OX40-L comprising at least one (e.g., one, two, or three) nanobodies of the present invention of sequence numbers 212-225. Again, such a compound of the present invention for OX40-L may be as further described herein and for this purpose may, for example, include a suitable linker, may include the C-terminal extension described herein, and may extend the half-life (for example, since the compound of the present invention comprises a nanobodies for human serum albumin, for example (preferably), the nanobodies of the present invention for human serum albumin). See Table S below.

[0270] Some specifically preferred examples of the compounds of the present invention for OX40-L are provided in Figure 23 as SEQ ID NOs: 550-585. Each of these compounds constitutes a further aspect of the present invention. In other aspects, the present invention relates to polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 550-585 for OX40-L. More generally, the compounds of the present invention for OX40-L can be as described in International Publication No. 2011 / 073180 and include the ISV of the present invention. The compounds of the present invention can also be used for the purposes described in International Publication No. 2011 / 073180.

[0271] [Table 27] TIFF0007881627000037.tif250168 TIFF0007881627000038.tif249168 TIFF0007881627000039.tif249168 TIFF0007881627000040.tif245170

[0272] Example 10: VH domains (and in particular nanobodies) relative to IgE and compounds of the present invention containing the same In one specific embodiment, the VH domain of the present invention (and in particular the ISVD of the present invention, and especially the nanobody of the present invention) and the compounds of the present invention can target IgE.

[0273] Such a VH domain of the present invention for IgE would generally comprise (i) a suitable framework sequence appropriately comprising the amino acid residues / mutations of the present invention as described herein, and (ii) a CDR sequence that enables the VH domain of the present invention to specifically bind to IgE. In addition, such a VH domain of the present invention for IgE may, in particular, be monovalent, or may appropriately have the C-terminal extension described herein if the VH domain forms the C-terminus of the compound of the present invention (as again further described herein). Furthermore, such a VH domain of the present invention for IgE may be as further described herein, and in particular may be ISVD.

[0274] Again, as with other aspects and embodiments of the Invention described herein, where a specific ISVD (e.g., an ISVD for IgE described in this example) or a compound containing the same is referred to as “the Invention” or “further described herein,” the preferred aspects / embodiments and selections of the ISVD or compound of the Invention described herein as a whole also apply specifically to each of the said specific ISVD or compound, unless otherwise explicitly indicated or otherwise required by specific technical context.

[0275] Therefore, in certain embodiments, the present invention relates to a VH domain (and in particular, ISVD) for IgE, where (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 such a VH domain for IgE, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, one of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v).

[0276] Furthermore, the VH domain of the present invention with respect to IgE may be as described herein, and again in particular, an ISVD (and especially a nanobody) with respect to IgE, or a protein, polypeptide, or other compound or construct comprising at least one such ISVD. Such a protein, polypeptide, or other compound or construct may also be as described herein, and for example, may have an extended half-life (i.e., as described herein, for example, at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example, a half-life of at least 10 days in human subjects - expressed as t1 / 2 beta), and for this purpose may include, for example, a serum albumin-binding nanobody. The nanobody may also be the serum albumin-binding nanobody of the present invention (again, as described herein).

[0277] Furthermore, such ISVDs can appropriately have C-terminal elongation (as further described herein and in International Publication No. 12 / 175741) in particular when the ISVD forms the C-terminus of a protein, polypeptide, or other compound or construct containing it (as further described herein).

[0278] In one preferred embodiment, the VH domain of the present invention relative to IgE includes (i) a CDR1 sequence which is the sequence of SEQ ID NO: 227 or an amino acid sequence having only one amino acid difference from the sequence of SEQ ID NO: 227 (the sequence of SEQ ID NO: 227 is preferred), (ii) a CDR2 sequence which is the sequence of SEQ ID NO: 228 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 228 (the sequence of SEQ ID NO: 228 is preferred), and (iii) a CDR3 sequence which is the sequence of SEQ ID NO: 229 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 229 (the sequence of SEQ ID NO: 229 is preferred).

[0279] More preferably, in the VH domain of the present invention for IgE in this embodiment, (i) CDR1 is sequence number: 227, (ii) CDR2 is sequence number: 228, and (iii) CDR3 is sequence number: 229.

[0280] In one specific embodiment, the nanobody of the present invention with respect to IgE is a variant of the nanobody of SEQ ID NO: 226 (having at least 90% sequence identity, e.g., at least 95% sequence identity with respect to SEQ ID NO: 226), where, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, any of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v). The CDR of such an ISV is preferably as defined in the previous two paragraphs.

[0281] Some specific, preferred, and non-limiting examples of the nanobodies of the present invention in relation to IgE are listed in Figure 14 as Sequence IDs: 230-243. Each of these nanobodies constitutes a further embodiment of the present invention.

[0282] Furthermore, the present invention relates to a compound for IgE comprising at least one (e.g., one, two, or three) nanobodies of the present invention of sequence numbers 230-243. Again, such a compound for IgE of the present invention may be as further described herein and for this purpose may, for example, include a suitable linker, may include the C-terminal extension described herein, and may extend the half-life (for example, since the compound of the present invention comprises a nanobody for human serum albumin, for example (preferably), the nanobody of the present invention for human serum albumin). See Table T below.

[0283] Some specific preferred examples of the compounds of the present invention for IgE are provided in Figure 24 as SEQ ID NOs: 586-594. Each of these compounds constitutes a further aspect of the present invention. In another aspect, the present invention relates to polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 586-594 for IgE.

[0284] More generally, the compounds of the present invention against IgE may be as described in the relevant parts of International Publication Nos. 2012 / 175740 and 2012 / 175400, and include the ISVs of the present invention. Furthermore, the compounds of the present invention may also be used for the purposes described in International Publication No. 2012 / 175740.

[0285] [Table 28] TIFF0007881627000042.tif245168 TIFF0007881627000043.tif245168 TIFF0007881627000044.tif245168 TIFF0007881627000045.tif241170

[0286] Example 11: VH domain (and in particular nanobody) relative to CXCR-4 and the present invention compound containing the same In one specific embodiment, the VH domain of the present invention (and in particular the ISVD of the present invention, and especially the nanobody of the present invention) and the compounds of the present invention can be used with CXCR-4.

[0287] Such a VH domain of the present invention for CXCR-4 would generally comprise (i) a suitable framework sequence appropriately comprising the amino acid residues / mutations of the present invention as described herein, and (ii) a CDR sequence that enables the VH domain of the present invention to specifically bind to CXCR-4. In addition, such a VH domain of the present invention for CXCR-4 may also appropriately have the C-terminal extension described herein if the VH domain is monovalent or if the VH domain forms the C-terminus of the compound of the present invention (as again further described herein). Furthermore, such a VH domain of the present invention for CXCR-4 may be as further described herein, and in particular may be ISVD.

[0288] Again, as with other aspects and embodiments of the Invention described herein, where a specific ISVD (e.g., the ISVD for CXCR-4 described in this example) or a compound containing the same is referred to as “the Invention” or “further described herein,” the preferred aspects / embodiments and selections of the ISVD or compound of the Invention described herein as a whole also apply specifically to each of the said specific ISVD or compound, unless otherwise explicitly indicated or otherwise required by specific technical context.

[0289] Therefore, in certain embodiments, the present invention relates to a VH domain (and in particular, an ISVD) for CXCR-4, where (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 such a VH domain for CXCR-4, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, one of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v).

[0290] Furthermore, the VH domain of the present invention relative to CXCR-4 may be as described herein, and again in particular, an ISVD (and especially a nanobody) relative to CXCR-4, or a protein, polypeptide, or other compound or construct comprising at least one such ISVD. Such a protein, polypeptide, or other compound or construct may also be as described herein, and for example, may have an extended half-life (i.e., as described herein, for example, at least 1 day, preferably at least 3 days, more preferably at least 7 days, for example, a half-life of at least 10 days in human subjects - expressed as t1 / 2 beta), and for this purpose may include, for example, a serum albumin-binding nanobody. The nanobody may also be the serum albumin-binding nanobody of the present invention (again, as described herein).

[0291] Furthermore, such ISVDs can appropriately have C-terminal elongation (as further described herein and in International Publication No. 12 / 175741) in particular when the ISVD forms the C-terminus of a protein, polypeptide, or other compound or construct containing it (as further described herein).

[0292] In a preferred embodiment, the VH domain of the present invention relative to CXCR-4 includes (i) a CDR1 sequence which is the sequence of SEQ ID NO: 245 or an amino acid sequence having only one amino acid difference from the sequence of SEQ ID NO: 245 (the sequence of SEQ ID NO: 245 is preferred), (ii) a CDR2 sequence which is the sequence of SEQ ID NO: 246 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 246 (the sequence of SEQ ID NO: 246 is preferred), and (iii) a CDR3 sequence which is the sequence of SEQ ID NO: 247 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 247 (the sequence of SEQ ID NO: 247 is preferred).

[0293] More preferably, in the VH domain of the present invention for CXCR-4 in this embodiment, (i) CDR1 is sequence number: 245, (ii) CDR2 is sequence number: 246, and (iii) CDR3 is sequence number: 247.

[0294] In one specific embodiment, the nanobody of the present invention relative to CXCR-4 is a variant of the nanobody of SEQ ID NO: 244 (having at least 90% sequence identity, e.g., at least 95% sequence identity, relative to SEQ ID NO: 244), where, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, any of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v). The CDR of such an ISV is preferably as defined in the previous two paragraphs.

[0295] In another preferred embodiment, the VH domain of the present invention relative to CXCR-4 includes (i) a CDR1 sequence which is the sequence of SEQ ID NO: 263 or an amino acid sequence having only one amino acid difference from the sequence of SEQ ID NO: 263 (the sequence of SEQ ID NO: 263 is preferred), (ii) a CDR2 sequence which is the sequence of SEQ ID NO: 264 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 264 (the sequence of SEQ ID NO: 264 is preferred), and (iii) a CDR3 sequence which is the sequence of SEQ ID NO: 265 or an amino acid sequence having only one or two amino acid differences from the sequence of SEQ ID NO: 265 (the sequence of SEQ ID NO: 265 is preferred).

[0296] More preferably, in the VH domain of the present invention for CXCR-4 in this embodiment, (i) CDR1 is sequence number: 263, (ii) CDR2 is sequence number: 264, and (iii) CDR3 is sequence number: 265.

[0297] In one specific embodiment, the nanobody of the present invention relative to IL-23 is a variant of the nanobody of SEQ ID NO: 262 (having at least 90% sequence identity, e.g., at least 95% sequence identity, relative to SEQ ID NO: 262), where, The amino acid residue at position -11 is preferably selected from L, V, or K (and most preferably V), and The amino acid residue at position -14 is preferably appropriately selected from A or P, and The amino acid residue at position -41 is preferably appropriately selected from A or P, and The amino acid residue at position -89 is preferably appropriately selected from T, V, or L, and The amino acid residue at position -108 is preferably appropriately selected from Q or L, and The amino acid residue at position -110 is preferably appropriately selected from T, K, or Q, and The amino acid residue at position -112 is preferably appropriately selected from S, K, or Q. Therefore, any of the following is possible: (i) the 112th position is K or Q, or (ii) the 110th position is K or Q and the 11th position is V, or (iii) the 89th position is T, or (iv) the 89th position is L and the 110th position is K or Q, or (v) the 11th position is V and the 89th position is L, or any appropriate combination of (i) to (v). Again, the CDR of such an ISV is preferably as defined in the previous two paragraphs.

[0298] Some specific, preferred, and non-limiting examples of the nanobodies of the present invention relative to CXCR-4 are listed in Figure 15A as Sequence IDs 248-261 and in Figure 15B as Sequence IDs 266-279, respectively. Each of these nanobodies constitutes a further embodiment of the present invention.

[0299] Furthermore, the present invention relates to a compound for CXCR-4 comprising at least one (e.g., one, two, or three) nanobodies of the present invention of sequence numbers 248-261 and / or 266-279. Again, such a compound for CXCR-4 of the present invention may be as further described herein and for this purpose may, for example, include a suitable linker, may include the C-terminal extension described herein, and may extend the half-life (for example, since the compound of the present invention comprises a nanobodies for human serum albumin, for example (preferably), the nanobodies of the present invention for human serum albumin). See Table U below.

[0300] For example, as described in International Publication Nos. 2009 / 138519, 2011 / 042398, and 2011 / 161266, a particularly preferred class of nanobody compounds for CXCR-4 are biantigen-binding compounds. Therefore, in one aspect of the present invention, the compound for CXCR-4 is a biantigen-binding construct comprising one ISV of SEQ ID NO: 244 or (preferably) an ISV of the present invention obtained from SEQ ID NO: 244 (as described in this Example 11), and one ISV of SEQ ID NO: 262 or (preferably) an ISV of the present invention obtained from SEQ ID NO: 262 (as described in this Example 11), provided that at least one (and preferably both) of these ISVs are ISVs of the present invention. Furthermore, such a biantigen-binding construct can also have an extended half-life (i.e., by a serum albumin-binding ISV). Some specific examples of such biantigen-binding constructs are provided in Sequence IDs: 595-603.

[0301] Some specifically preferred examples of the compounds of the present invention for CXCR-4 are provided in Figure 25 as SEQ ID NOs: 595-603, each of which constitutes a further aspect of the present invention. Thus, in another aspect, the present invention relates to polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 595-603 for CXCR-4. More generally, the compounds of the present invention for CXCR-4 can be as described in International Publication Nos. 2009 / 138519, 2011 / 042398, 2011 / 161266, and 2011 / 144749, and include the ISVs of the present invention. The compounds of the present invention can also be used for the purposes described in International Publication Nos. 2009 / 138519, 2011 / 042398, and 2011 / 161266.

[0302] [Table 29] TIFF0007881627000047.tif242168 TIFF0007881627000048.tif241170 TIFF0007881627000049.tif240168 TIFF0007881627000050.tif246170

[0303] Example 12: VH domain (and in particular nanobody) relative to HER-3 and compounds of the present invention containing the same In one specific embodiment, the VH domain of the present invention (and in particular the ISVD of the present invention, and especially the nanobody of the present invention) and the compounds of the present invention can be used against HER-3.

[0304] Such a VH domain of the present invention for HER-3 would generally comprise (i) a suitable framework sequence appropriately comprising the amino acid residues / mutations of the present invention as described herein, and (ii) a CDR sequence that enables the VH domain of the present invention to bind specifically to HER-3. In addition, such a VH domain of the present invention for HER-3 may also appropriately have the C-terminal extension described herein if the VH domain is monovalent or if it forms the C-terminus of the compound of the present invention in which the VH domain is present (again, as further described herein). Furthermore, such a VH domain of the present invention for HER-3 may be as further described herein, and in particular may be ISVD.

[0305] Again, as with other aspects and embodiments of the Invention described herein, where a specific ISVD (e.g., the ISVD for HER-3 described in this example) or a compound containing the same is referred to as "the Invention" or "further described herein," the preferred aspects / embodiments and selections of the ISVD or compound of the Invention described herein as a whole also apply specifically to each of the said specific ISVD or compound, unless otherwise explicitly indicated or otherwise required by specific technical context.

[0306] Therefore, in certain embodiments, the present invention relates to a VH domain (and in particular an ISVD) for HER-3, where (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 such a VH domain for HER-3, The amino acid residue at position -11 is preferably selected from L, ...

Claims

1. A heavy chain immunoglobulin single variable domain (ISVD) which is a VHH domain, a humanized VHH domain, or a camelized VH domain, wherein the amino acid residue at position 89 is T, and the amino acid residue / position is indicated by numbering according to Kabat.

2. The heavy chain ISVD according to claim 1, comprising an amino acid residue at position 11 selected from L, E, K, V, and Y.

3. The heavy chain ISVD according to claim 1 or 2, wherein the amino acid residue at position 89 is T, and the amino acid residue at position 11 is V.

4. The heavy chain ISVD according to claim 1 or 2, wherein the amino acid residue at position 89 is T, and the amino acid residue at position 11 is E.

5. (i) containing an amino acid residue at position 110 selected from T, K, and Q; and (ii) containing an amino acid residue at position 112 selected from S, K, and Q, according to any one of claims 1 to 4.

6. (i) containing an amino acid residue at position 110 which is T; and (ii) containing an amino acid residue at position 112 which is S, according to claim 5.

7. (i) containing an amino acid residue at position 110 selected from K and Q; and (ii) containing an amino acid residue at position 112 which is S, according to claim 5.

8. (i) containing an amino acid residue at position 110 which is T; and (ii) containing an amino acid residue at position 112 selected from K and Q, according to claim 5.

9. The heavy chain ISVD according to any one of claims 1 to 8, further comprising the Q108L substitution.

10. The heavy chain ISVD according to any one of claims 1 to 9, wherein the heavy chain ISVD contains C-terminal extension (X)n [during the extension, (i)n is 1 to 10; and (ii) each X is an independently selected amino acid residue].

11. The heavy chain ISVD according to claim 10, comprising C-terminal extension (X)n [wherein n is 1 to 5].

12. The heavy chain ISVD according to claim 11, comprising C-terminal extension (X)n [wherein n is 1, 2, 3, 4, or 5].

13. The heavy chain ISVD according to claim 11, comprising C-terminal extension (X)n [wherein n is 1].

14. The heavy chain ISVD according to claim 11, comprising C-terminal extension (X)n [wherein n is 2].

15. A heavy chain ISVD according to any one of claims 10 to 14, comprising C-terminal extension (X)n [wherein each X is an independently selected native amino acid residue].

16. The heavy chain ISVD according to claim 15, comprising C-terminal extension (X)n [during the extension, each X is a naturally occurring amino acid residue independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)].

17. The C-terminus of the heavy chain ISVD is 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: 96) The heavy chain ISVD according to any one of claims 1 to 16, wherein the sequence is one of (Sequence No.: 98), VZVZS (Sequence No.: 107, in which each amino acid residue Z is independently K or Q), and VZVZS(X)n (Sequence No.: 108, in which each amino acid residue Z is independently K or Q), where n is 1 to 10; and each X is an independently selected amino acid residue.

18. The heavy chain ISVD according to claim 17, comprising C-terminal extension (X)n [wherein n is 1 to 5].

19. The heavy chain ISVD according to claim 18, comprising C-terminal extension (X)n [wherein n is 1, 2, 3, 4, or 5].

20. The heavy chain ISVD according to claim 18, comprising C-terminal extension (X)n [wherein n is 1].

21. The heavy chain ISVD according to claim 18, comprising C-terminal extension (X)n [wherein n is 2].

22. A heavy chain ISVD according to any one of claims 17 to 21, comprising C-terminal extension (X)n [wherein each X is an independently selected native amino acid residue].

23. The heavy chain ISVD according to claim 22, comprising C-terminal extension (X)n [during the extension, each X is a naturally occurring amino acid residue independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)].

24. The heavy chain ISVD according to any one of claims 17 to 23, wherein the C-terminus of the heavy chain ISVD is one of VTVSS (SEQ ID NO: 76), VKVSS (SEQ ID NO: 95), VQVSS (SEQ ID NO: 96), VTVSS(X)n (SEQ ID NO: 77), VKVSS(X)n (SEQ ID NO: 97), and VQVSS(X)n (SEQ ID NO: 98), where X and n are as defined in claim 17.

25. The heavy chain ISVD according to any one of claims 17 to 24, wherein the C-terminus of the heavy chain ISVD is one of VTVSS (SEQ ID NO: 76) and VTVSS(X)n (SEQ ID NO: 77), where X and n are as defined in claim 17.

26. A heavy chain immunoglobulin single variable domain (ISVD) which is a VHH domain, a humanized VHH domain, or a camelized VH domain, The amino acid residue at position 11 is V; and The amino acid residue at position 14 is either A or P; and The amino acid residue at position 41 is either A or P; and The amino acid residue at position 89 is T; and The amino acid residue at position 108 is either Q or L; and The amino acid residue at position 110 is one of T, K, and Q; and The amino acid residue at position 112 is one of S, K, and Q. Here, amino acid residues / positions are indicated by numbering according to Kabat, in the heavy chain ISVD.

27. The heavy chain ISVD according to claim 26, wherein the amino acid residue at position 110 is T, and the amino acid residue at position 112 is S.

28. The heavy chain ISVD according to claim 26, wherein the amino acid residue at position 110 is K or Q, and the amino acid residue at position 112 is S.

29. The heavy chain ISVD according to claim 26, wherein the amino acid residue at position 110 is T, and the amino acid residue at position 112 is K or Q.

30. The heavy chain ISVD according to any one of claims 26 to 29, wherein the heavy chain ISVD contains a C-terminal extension (X)n [wherein n is 1 to 10, and each X is an independently selected amino acid residue].

31. The heavy chain ISVD according to claim 30, comprising C-terminal extension (X)n [wherein n is 1 to 5].

32. The heavy chain ISVD according to claim 31, comprising C-terminal extension (X)n [wherein n is 1, 2, 3, 4, or 5].

33. The heavy chain ISVD according to claim 31, comprising C-terminal extension (X)n [wherein n is 1].

34. The heavy chain ISVD according to claim 31, comprising C-terminal extension (X)n [wherein n is 2].

35. A heavy chain ISVD according to any one of claims 30 to 34, comprising C-terminal extension (X)n [wherein each X is an independently selected native amino acid residue].

36. The heavy chain ISVD according to claim 35, comprising C-terminal extension (X)n [during the extension, each X is a naturally occurring amino acid residue independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I)].

37. A heavy chain ISVD according to any one of claims 1 to 36, for Kv1.3, IL-23, OX40L, IgE, CXCR4, HER3, TNF, c-Met, RANKL, CXCR7, or A-beta.

38. A heavy chain ISVD according to any one of claims 1 to 36, which can specifically bind to human serum albumin.

39. CDR1 has the amino acid sequence SFGMS (SEQ ID NO: 41); CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 42); CDR3 has the amino acid sequence GGSLSR (SEQ ID NO: 43). The heavy chain ISVD according to claim 38.

40. The heavy chain ISVD according to claim 39, having at least 90% sequence identity with respect to at least one of Alb-1 (sequence number: 767), Alb-8 (sequence number: 46), and Alb-23 (sequence number: 61).

41. The heavy chain ISVD according to claim 39, having at least 95% sequence identity with respect to at least one of Alb-1 (sequence number: 767), Alb-8 (sequence number: 46), and Alb-23 (sequence number: 61).

42. A heavy chain ISVD according to any one of claims 38 to 41, comprising or consisting of one of sequence numbers 78 to 91.

43. A library of heavy-chain ISVDs according to any one of claims 1 to 36.

44. A library of nucleic acids encoding heavy chain ISVD according to any one of claims 1 to 36.

45. The library according to claim 44, which is an expression library.

46. A synthetic library, as described in any one of claims 43 to 45.

47. A library according to any one of claims 43 to 46, comprising at least 100 different sequences.

48. 10 ６ The library according to claim 47, which contains sequences of more than one type.

49. A polypeptide, construct, or compound comprising a heavy chain ISVD according to at least one of claims 1 to 42.

50. A nucleic acid encoding a heavy chain ISVD according to any one of claims 1 to 42, or a polypeptide, construct, or compound according to claim 49.

51. A method for producing a heavy chain ISVD according to any one of claims 1 to 42 or a polypeptide, construct, or compound according to claim 49, comprising expressing the nucleic acid according to claim 50 in a suitable host organism other than a human.

52. A composition comprising a heavy chain ISVD according to any one of claims 1 to 42, a polypeptide, construct, or compound according to claim 49, or a nucleic acid according to claim 50.

53. The composition according to claim 52, which is a pharmaceutical composition.

54. A heavy chain immunoglobulin single variable domain according to any one of claims 1 to 42, or a polypeptide, construct, or compound according to claim 49, for use in pharmaceuticals.

Images