Serum Albumin Binding Proteins

Abstract

The present invention relates to amino acid sequences that are capable of binding to serum albumin; to proteins and polypeptides comprising or essentially consisting of such amino acid sequences; to nucleic acids that encode such amino acid sequences, proteins or polypeptides; to compositions, and in particular pharmaceutical compositions, that comprise such amino acid sequences, proteins and polypeptides; and to uses of such amino acid sequences, proteins and polypeptides.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. provisional application Ser. No. 14/128,719, filed Mar. 19, 2014, which is a national stage filing under 35 U.S.C. .sctn.371 of international application PCT/EP2012/061304, filed Jun. 14, 2012, which was published under PCT Article 21(2) in English, and claims the benefit under 35 U.S.C. .sctn.119(e) of U.S. provisional application Ser. No. 61/500,464, filed Jun. 23, 2011, the disclosures of which are incorporated by reference herein in their entireties.

[0002] The present invention relates to amino acid sequences that are capable of binding to serum albumin; to proteins and polypeptides comprising or essentially consisting of such amino acid sequences; to nucleic acids that encode such amino acid sequences, proteins or polypeptides; to compositions, and in particular pharmaceutical compositions, that comprise such amino acid sequences, proteins and polypeptides; and to uses of such amino acid sequences, proteins and polypeptides.

[0003] Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

[0004] If any terms are not specifically defined herein, these terms have the meaning given to them in WO 2009/068627 or WO 06/122787. If any terms used herein are not specifically defined herein or in WO 2009/068627/WO 06/122787, then they have their usual meaning in the art, for which reference is for example made to the standard handbooks.

[0005] Amino acid sequences that are capable of binding to human serum albumin and uses thereof in polypeptide constructs in order to increase the half-life of therapeutically relevant proteins and polypeptides are known in the art.

[0006] For example, WO 04/041865 by applicant describes Nanobodies directed against serum albumin (and in particular against human serum albumin) that can be linked to other proteins (such as one or more other Nanobodies directed against a desired target) in order to increase the half-life of said protein.

[0007] The international application WO 06/122787 describes a number of Nanobodies against (human) serum albumin. These Nanobodies include the Nanobody called Alb-1 (SEQ ID NO: 52 in WO 06/122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 06/122787). [Nanobody.RTM. and Nanobodies.RTM. are trademarks of Ablynx N.V.]. Again, these can be used to extend the half-life of therapeutic proteins and polypeptide and other therapeutic entities or moieties.

[0008] As of the date of first filing of the present application, the use of Nanobodies against (human) serum albumin for extending the half-life of therapeutic moieties such as Nanobodies has been validated by means of clinical trials. For example, the safety, tolerability, immunogenicity and pharmacokinetics (PK) of ALX-0141, a protein construct that comprises two Nanobodies against RANK-L and one Nanobody against human serum albumin, has been confirmed in phase I clinical trials (data presented by Ablynx N.V. on May 27, 2011 at the Annual European Congress of Rheumatology (EULAR) in London). Also, numerous published patent applications of Ablynx N.V. give examples of constructs with increased half-life that comprise one or more Nanobodies against a therapeutic target and one or more Nanobodies against serum albumin (such as Alb-8). Reference is for example made to WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164, WO 2009/068627 and WO 2009/147248.

[0009] Although it has been established that the use of Nanobodies against (human) serum albumin (such as those described in WO 04/041865 and WO 06/122787, and in particular the humanized variants of Alb-1 described in WO 06/122787) provide a good and broadly applicable methodology for extending the half-life of Nanobodies and of other therapeutic moieties and entities, this does not mean that the skilled person would not benefit from having further improved Nanobodies against human serum albumin at his disposal for this purpose.

[0010] The present invention provides such an improved Nanobody that is directed against human serum albumin (called "Alb-23"; see SEQ ID NO:1), as well as a number of variants of this Nanobody (also referred to herein as "Alb-23 like sequences" or "Alb-23 variants", see SEQ ID NO's 3 to 11 for some non-limiting examples), as well as as compounds, polypeptides and other (protein) constructs comprising the same (as further described herein).

[0011] The advantages that the improved Nanobody of SEQ ID NO:1 and its variants can provide over the Nanobodies described in WO 04/041865 and WO 06/122787 will become clear from the further description herein. For example and without limitation, these advantages may include [0012] improved stability (such as improved thermal stability as determined by measuring the Tm); and/or [0013] and/or improved storage stability, as for example measured in the SEC experiment described in Example 5); and/or [0014] a reduced tendency to form dimers under certain formulation conditions (for example, at high concentrations in certain aqueous formulation buffers--see again for example Example 5).

[0015] In addition, it has been found that the improved Nanobody of SEQ ID NO:1 and its variants are particularly suited for extending the half-life of immunoglobulin single variable domains that contain more than one disulphide bridge, such as VHH's and Nanobodies belonging to the "VHH-1 class" (which as further described herein may comprise two or even three disulphide bridges). For example and without limitation, it has been found that polypeptides that comprise one or more therapeutic VHH's/Nanobodies of the VHH-1 class and the Nanobody Alb-23 (or an Alb-23 variant) may have better expression levels in certain hosts or host cells (and/or other advantageous properties when it comes to expression, purification and/or production/manufacture generally) than corresponding polypeptides that contain a serum albumin-binding Nanobody according to WO 06/122787 such as "Alb-8" (SEQ ID NO:62 in WO 06/122787) instead of the amino acid sequence of SEQ ID NO:1. Again, this will become clear from the further description and the Experimental Part herein.

[0016] These and other advantages, as well as the various aspects, embodiments, uses and applications of the invention, will become clear from the further description herein.

[0017] It is known that immunoglobulin single variable domains such as Nanobodies, VHH's, (single) domain antibodies, dAbs, IgNAR domains and microbodies (as for example described in WO 00/29004) can be expressed in a number of host cells and host organisms, such as bacterial cells such as E. coli, yeast strains such as Pichia and Saccharomyces, and various mammalian cells or cell lines. Reference is for example made to EP 0 656 946 and EP 0 698 097, as well as the various published patent applications from Ablynx N.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164, WO 2009/068627 or WO 2009/147248.

[0018] Similarly, it is known that when one or more immunoglobulin single variable domains that are directed against a therapeutic target are linked to a Nanobody that is directed to (human) serum albumin (such as those described in WO 04/041865 and WO 06/122787) in order to provide a construct that has increased half-life (i.e., compared to the therapeutic domain(s) alone), that the resulting polypeptides and constructs can also be expressed in a number of host cells and host organisms, such as bacterial cells such as E. coli, yeast strains such as Pichia and Saccharomyces, and various mammalian cells or cell lines. Reference is again made to WO 04/041865, WO 06/122787, to WO 2010/056550 and to the various published patent applications from Ablynx N.V. mentioned herein.

[0019] It is also generally known all VHH's and Nanobodies contain at least one disulphide bridge, between the cysteine residue at position 22 and the cysteine residue at position 92 (numbering according to Kabat, see the patent applications of Ablynx N.V.).

[0020] Although most VHH's contain only this single disulphide bridge, it is also known that some VHH's contain a total of two (or in exceptional cases three) disulphide bridges. For example, a class of VHH's and Nanobodies referred to as the "VHH-1 type" or "VHH-1 class" commonly has a second disulphide bridge between a cysteine residue at position 50 (the first amino acid residue of CDR2) and a cysteine residue present in CDR3 (such VHH's and Nanobodies also often have the sequence motif EREG at positions 44 to 47). Also, some VHH's derived from camels sometimes have a disulphide bridges between a cysteine residue present in CDR1 and a cysteine residue present in CDR3.

[0021] Some non-limiting examples of such VHH-1 type Nanobodies (given as illustration only; other VHH-1 type sequences against other targets can be found in some of the other patent applications from Ablynx N.V.) are the sequences P23ILPMP37D5 (SEQ ID NO: 2490) and P12ILPMP80F10 (SEQ ID NO: 1954) from WO 2009/068627; the sequences PMP30A2 (SEQ ID NO:419), PMP31C5 (SEQ ID NO: 413) and PMP30G11 (SEQ ID NO:416) from WO 2008/020079; and the sequences RSVPMP5A2 (SEQ ID NO: 262), RSVPMP5B2 (SEQ ID NO: 263) and RSVPMP5C3 (SEQ ID NO: 264) from WO 2009/147248.

[0022] The non-prepublished U.S. applications 61/388,172 (filed Sep. 30, 2010 and entitled "Biological materials related to c-Met") and U.S. 61/451,869 (filed Mar. 11, 2011 and entitled "Biological materials related to c-Met"), both assigned to Ablynx N.V., describe VHH's and Nanobodies that are directed against the therapeutic target c-Met; and some of these VHH's and Nanobodies (such as 4E09, SEQ ID NO:26 in U.S. 61/451,869 and SEQ ID NO: 12 herein, and some of the variants of 4E09 described in in U.S. 61/451,869) also belong to the VHH-1 class.

[0023] U.S. 61/388,172 and U.S. 61/451,869 also describe that such VHH-1 type Nanobodies can be linked to a Nanobody against human serum albumin in order increase its half-life (the Nanobody against human serum albumin used in U.S. 61/388,172 and U.S. 61/451,869 is called "Alb-11" and is given in SEQ ID NO: 5 in U.S. 61/451,869. Alb-11 has the same amino acid sequence as Alb-8, which is SEQ ID NO: 62 in WO 06/122787). An example of such a polypeptide comprising Alb-11 and 4E09 is given in SEQ ID NO: 9 of U.S. 61/451,869.

[0024] Although VHH's/Nanobodies of the VHH-1 type (or more generally, VHH's/Nanobodies that contain two or three disulphide bridges), as well as polypeptides and other protein constructs comprising the same (including such polypeptides that further comprise at least one Nanobody against serum albumin in order to provide increased half-life), can be expressed in any suitable host or host organism, it has been found that the expression levels that are obtained for such VHH, Nanobodies, polypeptides or constructs may be significantly low(er) than the expression levels that are obtained for similar/comparable VHH's, Nanobodies, polypeptides or constructs that contain VHH's or Nanobodies that do not belong to the VHH-1 class (or more generally, that contain VHH's/Nanobodies with only one disulphide bridge). For example and without limitation, whereas expression levels of more than 0.8 g/l or more (such as 1 g/l or more) can routinely be achieved in Pichia for polypeptides that comprise Alb-8 and one or more therapeutic Nanobodies that are not of the VHH-1 type (see again some of the published patent applications of Ablynx N.V.), it has been found that it is difficult to achieve expression levels of more than 0.5 g/l for polypeptides that comprise the anti-c-Met VHH-1 type Nanobody 4E09 (SEQ ID NO:26 in U.S. 61/451,869 and SEQ ID NO: 12 herein) or a humanized variant thereof and Alb-8 (see the Experimental Section below). Surprisingly, it has now been found that when the Nanobody of SEQ ID NO:1 is used in such polypeptides (i.e., instead of Alb-8), that significantly higher expression levels can be obtained (see the Experimental Section below).

[0025] As mentioned, in a first aspect, the invention relates to an amino acid sequence that is directed against (human) serum albumin, which essentially consists of or is the following amino acid sequence:

TABLE-US-00001 [SEQ ID NO: 1] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSS

[0026] This amino acid sequence is also referred to herein as "Alb-23" or the "amino acid sequence of the invention". Alb-23 is a humanized version of the amino acid sequence "Alb-1" (SEQ ID NO: 52 from WO 06/122787, and also referred to in WO 06/122787 as "PMP6A6").

[0027] For the sake of convenience, FIG. 1 gives an alignment of Alb-23 with Alb-1 and the humanized versions of Alb-1 disclosed in Table III of WO 06/122787 (called "Alb-3" to "Alb-10" in WO 06/122787, see also SEQ ID NO's: 57 to 64 of WO 06/122787). The amino acid sequence of Alb-1 is:

TABLE-US-00002 [SEQ ID NO: 2] AVQLVESGGGLVQPGNSLRLSCAAGFTFRSFGMSWVRQAPGKEPEWVSSI SGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGS LSRSSQGTQVTVSS

[0028] In the above sequence, the main differences between Alb1 and Alb-23 have been indicated in bold and underlined. These are (with the numbering according to Kabat, see for example Tables A-5 to A-8 of WO 2008/020079; and with each letter denominating an amino acid residue in accordance with the standard one-letter amino acid code, for which reference is made to Table A-2 of WO 2008/020079): position 5: V to L; position 16: N to G; positions 44 and 45: EP to GP; positions 74 to 76: AKT to SKN; position 83: K to R.

[0029] Thus, in a further aspect, the invention relates to an amino acid sequence that is a variant of the sequence Alb-1 (SEQ ID NO:2), which variant comprises:

(i) the amino acid motif GP on positions 44 and 45; (ii) the amino acid motif SKN on positions 74 to 76; (iii) a CDR1 that is the amino acid sequence SFGMS (SEQ ID NO:29); (iv) a CDR2 that is the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO:30); (v) a CDR3 that is the amino acid sequence GGSLSR (SEQ ID NO:31); and that preferably also comprises (vi) a G at position 16; and in which preferably (but without limitation): (vii) position 83 is an R (but may optionally also be a K; and that further comprises (i.e., in addition to the aforementioned amino acid differences at positions 16, 44 and 45, 74 to 76 and 83, with the amino acid differences at positions 16 and 83 being optional but preferred) between 1 and 7, such as between 1 and 5 further "amino acid differences" (as defined in WO 2008/020079) with the sequence given in SEQ ID NO:2, which may for example be one or more humanizing substitutions (as defined in WO 2008/020079; see for example again Tables A-5 to A-8) and/or other substitutions (with non-limiting examples of such humanizing or other substitutions being: position 1: A to E, position 14 P to A, or position 108: Q to L).

[0030] Similarly, the invention relates to an amino acid sequence that is a variant of the sequence Alb-23 (SEQ ID NO:1), which variant comprises:

(i) the amino acid motif GP on positions 44 and 45; (ii) the amino acid motif SKN on positions 74 to 76; (iii) a CDR1 that is the amino acid sequence SFGMS (SEQ ID NO:29); (iv) a CDR2 that is the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO:30); (v) a CDR3 that is the amino acid sequence GGSLSR (SEQ ID NO:31); and that preferably also comprises (vi) a G at position 16; and in which preferably (but without limitation): (vii) position 83 is an R (but may optionally also be a K); and that further comprises between 1 and 7, such as between 1 and 5 further "amino acid differences" (as defined in WO 2008/020079) with the sequence given in SEQ ID NO:1, which may for example be one or more humanizing substitutions (as defined in WO 2008/020079; see for example again Tables A-5 to A-8) and/or other substitutions (with non-limiting examples of such humanizing or other substitutions being: position 1: A to E, position 14: P to A, or position 108: Q to L).

[0031] As will be clear to the skilled person, and although (the use of) Alb-23 is generally preferred within the context of the present invention, the variants described in the preceding paragraphs share with Alb-23 some of the same amino acid substitutions (i.e., compared to Alb-1) that are characteristic of Alb-23 (also compared to the humanized variants of Alb-1 described in WO 06/122787), and thus are expected to provide at least some or even all of the advantages described herein for Alb-23. For these reasons, these variants are also referred to herein as "Alb-23-like sequences" or "Alb-23 variants".

[0032] In one specific, but non-limiting aspect, an Alb-23 variant is such that, when it is used in the storage stability assay described in Example 5 (i.e., as part of a construct that further comprises IGE045 and a 9GS linker), that the pre-peak on SE-HPLC for the construct comprising the Alb-23 variant after 1 month storage at 25.degree. C. (under the further conditions given in Example 5) is less than 10%, preferably less than 5%; and/or that the pre-peak on SE-HPLC for the construct comprising the Alb-23 variant after 1 month storage at 40.degree. C. (under the further conditions given in Example 5) is less than 20%, preferably less than 15%. Reference is for example made to the comparative results in Table 8.

[0033] Some non-limiting examples of some Alb-23 like sequences are given in SEQ ID NO's: 3 to 11. The variants of SEQ ID NO's 6 to 11 (or other Alb-23 variants with 1 to 3 amino acid residues at the C-terminus, which may each be independently chosen from naturally occurring amino acid residues and may for example be independently chosen from A, G, V, L and I) may in particular be used when the albumin-binding Nanobody is provided at the C-terminal end of the polypeptide or protein construct. An alignment of Alb-23 with the sequences of SEQ ID NO's: 6 to 11 is given in FIG. 2.

TABLE-US-00003 Alb-23A: [SEQ ID NO: 3] AVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSS Alb-23B: [SEQ ID NO: 4] AVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTQVTVSS Alb-23C: [SEQ ID NO: 5] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTQVTVSS Alb-23D: [SEQ ID NO: 6] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSSA Alb-23E: [SEQ ID NO: 7] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSSAA Alb-23F: [SEQ ID NO: 8] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSSAAA Alb-23G: [SEQ ID NO: 9] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSSG Alb-23H: [SEQ ID NO: 10] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSSGG Alb-231: [SEQ ID NO: 11] EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSS ISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGG SLSRSSQGTLVTVSSGGG

[0034] Thus, in one specific, but non-limiting aspect, the invention provides proteins or polypeptides that essentially consist of the amino acid sequence Alb-23 (or of one of the Alb-23 variants described herein).

[0035] As further described herein, the amino acid sequence Alb-23 and the further Alb-23 variants described herein can be used with advantage as a moiety, binding unit or fusion partner in order to increase the half-life of therapeutic moieties such as polypeptides, proteins, compounds (including, without limitation, small molecules) or other therapeutic entities.

[0036] Thus, in another aspect, the invention provides polypeptides, proteins, constructs, compounds or other chemical entities that comprise or essentially consist of the amino acid sequence Alb-23 (or of one of the Alb-23 variants described herein) and one or more other amino acid sequences, (binding) domains, binding units or other moieties or chemical entities.

[0037] In particular, the invention provides polypeptides, proteins, constructs, compounds or other chemical entities that comprise the amino acid sequence Alb-23 (or of one of the Alb-23 variants described herein) and one or more (such as one or two) therapeutic moieties (which may be the same or different, and may for example be directed against the same target or to different targets, and when they are directed to the same target may be directed towards the same or different epitopes, parts, domains or subunits of said target), suitably linked to each other either directly or via one or more suitable linkers or spacers. Such polypeptides, proteins or constructs may for example and without limitation be a fusion protein, as further described herein.

[0038] The invention further relates to therapeutic uses of such polypeptides, proteins, constructs or compounds and to pharmaceutical compositions comprising such polypeptides, proteins, constructs or compounds.

[0039] In one aspect, the at least one therapeutic moiety comprises or essentially consists of a therapeutic protein, polypeptide, compound, factor or other entity. In a preferred embodiment the therapeutic moiety is directed against a desired antigen or target, is capable of binding to a desired antigen (and in particular capable of specifically binding to a desired antigen), and/or is capable of interacting with a desired target. In another embodiment, the at least one therapeutic moiety comprises or essentially consists of a therapeutic protein or polypeptide. In a further embodiment, the at least one therapeutic moiety comprises or essentially consists of a binding domain or binding unit, such as an immunoglobulin or immunoglobulin sequence (including but not limited to a fragment of an immunoglobulin), such as an antibody or an antibody fragment (including but not limited to an ScFv fragment), or of another suitable protein scaffold, such as protein A domains (such as Affibodies.TM.), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

[0040] In yet another aspect, the at least one therapeutic moiety comprises or essentially consists of an antibody variable domain, such as a heavy chain variable domain or a light chain variable domain.

[0041] In a preferred aspect, the at least one therapeutic moiety comprises or essentially consists of at least one immunoglobulin single variable domain, such as a domain antibody, single domain antibody, "dAb" or Nanobody (such as a VHH, a humanized VHH or a camelized VH) or an IgNAR domain.

[0042] In a specific embodiment, the at least one therapeutic moiety comprises or essentially consists of at least one monovalent Nanobody or a bivalent, multivalent, bispecific or multispecific Nanobody construct.

[0043] The polypeptides, (fusion) proteins, constructs or compounds that comprise Alb-23 (or an Alb-23) variant and one or more therapeutic moieties can generally be (prepared and used) as described in the prior art cited above (such as WO 04/041865 and WO 06/122787), but with Alb-23 or an Alb-23 variant instead of the half-life increasing moieties described in said prior art.

[0044] The polypeptides, (fusion) proteins, constructs or compounds that comprise Alb-23 (or an Alb-23) variant and one or more therapeutic moieties will generally and preferably have an increased half-life, compared to the therapeutic moiety or moieties per se.

[0045] Generally, the constructs or fusion proteins described herein preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding therapeutic moiety per se (as measured in either in man or a suitable animal, such as mouse or cynomolgus monkey).

[0046] Also, preferably, any such fusion protein or construct has a half-life in man that is increased with more than 1 hour, preferably more than 2 hours, more preferably of more than 6 hours, such as of more than 12 hours, compared to the half-life of the corresponding therapeutic moiety per se.

[0047] Also, preferably, any fusion protein or construct has a half-life in man that is more than 1 hour, preferably more than 2 hours, more preferably of more than 6 hours, such as of more than 12 hours, and for example of about one day, two days, one week, two weeks or three weeks, and preferably no more than 2 months, although the latter may be less critical.

[0048] Half-life can generally be defined as the time taken for the serum concentration of the polypeptide to be reduce by 50%, in vivo, for example due to degradation of the ligand and/or clearance or sequestration of the ligand by natural mechanisms. In particular, half-life may be as defined in WO 2009/068627.

[0049] Methods for pharmacokinetic analysis and determination of half-life are familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd revised edition (1982).

[0050] As mentioned, in one aspect, the amino acid sequence Alb-23 (or of one of the Alb-23 variants described herein) can be used to increase the half-life of (one or more) immunoglobulin single variable domains, such as domain antibodies, single domain antibodies, "dAb's", VHH's or Nanobodies (such as VHH's, humanized VHH's or camelized VH's such as camelized human VH's).

[0051] In particular, as mentioned herein, the amino acid sequence Alb-23 (or of one of the Alb-23 variants described herein) can be used with advantage to increase the half-life of immunoglobulin single variable domains that comprise two or more (such as two or three) disulphide bridges, such as VHH's/Nanobodies of the VHH-1 class.

[0052] Thus, one embodiment of the invention relates to a polypeptide, construct or fusion protein that comprises the amino acid sequence Alb-23 (or one of the Alb-23 variants described herein) and one or more (such as one or two) immunoglobulin single variable domain sequences, which are suitably linked to each other, either directly or optionally via one or more suitable linkers or spacers. As mentioned herein, each such immunoglobulin single variable domain present in such a polypeptide, construct or fusion protein may independently be a domain antibody, single domain antibody, "dAb'" or Nanobody (such as a VHH, humanized VHH or camelized VH, such as a camelized human VH); and according to one specific but non-limiting aspect, at least one (and up to all) of these immunoglobulin single variable domains comprises two or three disulphide bridges.

[0053] Preferably, each such immunoglobulin single variable domain is a Nanobody; and according to one specific but non-limiting aspect, at least one (and up to all) of these immunoglobulin single variable domains is a Nanobody of the VHH-1 class.

[0054] For example and without limitation, such a construct, fusion protein or polypeptide may comprise: [0055] one copy of Alb-23 (or of one of the Alb-23 variants described herein) and one such immunoglobulin single variable domain sequence; [0056] one copy of Alb-23 (or of one of the Alb-23 variants described herein) and two such immunoglobulin single variable domain sequences (which may be the same or different); or even (although usually not required and less preferred because the resulting protein is bigger) [0057] two copies of Alb-23 (or two copies of the Alb-23 variants described herein, which may be the same or different) and one such immunoglobulin single variable domain sequence; [0058] two copies of Alb-23 (or two copies of the Alb-23 variants described herein, which may be the same or different) and two such immunoglobulin single variable domain sequence (which may be the same or different); [0059] one copy of Alb-23 and three such immunoglobulin single variable domain sequence (which may be the same or different).

[0060] Some non-limiting examples of constructs, fusion proteins or polypeptides of the invention can be schematically represented as follows, in which "[Alb-23]" represents Alb-23 (or of one of the Alb-23 variants described herein), "[therapeutic moiety 1]" and "[therapeutic moiety 2]" represent the therapeutic moieties (which as mentioned may each independently be an immunoglobulin single variable domain), "-" represents a suitable linker (which is optional) and the N-terminus is on the left hand side and the C-terminus is on the right hand side: [0061] [Alb-23]-[therapeutic moiety 1] [0062] [therapeutic moiety 1]-[Alb-23] [0063] [Alb-23]-[therapeutic moiety 1]-[therapeutic moiety 1] [0064] [therapeutic moiety 1]-[therapeutic moiety 1]-[Alb-23] [0065] [therapeutic moiety 1]-[Alb-23]-[therapeutic moiety 1] [0066] [Alb-23]-[therapeutic moiety 1]-[therapeutic moiety 2] [0067] [therapeutic moiety 1]-[therapeutic moiety 2]-[Alb-23] [0068] [therapeutic moiety 1]-[Alb-23]-[therapeutic moiety 2]

[0069] When two or more different therapeutic moieties (such as two or more different immunoglobulin single variable domains) are present in the constructs or polypeptides of the invention, they may be the same or different, and when they are different they may be directed towards the same target (for example, to the same or different parts, domains, subunits or epitopes of said target) or to different targets.

[0070] Thus, in another aspect, the invention relates to a multispecific (and in particular bispecific) Nanobody construct that comprises Alb-23 (or of one of the Alb-23 variants described herein) and at least one other Nanobody (such as one or two other Nanobodies, which may be the same or different), in which said at least one other Nanobody is preferably directed against a desired target (which is preferably a therapeutic target) and/or another Nanobody that useful or suitable for therapeutic, prophylactic and/or diagnostic purposes. Again, Alb-23 and the other Nanobodies may be suitably linked to each other either directly or optionally via one or more suitable linkers or spacers, and according to one specific but non-limiting aspect at least one (and up to all) of the other Nanobodies may be of the VHH-1 class.

[0071] For a general description of multivalent and multispecific polypeptides containing one or more Nanobodies and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to for example WO 96/34103, WO 99/23221, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

[0072] Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by Ablynx N.V. mentioned herein. In particular, for a general description of multivalent and multispecific constructs comprising at least one Nanobody against a serum protein for increasing the half-life, of nucleic acids encoding the same, of compositions comprising the same, of the preparation of the aforementioned, and of uses of the aforementioned, reference is made to the International applications WO 04/041865 and WO 06/122787 mentioned above (Alb-23 and the Alb-23 variants described herein can generally be used analogously to the half-life extending Nanobodies described therein such as Alb-8), as well as to the general description and specific examples of such constructs given in WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

[0073] In one non-limiting embodiment, the one or more other Nanobodies present in such a polypeptide or protein construct may be directed against c-Met, and may in particular be Type I Nanobodies directed against c-Met. Some non-limiting examples of Nanobodies against c-Met that may be present in such a polypeptide or protein construct may for example be found in the non-prepublished US applications U.S. 61/388,172 and U.S. 61/451,869 mentioned herein.

[0074] One particularly preferred Type I Nanobody against c-Met that may be present in such a multivalent and/or multispecific polypeptide (next to Alb-23 or an Alb-23 variant) is 4E09 (SEQ ID NO:26 in U.S. 61/451,869 and SEQ ID NO: 12 herein) or variant thereof. Such a variant of 4E09 may generally be as described in U.S. 61/451,869 (and will generally have at least 80%, such as at least 85%, for example at least 90% or more such as 95% or more sequence identity with 4E09) and is preferably such that (i) it competes with 4E09 for binding to c-Met (in a suitable binding assay, such as the alphascreen assay described in Example 7, but using 4E09 instead of HGF as used in Example 7); and/or (ii) it binds to the same epitope on c-Met as 4E09; and/or (iii) cross-blocks (as defined in WO 2009/068627) the binding of 4E09 to c-Met. Such a variant of 4E09 may for example be a humanized and/or sequence-optimized variant of 4E09 (as further described in U.S. 61/451,869). Some preferred, but non-limiting examples of variants of 4E09 that could be present in such proteins or polypeptides are the following, which are also described in U.S. 61/451,869: 04E09 (L49S); 04E09 (C50S/C100bG); 04E09 (C22A/C92S); A00790067=4E09 (Q108L); A00790068=4E09 (A74S, K83R, Q108L); A00790069=4E09 (A74S, K83R, G88A, Q108L) and A00790105=4E09 (E1D, A74S, K83R, G88A, Q108L), of which the latter is especially preferred. The amino acid sequences of 4E09 and these variants are given in SEQ ID NO's: 12 to 19.

[0075] Thus, in one specific but non-limiting aspect, the invention relates to a polypeptide or protein construct that comprises or essentially consists of Alb-23 (preferred) or an Alb-23 variant (as described herein), which is suitably linked (either directly or via one or more suitable linkers) to one or two Nanobodies against c-Met. As mentioned, according to a specific but non-limiting aspect, said one or two Nanobodies against c-Met comprise two disulphide bridges (i.e. are of "Class I").

[0076] In particular, the invention relates to a polypeptide or protein construct that comprises or essentially consists of Alb-23 (preferred) or an Alb-23 variant (as described herein), which is suitably linked (either directly or via one or more suitable linkers) to one or two (and preferably only one) Nanobodies against c-Met, which are 4E09 (SEQ ID NO 12) or a variant of 4E09 (as described herein and in U.S. 61/451,869), and preferably a humanized or sequence optimized variant of 4E09 and more preferably A00790105 (SEQ ID NO: 19).

[0077] Some specific but non-limiting examples of such proteins and polypeptides are the constructs Alb23-9GS-4E09, 4E09-9GS-Alb23, Alb23-9GS-A00790105, A00790105-9GS-Alb23, Alb23-35GS-4E09, 4E09-35GS-Alb23, Alb23-35GS-A00790105, A00790105-35GS-Alb23, and A00790105-35GS-A00790105-35GS-Alb23. The sequences of these are given in SEQ ID NO's: 20 to 28, respectively. Of these, the construct A00790105-9GS-Alb23 (SEQ ID NO: 23) is particularly preferred, and thus one aspect of the invention also relates to a polypeptide that has at least 80%, such as at least 85%, for example at least 90%, such as at least 95% or more sequence identity with the polypeptide of SEQ ID NO: 23.

[0078] The invention also relates to nucleotide sequences or nucleic acids that encode amino acid sequences, fusion proteins and constructs described herein. The invention further includes genetic constructs that include the foregoing nucleotide sequences or nucleic acids and one or more elements for genetic constructs known per se. The genetic construct may be in the form of a plasmid or vector. Again, such constructs can be generally as described in the published patent applications of Ablynx N.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

[0079] The invention also relates to hosts or host cells that contain such nucleotide sequences or nucleic acids, and/or that express (or are capable of expressing), the amino acid sequences, fusion proteins and constructs described herein. Again, such host cells can be generally as described in the published patent applications of Ablynx N.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

[0080] The invention also relates to a method for preparing an amino acid sequence, fusion protein or construct as described herein, which method comprises cultivating or maintaining a host cell as described herein under conditions such that said host cell produces or expresses an amino acid sequence, fusion protein or construct as described herein, and optionally further comprises isolating the amino acid sequence, fusion protein or construct so produced. Again, such methods can be performed as generally described in the published patent applications of Ablynx N.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

[0081] The invention also relates to a pharmaceutical composition that comprises at least one amino acid sequence, fusion protein or construct as described herein, and optionally at least one pharmaceutically acceptable carrier, diluent or excipient. Such preparations, carriers, excipients and diluents may generally be as described in the published patent applications of Ablynx N.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

[0082] However, since the amino acid sequences, fusion proteins or constructs described herein have an increased half-life, they are preferably administered to the circulation. As such, they can be administered in any suitable manner that allows the amino acid sequences, fusion proteins or constructs to enter the circulation, such as intravenously, via injection or infusion, or in any other suitable manner (including oral administration, subcutaneous administration, intramuscular administration, administration through the skin, intranasal administration, administration via the lungs, etc.) that allows the amino acid sequences, fusion proteins or constructs to enter the circulation. Suitable methods and routes of administration will be clear to the skilled person, again for example also from the teaching of the published patent applications of Ablynx N.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

[0083] Thus, in another aspect, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented or treated by the use of a fusion protein or construct as described herein, which method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a fusion protein or construct of the invention, and/or of a pharmaceutical composition comprising the same. The diseases and disorders that can be prevented or treated by the use of a fusion protein or construct as described herein will generally be the same as the diseases and disorders that can be prevented or treated by the use of the therapeutic moiety that is present in the fusion protein or construct of the invention.

[0084] In the context of the present invention, the term "prevention and/or treatment" not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

[0085] The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk from, the diseases and disorders mentioned herein.

[0086] In another embodiment, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of a fusion protein or construct of the invention, and/or of a pharmaceutical composition comprising the same.

[0087] The fusion protein or construct and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific polypeptide of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.

[0088] Generally, the treatment regimen will comprise the administration of one or more fusion proteins or constructs of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to be administered can be determined by the clinician, again based on the factors cited above.

[0089] Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency and/or the half-life of the specific fusion proteins or constructs to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the Nanobodies and polypeptides of the invention will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1, 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g., by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, taking into account however differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person.

[0090] Usually, in the above method, a single polypeptide of the invention will be used. It is however within the scope of the invention to use two or more polypeptides of the invention in combination.

[0091] The polypeptides of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e., as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.

[0092] In particular, the polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders that can be prevented or treated with the fusion proteins or constructs of the invention, and as a result of which a synergistic effect may or may not be obtained.

[0093] The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and or a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.

[0094] Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

[0095] The subject to be treated may be any warm-blooded animal, in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk from, the diseases and disorders mentioned herein.

[0096] Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

[0097] The invention will now be further illustrated by means of the non-limiting Experimental Part and Figures, in which:

[0098] FIG. 1 is an alignment of Alb-23 (SEQ ID NO: 1) with Alb-1 and the various humanized variants thereof described in WO 06/122787;

[0099] FIG. 2 is an alignment of Alb-23 (SEQ ID NO: 1) with some of the Alb-23 variants described herein;

[0100] FIG. 3 shows the various sequences referred to in the present specification;

[0101] FIG. 4 shows an expression profile using SDS-PAGE analysis of A007900009 produced by Pichia clones with a low (L=1) and high (H=more than 1) copy number (CN) of the expression cassette.

[0102] FIG. 5 shows an expression profile using SDS-PAGE analysis of A007900171 produced by Pichia clones with a low (L=1) and high (H=more than 1) copy number (CN) of the expression cassette.

[0103] FIG. 6 shows an expression profile using SDS-PAGE analysis of A007900057 and A007900058 produced by Pichia clones with a low (L=1) and high (H=more than 1) copy number (CN) of the expression cassette.

[0104] FIG. 7 shows an expression profile using SDS-PAGE analysis of A007901219 variant produced by Pichia clones with a low (L=1) and high (H=more than 1) copy number (CN) of the expression cassette.

[0105] FIG. 8 shows SDS-Page analysis (non-reducing conditions) of end of induction fermentation samples of Pichia pastoris expressions of A00700171 and A00701219. 5 .mu.L cell free medium is loaded onto the gel (Lane 1-4). Lanes 5 to 7 contain a control Nanobody loaded in different amounts. Left lane contains the molecular weight marker.

EXPERIMENTAL PART

Example 1: Expression Profile of 4E09 in Combination with Alb11 or Alb23

[0106] Table 1 gives an overview of the formats based on the anti-c-Met 4E09 VHH1 Nanobody building block (SEQ ID NO:12) in combination with Alb11 and Alb23. The different Nanobodies were cloned into the pPiczalpha expression vector and transformed in Pichia pastoris X-33 strain (commercially available expression system from Invitrogen/RCT). Clones were selected on zeocin containing plates and a qPCR was performed to rank the clones according to their copy numbers. Expression levels were compared between low and high copy number clones in shake flask experiments. An inverse correlation between expression level and copy numbers was observed for Alb11 containing formats (FIGS. 4 and 5). In contrast, a positive correlation between expression level and copy numbers was observed with Nanobody formats containing the Alb23 Nanobody (FIGS. 6 and 7).

TABLE-US-00004 TABLE 1 Overview of 5 formats of the VHH1 4E09 Nanobody building block or sequence optimized version fused via a 9GS or 35GS linker to the Alb11 or Alb23 Nanobody building block. Format Construct SEQ ID NO: A007900009 4E09-9GS-ALB11-Flag3-His6 39 A007900057 4E09-9GS-ALB23 21 A007900058 4E09-35GS-ALB23 25 A007900171 A00790105-9GS-Alb11 40 A007901219 A00790105-9GS-Alb23 23

Example 2: Fermentation Yield of 4E09 in Combination with Alb11 or Alb23

[0107] Expression of the A007900171 (4E9 sequence optimized-9GS-Alb11) and A007901219 (4E9 sequence optimized-9GS-Alb23) Nanobody.RTM. was evaluated using Pichia pastoris X33 as host organism. Both the AOX promoter (MeOH inducible promoter) and GAP promoter (constitutive induced promoter) were evaluated.

[0108] A complex, peptone based medium was used for the fermentation processes. For the fermentations with the X33 clone using the AOX promoter, MeOH was used for expression of the Nanobody. In short, this process can be divided in 3 phases: a batch phase, a glycerol fed batch and a MeOH induction phase. During the batch and glycerol fed batch phase, biomass was build up to approx. 40% (wet weight per volume). Then a MeOH adaptation and induction phase was started during which the Nanobody.RTM. was expressed and secreted into the medium.

[0109] For the fermentations with the X33 clone using the GAP promoter, glucose was used to induce expression of the Nanobody.RTM.. This process can also be divided in 3 phases: a batch phase on glycerol, a glucose fed batch using a high feed rate to build-up biomass to approx. 40% (wet weight per volume) and a second glucose fed batch phase at a lower feed rate to further induce expression/secretion of the Nanobody.RTM..

[0110] All Fermentations were carried out at 2 L fermenter scale using Sartorius equipment (Biostat.RTM. Aplus, Biostat.RTM. Bplus and Biostat.RTM. Bplus twin controllers, 2 L Univessel.RTM. reactors, control via MFCSwin software). During fermentations the following parameters were monitored/steered: DO (dissolved oxygen), pH, foaming, biomass (OD.sub.600 for E. coli and Wet Cell Weight (WCW) for Pichia pastoris), expression level and quality of the product. The DO was typical 30% and controlled via a stirring cascade, and further compensated by addition of pure oxygen on a need basis. The pH was monitored via a pH electrode and adjusted via ammonia additions via a base pump. Off line pH measurements were done to check pH electrode functioning following autoclaving.

[0111] Table 2 gives an overview of the fermentation runs performed for expression of A007900171 and A007901219 using Pichia pastoris as host organism.

[0112] FIG. 8 shows the SDS-Page analysis (non-reducing conditions) of the end of induction samples of the different fermentations with Pichia pastoris.

[0113] In Table 3 an overview of the different Pichia pastoris fermentations with their respective parameter settings, fermentation characteristics, and the estimated expression yield of the end of induction samples based on SDS-Page analysis, is shown.

[0114] The titer and purity of the produced Nanobody.RTM. at end of fermentation was determined via a small scale protA clean-up procedure followed by RP-HPLC analysis. In short, sample preparation is based on protA affinity chromatography which purifies the Nanobody.RTM. from the medium. The protein concentration of the elution fraction is determined using UV spectrophotometry and is followed by Reversed Phase High Performance Liquid Chromatography (RP-HPLC) allowing determination of product titer and product purity/heterogeneity. Table 4 summarizes the RPC results.

TABLE-US-00005 TABLE 2 Overview of the fermentation runs performed for expression of A007900171 and A007901219 using P. pastoris as host organism Fed-batch Induction Fermentation ID Construct Strain Promoter Fed-batch feed rate Induction feed rate A1/110511 A007901219 X33 AOX 60% glycerol + 21.6 g/h l 100% MeOH 3.16 g/h l Alb23 10% peptone A1/110518 A007900171 X33 AOX 60% glycerol + 21.6 g/h l 100% MeOH 3.16 g/h l Alb11 10% peptone A2/110511 A007901219 X33 GAP 25% glucose + 50.8 g/h l 50% glucose 6 g/h l Alb23 5% peptone A4/110511 A007900171 X33 GAP 25% glucose + 50.8 g/h l 50% glucose 6 g/h l Alb11 5% peptone

TABLE-US-00006 TABLE 3 Overview of the different A007900035 fermentations with Pichia pastoris and their respective expression yield in clarified medium at end of fermentation determined via SDS-Page analysis. WCW Expression yield Fermentation Induction at end of (mg/L clarified Expression yield ID Construct Strain Promoter time induction (g/L) medium) (mg/L broth) A1/110511 A007901219 X33 AOX 95 hrs 420 g/L -500 mg/L -290 mg/L Alb23 A1/110518 A007900171 X33 AOX 92 hrs 423 g/L -200 mg/L -115 mg/L Alb11 A2/110511 A007901219 X33 GAP 91 hrs 421 g/L -350 mg/L -203 mg/L Alb23 A4/110511 A007900171 X33 GAP 93 hrs 374 g/L -100 mg/L -63 mg/L Alb11

TABLE-US-00007 TABLE 4 Overview of the total yield determined via OD.sub.280 and RPC analysis after a ProtA clean-up step, % main peak and the intact monomeric yield. All yields are expressed in mg/L cell free medium. Total Yield via Total Yield via Intact yield via RPC OD.sub.290 after ProtA RPC analysis after % Main peak analysis after ProtA Ferm. ID Construct Promoter clean-up ProtA clean-up (RPC analysis) clean-up A1/110511 A007901219 AOX 820 mg/L 670 mg/L 68.7% 460 mg/L Alb23 A1/110518 A007900171 AOX 250 mg/L 200 mg/L 88.0% 170 mg/L Alb11 A2/110511 A007901219 GAP 340 mg/L 290 mg/L 79.0% 230 mg/L Alb23 A4/110511 A007900171 GAP 180 mg/L 150 mg/L 86.0% 130 mg/L Alb11

Example 3: Purification of A007900171 and A007901219

[0115] Both A007900171 and A007901219 molecules were purified according to the scheme shown in Table 5:

TABLE-US-00008 TABLE 5 Overview of purification steps. Process step Description Aim Step 1: Centrifugation and TFF Clarification of the Clarification microfiltration (0.2 .mu.m fermentation broth Hydrosart Sartocon slice cassette, Sartorius) Step 2: HCIC (MEP Hypercel Capture product Capture step with elution at pH 3.5 + from the medium neutralization with 25 mM and removal Tris pH 8.5) + buffer of impurities exchange* (Sephadex G25 to 25 mM Tris buffer pH 8.5) Step 3: AIEX (POROS 50HQ or Q Removal of product Intermediate Sepharose HP) in 25 mM related variants step Tris pH 8.5 and impurities Step 4: Polish OGP treatment for LPS Removal of LPS step removal + SEC (Superdex 75) and product related variants and impurities *Only for A007901219

Example 4: Comparison of Properties of Alb-1, Alb-8 (=Alb-11) and Alb-23

[0116] Various properties of Alb-1, Alb-8 and Alb-23 were determined and compared. The results are shown in Table 6.

[0117] Also, for a side-by-side comparison of the tendency of Alb-8 and Alb-23 to form dimers, dimerization kinetics for monovalent Alb-8 (Ea=73.0 kcal/mol) and Alb-23 (Ea=50.8 kcal/mol) at various temperatures in PBS buffer were calculated from an Arrhenius plot. The results are shown in Table 7, and show that for Alb-11, the kinetics start to increase from room temperature upwards, in particular when compared to Alb-23.

TABLE-US-00009 TABLE 6 Comparison of some of the properties of Alb-1, Alb-8 and Alb-23. ALB1 ALB23 ALB8 K.sub.D (nM) Human SA 1.8 2.7 4.5 Cyno SA 1.9 3.0 4.6 Murine SA 17.3 16.5 68.8 Tm (.degree. C.) TSA 64.5 69.0 61.6 DSC 65.6 71.7 65.9 Reversibility of DSC @ 0.3 mg/ml & 64 92 16 unfolding (%) Tm + 5.degree. C. Dimerization (%) 3w @ 37.degree. C. & 20 mg/ml 2 17 12w @ 37.degree. C. & 20 mg/ml 6 9 44 Humanization % FR identity 86.5 93.3 91.0

TABLE-US-00010 TABLE 7 Side-by-side comparison of dimerization kinetics of monovalent ALB8/ALB11 and ALB23 over a broad concentration range, as calculated from an Arrhenius plot (max. predicted % dimers after 3 years at the indicated concentration in PBS buffer and indicated temperature). 10 mg/ml 25 mg/ml 50 mg/ml Conc. (mg/ml) ALB11 ALB23 ALB11 ALB23 ALB11 ALB23 Temp (.degree. C.) % dimers % dimers % dimers % dimers % dimers % dimers 5 0.00 0.03 0.01 0.09 0.02 0.17 10 0.04 0.15 0.09 0.37 0.18 0.74 15 0.31 0.63 0.77 1.60 1.50 3.20 20 2.50 2.70 6.30 6.60 12.50 13.30 25 19.00 10.60 47.50 26.60 95.10 53.20 30 >100 41.60 >100 >100 >100 >100

Example 5: Storage Stability of Constructs Comprising Alb-23 Compared to Alb-11

[0118] To compare the storage stability of bispecific constructs comprising Alb-8 and Alb-23, respectively, two bispecific Nanobody constructs comprising the same Nanobody against IgE (IGE045, SEQ ID NO:43) linked to Alb-23 (IGE045-9GS-ALB23; SEQ ID NO: 41) and Alb-11 (IGE045-9GS-ALB11; SEQ ID NO: 42) were prepared, formulated in D-PBS buffer at a concentration of 50 mg/ml and stored in plastic PCR tubes in the dark for 1 month at different temperatures. After that, the amount of pre-peak (corresponding to dimer formation) was determined and compared using SE-HPLC. The SE-HPLC analysis was performed using aBioSep SEC-2000 column (Phenomenex) and D-PBS as running buffer at a flow rate of 0.2 ml/min. 10 .mu.g material was injected and data was analysed using Chromeleon software. The results are shown in Table 8.

[0119] In a similar experiment, the storage stability of two trivalent bispecific constructs were compared (at a concentration of 50 mg/ml in 20 mM His buffer, pH6.5, 8% sucrose). The constructs were OX40L079 (SEQ ID NO:44), a trivalent bispecific Nanobody construct comprising Alb-11 and two Nanobodies against OX40L (see SEQ ID NO:229 of PCT/EP2010/069606 of Ablynx N.V., filed on 14, Dec. 2010) and OX40L089 (SEQ ID NO:45), a corresponding construct comprising the same two Nanobodies against OX40L, but with Alb-23 instead of Alb-11. The results are also shown in Table 8A.

TABLE-US-00011 TABLE 8A Comparative storage stability of Alb-23 and Alb-11 constructs (% Pre peak on SE-HPLC after storage for the indicated storage period at the indicated temperatures) Albumin Storage Temperature (.degree. C.) Construct binder period 4 5 25 40 IGE045-9GS-ALB23 Alb-23 1 month -- 0.0 3.7 7.6 (SEQ ID NO: 41) IGE045-9GS-ALB11 Alb-11 1 month -- 0.0 11.7 30.3 (SEQ ID NO: 42) OX40L079 Alb-11 2 weeks 0.4 -- 1.4 25 (SEQ ID NO: 44) OX40L089 Alb-23 2 weeks 0.3 -- 1.4 13 (SEQ ID NO: 45)

[0120] In another experiment, the fermentation yield storage stability of four different Nanobody constructs against c-Met (A007901222/SEQ ID NO: 46; A007901256/SEQ ID NO: 47; A007901259/SEQ ID NO: 48 and A007901260/SEQ ID NO: 49) was compared. Of these constructs, A007901222 contains the Alb-23 albumin-binding Nanobody (invention) as well as a "VHH-1 type" anti-c-Met Nanobody (4E09--see again the U.S. applications 61/388,172 and U.S. 61/451,869 referred to above) whereas the other constructs (used as comparative references) contain the Alb-11 albumin-binding Nanobody and anti c-Met Nanobodies which are not of the VHH-1 type. For a more detailed description of the constructs used, reference is made to the co-pending U.S. application Ser. No. 13/435,567 (Beste et al.) by Applicant filed on Mar. 30, 2012.

[0121] The different Nanobody constructs were produced in Pichia pastoris (see again U.S. Ser. No. 13/435,567, U.S. 61/388,172 and U.S. 61/451,869) and afforded broadly comparable fermentation yields (1.03 g/L for; 1.43 g/L for A007901256; 0.91 g/L for A007901259 and 1.45 g/L for A007901260), confirming that the present invention makes it possible to provide constructs comprising VHH-1 type Nanobodies with expression yields that are comparable to the expression yields of constructs that do not contain a VHH-1 type Nanobody.

[0122] The Nanobody constructs were then purified (>99% purity on SEC and >90% purity on RPC) and incubated at -70.degree. C., -20.degree. C., 5.degree. C., 25.degree. C. and 40.degree. C. at a concentration of about 15 mg/ml in D-PBS for 7.5 weeks. The samples were evaluated by turbidity measurement (OD500), RP-HLPC and SE-HLPC. The results are summarized in Table 8B. Stability was acceptable for all samples tested when stored at -70.degree. C., -20.degree. C. or 5.degree. C. for 7.5 weeks.

TABLE-US-00012 TABLE 8B comparative storage stability of different anti c-Met Nanobody constructs Storage stability (7.5 weeks incubation) Construct 25.degree. C. 40.degree. C. A007901222 0.3% HMW; 3.5% HMW; OD500 < 0.005 OD500 = 0.005 A007901256 0.5% HMW; 24.1% HMW; OD500 < 0.005 OD500 = 1.30 (*) A007901259 0.7% HMW; 23.7% HMW; OD500 < 0.005 OD500 = 1.91 (*) A007901260 0.5% HMW; 23.1% HMW; OD500 < 0.005 OD500 = 0.45 (*) HMW = high molecular weight (pre-peak) components (*) = sample was opalescent.

Example 6: Affinity Determination Using Surface Plasmon Resonance

[0123] Kinetic analysis of the anti-c-Met Nanobody-Alb fusion constructs A007900171 (SEQ ID NO:40) and A007901219 (SEQ ID NO:23) for human, cynomolgus and mouse serum albumin was performed using Surface Plasmon Resonance on the Biacore T100 instrument. HBS-EP+buffer (0.01M HEPES buffer containing 0.15M NaCl, 3 mM EDTA and 0.05% Surfactant p20, pH7.4) was used as the running buffer and experiments were performed at 25.degree. C. Serum albumin was chemically coupled on a Series S sensorchip chip CMS with carboxymethylated dextran surface by a manual immobilization run at a flow rate of 5 .mu.l/min. Surfaces were first activated with a 7 min injection of a 1:1 mixture of 75 mg/ml EDC and 11.5 mg/ml NHS (Biacore amine coupling kit). Serum albumin was injected at 10 .mu.g/ml in 10 mM acetate pH4.5, until a level of 970 RU (flow cell 2), 890 RU (flow cell 3) and 1360 RU (flowcell 4) for respectively human, cynomolgus and mouse serum albumin was reached. After immobilization, surfaces were deactivated with a 7 min injection of 1M ethanolamine pH8.5. A blank reference surface (flowcell 1) was activated and deactivated as described above. A series of Nanobody concentrations was prepared in HBS-EP+(i.e. 0 nM, 1.9 nM, 7.8 nM, 31.25 nM, 125 nM, 500 nM, 0 nM, 7.8 nM, 125 nM), were injected for 2 min at 45 .mu.l/min (flow path 1, 2, 3, and 4) and allowed to dissociate in running buffer for 10 min. Between different samples, the surfaces were regenerated with regeneration buffer 10 mM glycine-HCl pH1.5, 100 s at 45 .mu.l/min Data were double referenced by subtraction of the curves on the reference channel and of a blank running buffer injection curve. Processed curves were evaluated by fitting a 1:1 binding model onto the binding curves in the Biacore T100 Evaluation software. On-rates (ka), off-rates (kd) and affinities (KD) were reported and are shown in Table 9.

TABLE-US-00013 TABLE 9 Affinity determination of Nanobodies against serum albumin by surface plasmon resonance (SPR) measurement human serum albumin cynomolgus serum albumin mouse serum albumin ID k.sub.on k.sub.off K.sub.D k.sub.on k.sub.off K.sub.D k.sub.on k.sub.off K.sub.D Alb11 5.2 1.9 3.6 4.6 1.7 3.6 7.1 35 49 A007900171 (Alb11)* 1.0 6.2 60 0.93 6.1 66 0.93 66 710 Alb23 4.5 1.2 2.6 4.0 1.1 2.7 7.0 12 17 A007901219 (Alb23) 1.3 3.3 24 1.2 3.2 27 1.7 50 300 k.sub.on is given in 10.sup.5 M.sup.-1s.sup.-1, k.sub.off is given in 10.sup.-3 s.sup.-1, K.sub.D is given in nM (10.sup.-9 M) *Data obtained in a separate but equivalent experiment

Example 7: Binding of the MET-Ligand HGF in Alphascreen Assay

[0124] The anti-c-MET/anti-serum albumin Nanobody constructs were characterized in an HGF/c-MET competition AlphaScreen assay to evaluate their blocking potency and efficacy and compare this with a benchmark antibody fragment (5D5 Fab v2). A dilution series of anti-c-MET Nanobodies and benchmark 5D5 Fab v2 starting from 250 nM up to 0.9 pM was pre-incubated with 100 pM biotinylated hHGF during 15 minutes at RT. To this mixture the anti-human Fc conjugated acceptor beads and c-MET/Fc (100 pM final concentration) were added and incubated for 2 hours at RT. Next, streptavidin donor beads were added and the mixture was incubated for 1 additional hour. Fluorescence was measured by reading plates on the EnVision Multilabel Plate Reader using an excitation wavelength of 680 nm and an emission wavelength of 520 nm.

[0125] The two constructs effectively inhibit the HGF binding to c-MET receptor in a dose-dependent manner. The calculated IC.sub.50 values and corresponding 95% confidence intervals are shown in Table 10. A007900171 and the two batches of A007901219 have similar IC50 values; their 95% CI are overlapping, which suggests that the difference is statistically not significant. The Nanobodies showed an >5-fold improved potency as compared to the benchmark 5D5 Fab v2.

TABLE-US-00014 TABLE 10 Inhibition of HGF binding to cMET as determined by Alphascreen (IC50 values and 95% confidence intervals) ID IC.sub.50 [in pM] 95% CI [in pM] 5D5 Fab v2 380 330 to 440 A007900171 (Alb11) 58 50 to 66 A007901219 (Alb23) 66 57 to 78

Example 8: Blocking the HGF-Induced MET Phosphorylation in the A549 Cancer Cell Line

[0126] The purified anti-c-MET/anti-serum albumin Nanobody constructs were characterized in the HGF-dependent phosphorylation assay. A dilution series of the anti-cMET constructs or the anti-cMET benchmark 5D5 Fab v2 starting from 1 .mu.M up to 0.23 nM was co-incubated with 1 nM HGF on A549 cells during 15 min at 37.degree. C. 1/3 of the lysed cell solution was then applied to the phospho c-MET MSD assay plates. Two duplicates on cell culture level were pooled on MSD level. After washing away unbound material, a sulfo tagged detection cMET antibody detected both the phosphorylated as well as the unphosphorylated receptor. The read out was performed with the sector imager 2400.

[0127] The two anti-c-MET/anti-serum albumin Nanobody constructs effectively inhibit the HGF-dependent c-MET receptor phosphorylation in a dose-dependent manner. The calculated IC.sub.50 values and corresponding 95% confidence intervals are shown in 11. A007900171 and the two batches of A007901219 have similar IC.sub.50 values; their 95% CI are overlapping, which suggests that the differences are statistically not significant. The Nanobodies showed a ca. 2-fold improved potency as compared to the benchmark 5D5 Fab v2. Additionally, within 95% confidence intervals, the addition of human serum albumin to the stimulated cells did not alter IC.sub.50 values of the tested Nanobodies.

TABLE-US-00015 TABLE 11 Inhibition of HGF binding to cMET as determined by cMET phosphorylation assay (IC50 values and 95% confidence intervals) -HSA +HSA IC.sub.50 95% CI IC.sub.50 95% CI ID [in nM] [in nM] [in nM] [in nM] 5D5 Fab v2 11.9 8.57 to 16.5 n.d. A007900171 (Alb11) 5.97 5.08 to 7.00 6.28 5.35 to 7.36 A007901219 (Alb23) 5.41 4.61 to 6.35 4.20 3.60 to 4.91

[0128] The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced herein.

Sequence CWU 1

1

491115PRTArtificial Sequencenanobody or nanobody construct 1Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 2115PRTArtificial Sequencenanobody or nanobody construct 2Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 3115PRTArtificial Sequencenanobody or nanobody construct 3Ala Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 4115PRTArtificial Sequencenanobody or nanobody construct 4Ala Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 5115PRTArtificial Sequencenanobody or nanobody construct 5Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 6116PRTArtificial Sequencenanobody or nanobody construct 6Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala 115 7117PRTArtificial Sequencenanobody or nanobody construct 7Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala 115 8118PRTArtificial Sequencenanobody or nanobody construct 8Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ala Ala 115 9116PRTArtificial Sequencenanobody or nanobody construct 9Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly 115 10117PRTArtificial Sequencenanobody or nanobody construct 10Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly 115 11118PRTArtificial Sequencenanobody or nanobody construct 11Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly 115 12125PRTArtificial Sequencenanobody or nanobody construct 12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 13125PRTArtificial Sequencenanobody or nanobody construct 13Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 14125PRTArtificial Sequencenanobody or nanobody construct 14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Ser Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Gly Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 15125PRTArtificial Sequencenanobody or nanobody construct 15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Ala Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Ser 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 16125PRTArtificial Sequencenanobody or nanobody construct 16Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 17125PRTArtificial Sequencenanobody or nanobody construct 17Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 18125PRTArtificial Sequencenanobody or nanobody construct 18Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125

19125PRTArtificial Sequencenanobody or nanobody construct 19Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 20249PRTArtificial Sequencenanobody or nanobody construct 20Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr Ala Ile Gly Trp 145 150 155 160 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Leu Cys Ile Asp 165 170 175 Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn 195 200 205 Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Thr Pro Ile 210 215 220 Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 21249PRTArtificial Sequencenanobody or nanobody construct 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 130 135 140 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 145 150 155 160 Phe Thr Phe Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 165 170 175 Lys Gly Pro Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr 180 185 190 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 22249PRTArtificial Sequencenanobody or nanobody construct 22Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Asp Val Gln Leu 115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 130 135 140 Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr Ala Ile Gly Trp 145 150 155 160 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Leu Cys Ile Asp 165 170 175 Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 180 185 190 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn 195 200 205 Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Pro Ile 210 215 220 Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 23249PRTArtificial Sequencenanobody or nanobody construct 23Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 130 135 140 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 145 150 155 160 Phe Thr Phe Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 165 170 175 Lys Gly Pro Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr 180 185 190 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 24275PRTArtificial Sequencenanobody or nanobody construct 24Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 165 170 175 Phe Ile Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly 180 185 190 Lys Glu Arg Glu Gly Val Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr 195 200 205 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 210 215 220 Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 225 230 235 240 Thr Gly Val Tyr Tyr Cys Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys 245 250 255 Leu Leu Glu Tyr Asp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 260 265 270 Val Ser Ser 275 25275PRTArtificial Sequencenanobody or nanobody construct 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 165 170 175 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 180 185 190 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 195 200 205 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 210 215 220 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 225 230 235 240 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 245 250 255 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 260 265 270 Val Ser Ser 275 26275PRTArtificial Sequencenanobody or nanobody construct 26Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly Gly 145 150 155 160 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 165 170 175 Phe Ile Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly 180 185 190 Lys Glu Arg Glu Gly Val Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr 195 200 205 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 210 215 220 Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 225 230 235 240 Thr Ala Val Tyr Tyr Cys Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys 245 250 255 Leu Leu Glu Tyr Asp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 260 265 270 Val Ser Ser 275 27275PRTArtificial Sequencenanobody or nanobody construct 27Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 165 170 175 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 180 185 190 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 195 200 205 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 210 215 220 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 225 230 235 240 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 245 250 255 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 260 265 270 Val Ser Ser 275 28383PRTArtificial Sequencenanobody or nanobody construct 28Asp Val Gln

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly Gly 130 135 140 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 145 150 155 160 Phe Ile Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Gly Val Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr 180 185 190 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys 225 230 235 240 Leu Leu Glu Tyr Asp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu 260 265 270 Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 275 280 285 Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe Gly Met Ser Trp 290 295 300 Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Ser Ile Ser 305 310 315 320 Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe 325 330 335 Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 340 345 350 Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly 355 360 365 Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser 370 375 380 295PRTArtificial Sequencenanobody or nanobody construct 29Ser Phe Gly Met Ser 1 5 3017PRTArtificial Sequencenanobody or nanobody construct 30Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 316PRTArtificial Sequencenanobody or nanobody construct 31Gly Gly Ser Leu Ser Arg 1 5 32115PRTArtificial Sequencenanobody or nanobody construct 32Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 33115PRTArtificial Sequencenanobody or nanobody construct 33Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 34115PRTArtificial Sequencenanobody or nanobody construct 34Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 35115PRTArtificial Sequencenanobody or nanobody construct 35Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 36115PRTArtificial Sequencenanobody or nanobody construct 36Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 37115PRTArtificial Sequencenanobody or nanobody construct 37Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 38115PRTArtificial Sequencenanobody or nanobody construct 38Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 39283PRTArtificial Sequencenanobody or nanobody construct 39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 130 135 140 Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 145 150 155 160 Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 165 170 175 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr 180 185 190 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ala Ala Asp Tyr Lys Asp 245 250 255 His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp 260 265 270 Asp Lys Gly Ala Ala His His His His His His 275 280 40249PRTArtificial Sequencenanobody or nanobody construct 40Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 130 135 140 Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 145 150 155 160 Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 165 170 175 Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr 180 185 190 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser 245 41246PRTArtificial Sequencenanobody or nanobody construct 41Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Tyr 20 25 30 Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Arg Pro Glu Trp Val 35 40 45 Ser Ser Ile Asp Thr Gly Gly Asp Ile Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Trp Cys 85 90 95 Ala Thr Asp Glu Glu Tyr Ala Leu Gly Pro Asn Glu Phe Asp Tyr Tyr 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 145 150 155 160 Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro 165 170 175 Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 195 200 205 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser 245 42246PRTArtificial Sequencenanobody or nanobody construct 42Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Tyr 20 25 30 Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Arg Pro Glu Trp Val 35 40 45 Ser Ser Ile Asp Thr Gly Gly Asp Ile Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Trp Cys 85 90 95 Ala Thr Asp Glu Glu Tyr Ala Leu Gly Pro Asn Glu Phe Asp Tyr Tyr 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 145 150 155 160 Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala 180 185 190 Asp Ser Val Lys Gly Arg Phe Thr Ile

Ser Arg Asp Asn Ala Lys Thr 195 200 205 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr 225 230 235 240 Leu Val Thr Val Ser Ser 245 43122PRTArtificial Sequencenanobody or nanobody construct 43Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asn Tyr 20 25 30 Asp Met Ala Trp Val Arg Gln Ala Pro Gly Lys Arg Pro Glu Trp Val 35 40 45 Ser Ser Ile Asp Thr Gly Gly Asp Ile Thr His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Trp Cys 85 90 95 Ala Thr Asp Glu Glu Tyr Ala Leu Gly Pro Asn Glu Phe Asp Tyr Tyr 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 44433PRTArtificial Sequencenanobody or nanobody construct 44Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Ile 20 25 30 Tyr Ala Lys Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 35 40 45 Val Ala Ala Ile Ser Arg Ser Gly Arg Ser Thr Ser Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Val Gly Gly Ala Thr Thr Val Thr Ala Ser Glu Trp Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 165 170 175 Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Ile Tyr 180 185 190 Ala Lys Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 195 200 205 Ala Ala Ile Ser Arg Ser Gly Arg Ser Thr Ser Tyr Ala Asp Ser Val 210 215 220 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 225 230 235 240 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 245 250 255 Ala Ala Val Gly Gly Ala Thr Thr Val Thr Ala Ser Glu Trp Asp Tyr 260 265 270 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 275 280 285 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 290 295 300 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val 305 310 315 320 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu 325 330 335 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met 340 345 350 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser 355 360 365 Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly 370 375 380 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln 385 390 395 400 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile 405 410 415 Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser 420 425 430 Ser 45250PRTArtificial Sequencenanobody or nanobody construct 45Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 130 135 140 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 145 150 155 160 Phe Thr Phe Arg Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 165 170 175 Lys Gly Pro Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr 180 185 190 Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Ala 245 250 46249PRTArtificial Sequencenanobody or nanobody construct 46Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Gly Val 35 40 45 Ser Ser Ile Ser Ser Thr Tyr Gly Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Pro Ile Gly Leu Ile Gly Leu Asp Ala Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 145 150 155 160 Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys 165 170 175 Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 195 200 205 Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Ala 245 47249PRTArtificial Sequencenanobody or nanobody construct 47Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Leu Gly Val 35 40 45 Ser Ser Ile Ser Ser Thr Tyr Gly Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Pro Ile Gly Leu Ile Gly Leu Asp Ala Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 145 150 155 160 Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys 165 170 175 Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 195 200 205 Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Ala 245 48249PRTArtificial Sequencenanobody or nanobody construct 48Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Leu Gly Val 35 40 45 Ser Ser Ile Ser Ser Thr Tyr Gly Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Pro Ile Gly Leu Ile Gly Leu Asp Ala Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 145 150 155 160 Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys 165 170 175 Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 195 200 205 Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Ala 245 49249PRTArtificial Sequencenanobody or nanobody construct 49Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Asp Asp Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Leu Gly Val 35 40 45 Ser Ser Ile Ser Ser Thr Tyr Gly Leu Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Pro Ile Gly Leu Ile Gly Leu Asp Ala Tyr Glu Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 145 150 155 160 Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys 165 170 175 Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 195 200 205 Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser Ser Ala 245

Claims

1. Polypeptide, protein, construct, compound or other chemical entity that comprises or essentially consists of the amino acid sequence Alb-23 (SEQ ID NO:1) or a variant of Alb-23 which is capable of specifically binding to human serum albumin and in which the amino acid residue at position 5 is L, the amino acid residue at position 16 is G, the amino acid residues at positions 44 and 45 are GP, the amino acid residues at positions 74 to 76 are SKN, and the amino acid residue at position 83 is R, and one or more (such as one or two) other immunoglobulin single variable domains.

2. (canceled)

3. (canceled)

4. Polypeptide, protein, construct, compound or other chemical entity according to claim 1, in which said one or more (such as one or two) other immunoglobulin single variable domains are one or more (such as one or two) therapeutic immunoglobulin single variable domains.

5. (canceled)

6. (canceled)

7. Polypeptide, protein construct, compound or other chemical entity according to claim 1, in which said one or more (such as one or two other immunoglobulin single variable domains are immunoglobulin single variable domains that comprise at least two disulphide bridges.

8. Polypeptide, protein construct, compound or other chemical entity according to claim 1, in which at least one (and up to all) of the other immunoglobulin single variable domains is a Nanobody of VHH Class I.

9.-13. (canceled)