RU2797270C2 - Advanced serum albumin binding agents - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a single immunoglobulin variable domain (ISVD) capable of specifically binding to serum albumin, a method for its production, as well as to a protein construct and composition containing the above ISVD. Also a nucleic acid encoding the above ISVD, a nucleic acid encoding the above protein construct, and cells containing the above nucleic acids are disclosed. The invention also relates to a method for obtaining the above protein structure.

EFFECT: invention is effective for increasing the half-life of therapeutic compounds, fragments or substances.

24 cl, 7 dwg, 4 tbl, 4 ex

Description

The present invention relates to amino acid sequences that can bind to serum albumin.

In particular, the present invention relates to immunoglobulin single variable domains, and in particular heavy chain single variable domains, which can bind to serum albumin.

As described herein, the immunoglobulin single variable domains provided by the invention are preferably such that they can (at least) bind (and in particular bind specifically) to human serum albumin. More preferably, as further described herein, these immunoglobulin single variable domains are preferably also such that they cross-react (as described herein) with human serum albumin and serum albumin of at least one other mammalian species.

The invention also relates to proteins, polypeptides and other constructs, compounds, molecules or chemicals that contain at least one of the single immunoglobulin variable domains that bind to serum albumin, which are described in this document.

Single immunoglobulin variable domains are also generically referred to herein by the abbreviations "ISV" or "ISVD" (to be used interchangeably herein).

The serum albumin-binding immunoglobulin single variable domains described herein will also be referred to herein as "amino acid sequences of the invention" or "serum albumin binding agents of the invention". As further described herein, the albumin-binding agents of the invention may specifically be nanobodies (as further described herein).

Proteins, polypeptides and other constructs, compounds, molecules or chemicals that include at least one of the serum albumin binding agents of the invention will also be referred to herein as "compounds of the invention" or as "polypeptides of the invention". Preferably, the compounds of the invention are proteins or polypeptides, and in particular may be fusion proteins.

Other aspects, embodiments, features, applications, and advantages of the invention will be apparent to those skilled in the art based on the disclosures herein.

In this application, the amino acid residues/positions in the variable domain of an immunoglobulin heavy chain will be designated by Kabat numbering. For convenience, in Fig. 1 is a table listing some of the amino acid positions that will be specifically referred to in this document and their numbering in accordance with some alternative numbering systems (such as Aho and IMGT. Note: unless explicitly stated otherwise, in this description and Kabat numbering is decisive in the claims; other numbering systems are for reference only).

Regarding CDRs, as is well known in the art, there are many conventions for defining and describing a CDR from a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location structural loop regions). A link is made, for example, to the website http://www.bioinf.org.uk/abs/. For the purposes of this specification and claims, even though Kabat CDRs may also be mentioned, CDRs are most preferably determined based on the Abm determination (which is based on Oxford Molecular's AbM antibody modeling software), as this is considered optimal. a compromise between the definitions of Kabat and Chothia. The link is again made to the website http://www.bioinf.org.uk/abs/).

Accordingly, in the present description and claims, all CDRs are defined in accordance with the Abm convention, unless expressly stated otherwise.

ISVDs (and in particular nanobodies) that can bind to serum albumin and their use are well known in the art, e.g. PCT/EP2016/077973 which describe serum albumin binding ISVDs and their use to extend the serum half-life (as defined in these applications) of therapeutic compounds, fragments and substances. For example, WO 2006/122787 discloses a humanized serum albumin-binding nanobody designated as Alb-8 as SEQ ID NO: 62 (see SEQ ID NO: 1 herein). WO 2012/175400 discloses a humanized serum albumin-binding nanobody designated as Alb-23D as SEQ ID NO: 6 (see SEQ ID NO: 2 herein). The amino acid sequences of Alb-8 and Alb-23D and their CDRs (which are the same for Alb-8 and Alb-23D) are shown in Table A below as SEQ ID NOs: 1, 2 and 3-8, respectively.

Some other references that disclose ISVD against serum albumin include WO 2003/035694, WO 2004/003019, EP 2139918, WO 2011/006915 and WO 2014/111550.

In FIG. 3A and 3B show alignments of Alb-8 (reference), Alb-23D (reference), SEQ ID NO: 18 (according to the invention) and SEQ ID NO: 19 (according to the invention).

The present invention is directed to improved serum albumin binding agents, and in particular serum albumin binding agents, which have improved properties compared to serum albumin binding agents known in the art. In particular, it is an object of the invention to provide serum albumin binding agents that can bind to canine serum albumin and/or that have improved cross-reactivity between human serum albumin and canine serum albumin (i.e. compared to prior art albumin binding agents). techniques such as Alb-8 and/or Alb-23D).

Table A: Alb-8, Alb-23D and their CDRs

SEQ
ID NO Description Subsequence 1 Alb-8
(WO 2006/122787; SEQ ID NO: 62) EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 2 Alb-23D
(WO 2012/175400; SEQ ID NO: 6) EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA 3 CDR1 (Kabat) SFGMS 4 CDR2 (Kabat) SISGSGSDTLYADSVKG 5 CDR3 (Kabat/Abm) GGSLSSR 6 CDR1 (Abm) GFTFRSFGMS 7 CDR2 (Abm) SISGSGSDTL 8 CDR3 (Kabat/Abm) GGSLSSR Note:
- SEQ ID NO NO: 1 and 2 share the same CDRs according to Kabat. However, if the CDR is defined according to the Abm convention, then SEQ ID NO: 1 has a different CDR1 in SEQ ID NO: 2 compared to SEQ ID NO: 2, SEQ ID NO: 1 contains S at position 30 instead of R.
- SEQ ID NO:5 and SEQ ID NO:8 are identical.
- All CDRs are defined in accordance with the Abm convention, unless otherwise noted.

Table B: SEQ ID NOs: 18 and 19 and their CDRs.

SEQ
ID NO Description Subsequence 18 T0235005G01 EVQLVESGGGLVQAGGSLRLSCAASGSNISSYVMGWFRRAPGKEREFVAAISRSGGYTYYADSVKGRFTISRDNAKKTAYLQMNSLEPEDTAVYYCAAGRYSAWYSQSYEYDYWGQGTLVTVSS 19 T023500043 EVQLVESGGGVVQPGGSLRLSCAASGSTISSYVMGWFRRAPGKEREFVAAISRSGGYTYYADSVKGRFTISRDNSKKTAYLQMNSLRPEDTALYYCAAGRYSAWYSQSYEYDYWGQGTLVTVSS 9 CDR1 (Kabat) SYVMG 10 CDR2 (Kabat) AISRSGGYTYYADSVKG eleven CDR1 (Abm) GSNISSYVMG 12 CDR1 (Abm) GSTISSYVMG 13 CDR2 (Abm) AISRSGGYTY 14 CDR3 (Kabat/Abm) GRYSAWYSQSYEYDY Note:
- All CDRs are defined in accordance with the Abm convention, unless otherwise noted.

In general, the serum albumin-binding ISVDs provided by the present invention are variants of the sequences of SEQ ID NOS: 18 and 19 in which:

- they have the same CDRs (or essentially the same CDRs) as the sequence of SEQ ID NO: 18 or the sequence of SEQ ID NO: 19; And

- they have a certain degree of sequence identity with the sequence of SEQ ID NO: 18 and/or 19 (this degree of sequence identity is as described below).

In particular, the serum albumin-binding ISVDs provided by the present invention will typically have a (limited) set of "amino acid differences" (as described herein) compared to the sequence of SEQ ID NO: 18 and/or 19. These amino acid differences may be present. in CDRs (provided that the resulting amino acid sequences are such that they retain the additional properties of the amino acid sequences of the present invention as set forth in this application) and/or be present in framework regions, and may in particular be present in framework regions (as defined according to with Kabat and/or according to Abm). For example, and without limitation, these amino acid differences can be, for example, humanizing substitutions; substitutions that improve expression in the desired host cell or host organism; substitutions that improve stability and/or resistance to degradation and/or proteases; mutations that reduce binding by preexisting antibodies, and/or other mutations that are designed to optimize the sequence of the amino acid sequences of the invention; or any suitable combination of such amino acid differences. Reference is made to further disclosure in this document.

In a first aspect, the invention relates to an ISVD that can bind (and in particular bind specifically) to human serum albumin and which has:

- CDR1 (according to Kabat), which is the amino acid sequence of SYVMG (SEQ ID NO: 9) or an amino acid sequence that has a 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence of SEQ ID NO: : 9; And

- CDR2 (according to Kabat), which is the amino acid sequence AISRSGGYTYYADSVKG (SEQ ID NO: 10) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) (as defined herein) from the amino acid sequence SEQ ID NO: 10; And

- CDR3 (according to Kabat), which is the amino acid sequence GRYSAWYSQSYEYDY (SEQ ID NO: 14) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SEQ ID NO: 14.

In particular, the serum albumin binding agent according to this aspect of the invention may be (and preferably is) as further described herein.

In a more specific aspect, the invention relates to an ISVD that can bind (and in particular bind specifically) to human serum albumin, and which has:

- CDR1 (according to Kabat), which is the amino acid sequence of SYVMG (SEQ ID NO: 9); And

- CDR2 (according to Kabat), which is the amino acid sequence AISRSGGYTYYADSVKG (SEQ ID NO: 10); And

- CDR3 (according to Kabat), which is the amino acid sequence GRYSAWYSQSYEYDY (SEQ ID NO: 14).

Again, the serum albumin binding agent according to this aspect of the invention may be (and preferably is) as further described herein.

In another aspect, the invention relates to an ISVD that can bind (and in particular bind specifically) to human serum albumin, and which has:

- CDR1 (according to Abm), which is the amino acid sequence GSNISSYVMG (SEQ ID NO: 11) or GSTISSYVMG (SEQ ID NO: 12), or an amino acid sequence that has 2 or 1 amino acid difference(s) (as defined in herein) with the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; And

- CDR2 (according to Abm), which is the amino acid sequence AISRSGGYTY (SEQ ID NO: 13), or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) (as defined in this application) with the amino acid sequence SEQ ID NO:13; And

- CDR3 (according to Abm), which is the amino acid sequence GRYSAWYSQSYEYDY (SEQ ID NO: 14) or an amino acid sequence that has 3, 2 or 1 amino acid difference(s) (as defined herein) from the amino acid sequence SEQ ID NO: 14.

In particular, the serum albumin binding agent according to this aspect of the invention may be (and preferably is) as further described herein.

In a more specific aspect, the invention relates to an ISVD that can bind (and in particular bind specifically) to human serum albumin, and which has:

- CDR1 (according to Abm), which is the amino acid sequence of GSNISSYVMG (SEQ ID NO: 11) or GSTISSYVMG (SEQ ID NO: 12); And

- CDR2 (according to Abm), which is the amino acid sequence AISRSGGYTY (SEQ ID NO: 13); And

- CDR3 (according to Abm), which is the amino acid sequence GRYSAWYSQSYEYDY (SEQ ID NO: 14).

Again, the serum albumin binding agent according to this aspect of the invention can be (and preferably is) as further described herein.

In general, serum albumin binding agents according to various aspects of the invention are preferably such that they have:

- degree of sequence identity with the sequence of SEQ ID NO: 18 and/or 19 (in which the CDR and any C-terminal extension that may be present are not taken into account to determine the degree of sequence identity) of at least 85%, preferably at least 90% , more preferably at least 95%;

and/or those that have:

- and/or have no more than 7, preferably no more than 5, e.g. only 3, 2, or 1 "amino acid differences" (as defined herein, ignoring CDR extension and any C-terminal extension that may be present) with the sequence SEQ ID NO: 18 and/or 19.

Serum albumin binding agents according to various aspects of the invention are generally preferably such that they bind to human serum albumin with a dissociation constant (KD) of 10 ^-5 to 10 ^-12 mol/liter or less, and preferably 10 ^-7 up to ^10-12 mol/liter or less, and more preferably from 10 ^-8 to 10 ^-12 mol/liter, and/or with a binding affinity of at least 10 ⁷ M ^-1 , preferably at least 10 ⁸ M ^-1 , more preferably at least 10 ⁹ M ^-1 , such as at least 10 ¹² M ^-1 as determined using ProteOn (refer to Example 1). Preferably, the serum albumin binding agent of the invention will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM, again as determined using ProteOn (referring again to Example 1).

Serum albumin binding agents according to various aspects of the invention are preferably also such that they compete with the amino acid sequence of SEQ ID NO: 18 for binding to (human) serum albumin and/or that they "cross-block" (as defined herein). document) binding the amino acid sequence of SEQ ID NO: 18 to (human) serum albumin.

Preferably, the albumin binding agents of the invention are such that they bind to essentially the same amino acid residues and/or epitopes in human serum albumin as SEQ ID NO: 18, and even more preferably, they have essentially the same amino acid interactions. , as SEQ ID NO: 18. For this purpose, in accordance with a specific, but not limiting aspect, the albumin binding agents of the invention preferably either have the same CDRs as the sequence of SEQ ID NO: 18, or compared with the sequence of SEQ ID NO: 18 preferably contain within their CDRs only those mutations (such as conservative amino acid substitutions) that still allow them to undergo the same or substantially the same amino acid interactions with human serum albumin as for SEQ ID NO: 18.

The serum albumin binding agents according to various aspects of the invention are generally preferably also such that they cross-react with human serum albumin and serum albumin from at least one, preferably from at least two, more preferably from at least three, and substantially all of the following mammals: dog, rat, mouse, rabbit, guinea pig, pig, sheep, cow, and cynomolgus monkey. In particular, the serum albumin binding agents according to various aspects of the invention are such that they (at least) cross-react with human serum albumin and canine serum albumin, and preferably also with either human serum albumin and/or canine serum albumin, on one on the other hand, and at least one, preferably at least two, more preferably all three of rat serum albumin, mouse serum albumin and cynomolgus macaque serum albumin, on the other hand. In this regard, the serum albumin binding agents of the invention may have improved cross-reactivity (particularly with human serum albumin on the one hand and canine serum albumin on the other hand) compared to serum albumin binding agents which have (according to essentially) the same CDR. like Alb-11 and/or Alb-23D. A link is provided to the data in the experimental part below.

For reference in FIG. Figure 11 shows the alignment of serum albumin in different mammalian species (source: http://macromoleculeinsights.com/albumin.php, the amino acid numbering in Figure 11 is the numbering used on the referenced web page). For convenience, portions of amino acids have been highlighted in the human serum albumin sequence that are believed to be part of the putative epitope of the amino acid sequences of the invention. Without being limited to any particular mechanism or hypothesis, it is believed that the amino acid sequences of the invention are (substantially) capable of binding (one or more amino acid residues within) corresponding extensions from amino acid residues that are present within the amino acid sequence of those mammalian serum albumins with which cross-react amino acid sequences of the invention.

In general, serum albumin binding agents of the invention can be considered as cross-reactive with human serum albumin and serum albumin of one of these species when they can bind to human serum albumin with an affinity of less than 500 nM, preferably less than 200 nM. more preferably less than 10 nM; and also with serum albumin of said species with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, again as determined using ProteOn (reference is again made to Example 1).

Serum albumin binding agents according to various aspects of the invention are also preferably either such that:

they have a human serum half-life (expressed as t1/2 beta) that is greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours; for example, about one week, two weeks, and up to the half-life of human serum albumin (estimated to be about 19 days);

and/or such that:

- when they bind to a therapeutic moiety or substance, they confer a human serum half-life (expressed as t1/2 beta) to the resulting polypeptide of the invention, which is greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even greater preferably more than 72 hours; for example, about one week, two weeks, and up to the half-life of human serum albumin (estimated to be about 19 days).

The half-life in non-human mammalian species will, among other factors, depend primarily on the binding properties (such as affinity) of the albumin-binding agent of the invention for serum albumin from said mammalian species, as well as the half-life of intact serum albumin in said species. . In accordance with a preferred embodiment of the invention, when the serum albumin binding agent of the invention is cross-reactive (as defined herein) with human serum albumin and serum albumin from another mammalian species, then the half-life of the serum albumin binding agent of the invention (and/ or a compound of the invention containing said serum albumin binding agent) as defined for said species is preferably at least 5%, such as at least 10%, more preferably at least 25%, such as about 50%, and possibly up to 100% of the half-life of intact serum albumin in these species.

Compared to the sequence of SEQ ID NO: 18, the serum albumin binding agents of the invention preferably also contain (at least):

- one or more humanizing substitutions;

and/or

- one or more mutations (ie amino acid substitutions, deletions or additions, and in particular substitutions) that reduce binding to preexisting antibodies;

and may optionally contain one or more additional mutations as described herein.

For suitable humanizing substitutions (and suitable combinations thereof), for example, reference is made to WO 09/138519 (or the prior art cited in WO 09/138519) and WO 08/020079 (or the prior art cited in WO 08 /020079), as well as tables A-3 to A-8 of WO 08/020079 (which are lists showing possible humanizing substitutions). Some preferred, but non-limiting examples of such humanizing substitutions are Q108L and A14P, or a suitable combination thereof. Such humanizing substitutions may also be appropriately combined with one or more other mutations as described herein (eg, one or more mutations that reduce binding by preexisting antibodies).

With regard to suitable mutations that can reduce binding by preexisting antibodies (and suitable combinations of such mutations), reference is made to, for example, WO 2012/175741 and WO 2015/173325, and also to, for example, WO 2013/024059 and WO 2016/118733 . As described therein, such mutations may include (in a suitable combination) one or more amino acid substitutions, deletions or additions (and in particular substitutions), which will often be located in the so-called C-terminal region of the ISV. For example, such mutations may contain mutations (and in particular substitutions) at one or more positions 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89 , 103, 108 and/or mutations at one or more positions in the C-terminal sequence of VTVSS (i.e., positions 109, 110, 111, 112 and 113), where one or more mutations at positions 11, 89, 110 and/or 112 are particularly preferred. Some preferred, but non-limiting examples of such mutations are suitable substitutions (where appropriate) such that after the mutation, one of the following amino acid residues is present at the indicated position: 11L, 11K, 11V, 14A, 14P, 41A, 41L, 41P, 41S , 41T, 42E, 42G, 87A, 87T, 89A, 89L, 89T, 108L, 110K, 110Q, 112K and/or 112Q (where 11L, 89A, 89L, 89T, 110K, 110Q, 112K and/or 112Q are especially preferred ); or any suitable combination of such substitutions, such as, for example, 11V in combination with 89L or 89T; 11V combined with 110K or 110Q; or 11V in combination with but not limited to 89L and 110K or 110Q. Such mutations that reduce binding to preexisting antibodies may also be appropriately combined with one or more other mutations as described herein (eg, one or more humanizing substitutions).

Where appropriate (as further described herein, and in particular when a serum albumin binding agent of the invention is present and/or forms the C-terminal portion of a compound of the invention in which it is present), to reduce binding by preexisting antibodies, serum albumin binding agents of the invention (and, as further described herein, also compounds of the invention) may also contain a C-terminal extension (such as a C-terminal alanine residue). As described in WO 2012/175741, this C-terminal extension reduces binding by pre-existing antibodies. A suitable C-terminal extension may be generally further described herein, and in particular may be of the formula -(X)n, in which X may be any naturally occurring amino acid (but preferably not cysteine) and n may be 1, 2, 3, 4 or 5. Reference is again made to WO 2012/175741 and also, for example, to WO 2015/173325, WO 2013/024059 and WO 2016/118733. The presence of such a C-terminal extension may also be appropriately combined with one or more of the other mutations described herein (eg, one or more humanizing substitutions and/or one or more mutations that reduce binding by preexisting antibodies).

Other mutations that may be present in the serum albumin binding agents of the invention, for example, include one or more mutations (in particular substitutions) that improve expression in the desired host cell or host organism, one or more mutations (and in particular, substitutions) that improve stability and/or resistance to degradation and/or proteases, and/or one or more other mutations that are designed to optimize the sequence of the amino acid sequences of the invention (for example, one or more mutations that (additionally) reduce which - or the propensity of agents that bind albumin, to form dimers, but not limited to this); or any suitable combination of such mutations, but are not limited to.

Some non-limiting examples of such mutations are suitable substitutions (if necessary) such that after the mutation, one of the following amino acid residues is present at the indicated position: 28T, 39Q, 74S, 76N, 78L, and 83R; or any suitable combination of such substitutions (eg, to form the SKN motif at positions 74-76). In addition, if desired (as further described herein), the serum albumin binding agents of the invention may have a D at position 1 (i.e., an E1D mutation compared to the sequence of SEQ ID NOs: 18 and/or 19), in particular, when the serum albumin binding agent of the invention is present and/or forms the N-terminal portion of the compound of the invention in which it is present. Such mutations may again be appropriately combined with one or more other mutations as described herein (eg, one or more humanizing substitutions and/or one or more mutations that reduce binding by preexisting antibodies).

Other mutations that may be present in the amino acid sequences of the invention will be apparent to those skilled in the art based on the disclosure herein.

It is also possible that a single mutation (or a suitable combination of mutations) provides multiple functionalities or benefits. For example, and without limitation, a humanizing Q108L substitution can also reduce binding by preexisting antibodies.

Some preferred, but non-limiting examples of amino acid residues (i.e., mutations compared to the amino acid sequence of SEQ ID NO: 18) that may be present in the amino acid sequences of the invention (i.e., alone or in a suitable combination) include: 11V (i.e. L11V), 14P (i.e. A14P), 28T (i.e. N28T), 39Q (i.e. R39Q), 74S (i.e. A74S), 76N (i.e. K76N), 78L (i.e. A78L), 83R (i.e. E83R), 89L (i.e. V89L), 89T (i.e. V89T), 110K (i.e. T110K) or 110Q (i.e., T110Q); and, where appropriate (as further described herein), 1D (eg, E1D) and/or C-terminal extension (X)n as defined herein (eg, 114A). Reference is also made to the sequences and mutations shown in Figures 4A and 4B. For example, some preferred, but non-limiting examples of suitable combinations of such amino acid residues (i.e., mutations compared to the amino acid sequence of SEQ ID NO: 18) include:

- L11V, A14P, N28T, R39Q, A74S, E83R, V89L;

- L11V, A14P, N28T, A74S, K76N, E83R, V89L;

- L11V, A14P, N28T, A74S, A78V, E83R, V89L;

- L11V, A14P, N28T, R39Q, A74S, K76N, E83R, V89L;

- L11V, A14P, N28T, R39Q, A74S, K76N, E83R, V89L;

- L11V, A14P, N28T, R39Q, A74S, A78V, E83R, V89L;

- L11V, A14P, N28T, A74S, K76N, A78V, E83R, V89L;

- L11V, A14P, N28T, R39Q, A74S, K76N, A78V, E83R, V89L;

- L11V, A14P, N28T, A74S, A78L, E83R, V89L;

- L11V, A14P, N28T, R39Q, A74S, A78L, E83R, V89L;

- L11V, A14P, N28T, A74S, K76N, A78L, E83R, V89L; And

- L11V, A14P, N28T, R39Q, A74S, K76N, A78L, E83R, V89L;

and other suitable combinations that will be understood by those skilled in the art based on the disclosure herein.

Some preferred, but non-limiting, amino acid sequences of the invention are those in which (i) position 11 is V and position 89 is L; (ii) position 89 is T; (iii) provisions 74-76 form the SKN motif; (iv) position 11 is V, position 89 is L, and positions 74-76 form an SKN motif; where the amino acid sequences of the invention are as described hereinafter (and accordingly, may also appropriately contain one or more additional amino acid mutations, as described herein).

Some preferred, but non-limiting examples of the amino acid sequences of the invention are shown in FIG. 2 as:

- SEQ ID NO: 18-31, which are examples of amino acid sequences according to the invention without C-terminal alanine extension;

- SEQ ID NO: 32-45, which are examples of the amino acid sequences of the invention with a C-terminal extension (in each case, exemplified by a C-terminal alanine extension, which is usually the preferred C-terminal extension); And

- SEQ ID NO: 46-59, which are examples of amino acid sequences according to the invention with N-terminal mutation E1D).

Based on the further disclosure herein, it will be understood by those skilled in the art that, in practice:

albumin binding agents of the invention with C-terminal extension (such as those from SEQ ID NOs: 32-45) will often be used/present at the C-terminal portion of the polypeptides of the invention (as defined herein) in which they are present;

albumin binding agents of the invention with an E1D mutation (such as mutations of SEQ ID NOs: 46-59) will often be used/present at the N-terminal portion of the polypeptides of the invention in which they are present;

albumin binding agents of the present invention without a C-terminal extension and without an E1D mutation (such as SEQ ID NOs: 18-31 mutations) will often be present somewhere in the "middle" of the polypeptide of the invention.

Each of the amino acid sequences of SEQ ID NOs: 18-59, as well as proteins, polypeptides, and other compounds and constructs containing them (as further described herein), form additional aspects of the present invention.

In a further aspect, the invention relates to an amino acid sequence which is one of the amino acid sequences of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 , SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO :40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 , SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 or SEQ ID NO:59; and each of these amino acid sequences of the invention (as well as the polypeptides of the invention - as defined herein - which comprise such an amino acid sequence of the invention) form a further aspect of the invention.

As further described herein, the amino acid sequences provided by the invention are proteins that can bind, and in particular can specifically (as described herein) bind to human serum albumin. Thus, they can be used as binding units or binding domains for binding to (human) serum albumin, for example, to provide an increase in the half-life (as defined herein) of therapeutic compounds, fragments or substances. With regard to the use of serum albumin binding domains to increase the half-life of therapeutic compounds, fragments or substances, for example, reference is made to WO 2004/041865, WO 2006/122787, EP 2139918, WO 2011/006915, WO 2012/175400 and/or WO 2014/111550. The serum albumin binding agents of the invention can generally be used in the same manner and for the same purposes as the serum albumin binding agents described in these references.

In some additional non-limiting aspects, the invention also relates to:

- proteins, polypeptides and other constructs, molecules or chemicals that contain or essentially consist of at least one serum albumin binding agent of the invention, as described herein (again, also referred to herein as "compounds of the invention "or as "polypeptides according to the invention");

- methods for expressing/obtaining a serum albumin-binding agent of the invention and/or a compound of the invention;

a host cell, host organism, or other (expressing) system that can express or produce a serum albumin binding agent of the invention and/or a compound of the invention;

- compositions and products (such as pharmaceutical compositions and products) which contain a serum albumin binding agent according to the invention and/or a compound according to the invention;

- nucleotide sequences and nucleic acids, such as (expressing) vectors, which encode a serum albumin binding agent of the invention and/or compounds of the invention;

- the use of compounds of the invention and/or compounds of the invention, such as the use of a compound of the invention to increase the (serum) half-life of a therapeutic compound, moiety or substance and the therapeutic and/or prophylactic use of a compound of the invention.

These and additional aspects, embodiments, advantages, applications and uses of the invention will become apparent from the further description provided herein.

In the present description:

- the term "single immunoglobulin variable domain" (also called "ISV" or "ISVD") is commonly used to refer to immunoglobulin variable domains (which can be heavy chain or light chain domains, including VH, VHH or VL domains) that can form a functional an antigen-binding site without interaction with another variable domain (eg, without VH/VL interaction, as required between the VH and VL domains of a conventional 4-chain monoclonal antibody). Examples of ISVDs will be understood by those skilled in the art and, for example, include nanobodies (including VHH, humanized VHH and/or camelized VHs such as camelized human VHs), IgNARs, domains, (single domain) antibodies (such as dAbs™) that are VH domains. or derived from a VH domain, and (single domain) antibodies (such as dAbs™) that are VL domains or derived from a VL domain. Unless explicitly stated otherwise, ISVDs that are based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. Most preferably, unless explicitly stated otherwise, the ISVD will be a nanobody.

- the term "nanobody" is generally as defined in WO 2008/020079 or WO 2009/138519, and thus, in a specific aspect, generally means VHH, humanized VHH, or camelized VH (such as camelized human VH) or typically a sequence-optimized VHH (such as, for example, optimized for chemical stability and/or solubility, maximum overlap with known human framework regions, and maximum expression). It should be noted that the terms "nanobody" or "nanobody" are registered trademarks of Ablynx N.V. and therefore may also be referred to as Nanobody® and/or Nanobody®);

- in general, unless otherwise indicated herein, ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs mentioned herein will be for use in the prevention or treatment of diseases or disorders in humans (and/or, if appropriate, also in warm-blooded animals, and in particular mammals). Thus, in general, ISVDs, nanobodies, polypeptides, proteins, and other compounds and constructs described herein are preferably such that they can be used as and/or suitably be part of a (biological) drug or other pharmaceutical product. or a therapeutically active compound and/or a pharmaceutical product or composition. Such drug, compound, or product is preferably such that it is suitable for administration to a human, for example, for the prevention or treatment of a subject in need of such prevention or treatment, or, for example, as part of a clinical trial. As further described herein, for this purpose, such a drug or compound may contain moieties, substances, or binding units other than the ISVDs provided by the invention (which, as also described herein, may, for example, be one or more other additional therapeutic groups and/or one or more other moieties that affect the pharmacokinetic or pharmacodynamic properties of ISVD or nanobody biologicals, such as their half-life). Suitable examples of such additional therapeutic or other moieties will be understood by those skilled in the art and, for example, may typically include any therapeutically active protein, polypeptide, or other binding domain or binding unit, as well as, for example, such modifications as described on pages 149 -152 WO 2009/138159. The ISVD or nanobody biological is preferably a therapeutic agent or intended for use as a therapeutic agent (which includes prophylaxis and diagnosis) and for this purpose preferably contains at least one ISVD against a therapeutically relevant target (such as, for example, , RANK-L, vWF, IgE, RSV, CXCR4, IL-23 or other interleukins, etc.). For some specific, but non-limiting examples of such ISVD or nanobody biologicals, reference is made to Examples 8-18, as well as, for example, various uses of Ablynx N.V. (such as, for example, and without limitation, WO 2004/062551, WO 2006/122825, WO 2008/020079 and WO 2009/068627) and also, for example (and without limitation) for applications such as in WO 2006/ 038027, WO 2006/059108, WO 2007/063308, WO 2007/063311, WO 2007/066016 and WO 2007/085814. In addition, as further described herein, the additional fragment may be an ISVD or nanobody as described herein directed against a (human) serum protein such as (human) serum albumin, and such an ISVD or nanobody may also find therapeutic the use, in particular, for the purpose and/or to increase the half-life of the agents that bind TNF described in this document. Reference is made to, for example, WO 2004/041865, WO 2006/122787 and WO 2012/175400 which generally describe the use of serum albumin-binding nanobodies to increase half-life. In addition, in the present description, unless expressly stated otherwise, all terms mentioned in this document have the meaning given in WO 2009/138519 (or in the prior art cited in WO 2009/138519) or WO 2008/020079 (or in the prior art cited in WO 2008/020079). In addition, when a method or technique is not specifically described herein, it can be performed as described in WO 2009/138519 (or the prior art cited in WO 2009/138519) or WO 2008/020079 (or the prior art cited in WO 2008/020079). In addition, as described herein, any pharmaceutical product or composition containing any ISVD or compound of the invention may also contain one or more additional components known as such for use in pharmaceutical products or compositions (i.e., depending on intended pharmaceutical form) and/or, for example, one or more other compounds or active ingredients intended for therapeutic use (ie, to provide a combination product).

In addition, when used in the present specification or claims, the following terms have the same meaning as given and/or, where applicable, may be defined in the manner described on pages 62-75 of WO 2009/138519: "agonist", " antagonist", "inverse agonist", "non-polar uncharged amino acid residue", "polar uncharged amino acid residue", "polar charged amino acid residue", "sequence identity", "exactly the same" and "amino acid difference" (when referring to sequence comparison two amino acid sequences), “(in) a substantially isolated (form)”, “domain”, “binding domain”, “antigenic determinant”, “epitope”, “against” or “against” (antigen), “specificity” and "half-life". In addition, the terms "modulatory" and "modulate", "interaction site", "specific for", "cross-block", "cross-block" and "cross-block", and "substantially independent of pH" are as defined ( and/or may be defined as described) on pages 74-79 of WO 2010/130832 from Ablynx NV. In addition, when referring to a construct, compound, protein, or polypeptide of the invention, terms such as "univalent", "bivalent" (or "polyvalent"), "bispecific" (or "polyspecific"), and "biparatopic" (or " polyparatopic") may have the meaning given in WO 2009/138519, WO 2010/130832 or WO 2008/020079.

As used herein, the term "half-life" in relation to an ISVD, nanobody, ISVD biological substance, nanobody biological substance, or any other amino acid sequence, compound, or polypeptide referred to herein can generally be defined as described in paragraph o) on page 57 of WO 2008/020079, and as mentioned therein, refers to the time it takes for the serum concentration of an amino acid sequence, compound or polypeptide to decrease by 50% in vivo, e.g. due to degradation of the sequence or compound, and/ or clearance or sequestration of the sequence or connection by natural mechanisms. The in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined by any known method such as pharmacokinetic analysis. Suitable techniques will be understood by a person skilled in the art and may, for example, be generally as described in paragraph (o) on page 57 of WO 2008/020079. As also mentioned in paragraph (o) on page 57 of WO 2008/020079, half-life can be expressed using parameters such as t1/2-alpha, t1/2-beta and area under the pharmacokinetic curve (AUC). In this regard, it should be noted that the term "half-life" as used herein specifically refers to t1/2-beta or terminal half-life (in which t1/2-alpha and/or AUC or both may not be considered) . Reference is made, for example, to the experimental section below, as well as to standard reference books such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). Similarly, the terms "increased half-life" or "increased half-life" are also defined in paragraph (o) on page 57 of WO 2008/020079 and specifically refer to an increase in t1/2-beta, with or without an increase in t1/2 -alpha and/or AUC, or both.

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

Also, as already mentioned herein, the amino acid residues of the nanobody are numbered according to the general VH numbering given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), applied to camelid VHH domains in Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195; or referred to in this document. According to this numbering, nanobody FR1 contains amino acid residues at positions 1-30, nanobody CDR1 contains amino acid residues at positions 31-35, nanobody FR2 contains amino acids at positions 36-49, nanobody CDR2 contains amino acid residues at positions 50-65 , nanobody FR3 contains amino acid residues at positions 66-94, nanobody CDR3 contains amino acid residues at positions 95-102, and nanobody FR4 contains amino acid residues at positions 103-113. [In this regard, it should be noted that - as is well known in the art for VH domains and VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (then there are one or more positions according to Kabat numbering that cannot be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for Kabat numbering). This means that, in general, the Kabat numbering may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. In general, however, it can be said that according to the Kabat numbering and regardless of the number of amino acid residues in the CDR, position 1 according to the Kabat numbering corresponds to the beginning of FR1 and vice versa, position 36 according to the Kabat numbering corresponds to the beginning of FR2 and vice versa, position 66 according to the numbering Kabat corresponds to the beginning of FR3 and vice versa, and position 103 according to Kabat numbering corresponds to the beginning of FR4 and vice versa].

Alternative methods for numbering amino acid residues of VH domains, which can also be applied similarly to camelid VHH domains and to nanobodies, is the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, aspects and figures, the Kabat numbering for VHH domains proposed by Riechmann and Muyldermans will be used unless otherwise indicated.

It should also be noted that the figures, any sequence listing and experimental portion/examples are provided only to further illustrate the invention and should not be interpreted or construed as limiting the scope of the invention and/or the appended claims in any way, unless expressly stated otherwise.

As further described herein, the serum albumin binding agents of the invention can be used advantageously as a moiety, linker, or fusion partner to increase the half-life of therapeutic compounds, moieties, or substances such as polypeptides, proteins, compounds (including small molecules, but not limited to) or other therapeutic substances.

Thus, in another aspect, the invention relates to polypeptides, proteins, constructs, compounds, or other chemicals that include or essentially consist of a serum albumin binding agent of the invention and one or more other amino acid sequences, (binding) domains, binding units, or other fragments or chemicals.

In particular, the invention relates to polypeptides, proteins, constructs, compounds or other chemicals that contain the serum albumin binding agent of the invention and one or more (for example, one or two) therapeutic moieties (which may be the same or different, and may, for example, be directed against the same target or different targets, and when directed against the same target, they may be directed against 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.

The invention further relates to the therapeutic use of such polypeptides, proteins, constructs or compounds, and to pharmaceutical compositions containing such polypeptides, proteins, constructs or compounds.

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

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

In a preferred aspect, at least one therapeutic fragment contains or essentially consists of at least one immunoglobulin single variable domain, such as a domain antibody, single domain antibody, "dAb", or nanobody (such as VHH, humanized VHH, or camelized VH) or domain IgNAR.

In a particular embodiment, at least one therapeutic moiety comprises or essentially consists of at least one monovalent nanobody or a divalent, polyvalent, bispecific, or polyspecific nanobody construct.

Polypeptides, (fusion) proteins, constructs or compounds that contain a serum albumin binding agent of the invention and one or more therapeutic groups can generally be (obtained and used) as described in the prior art cited above (such as WO 04/041865, WO 06/122787, WO 2012/175400 and WO 2015/173325; also referred to, for example, WO 2004/003019, EP 2139918, WO 2011/006915 and WO 2014/111550), serum albumin agent according to the invention instead of the components that increase the half-life described in the above.

Polypeptides, (fusion) proteins, constructs or compounds that contain a serum albumin binding agent of the invention and one or more therapeutic moieties will generally and preferably have an extended half-life (as described herein and preferably expressed as t1/2- beta) compared to the therapeutic fragment or fragments as such.

In general, the compounds, polypeptides, constructs, or fusion proteins described herein preferably have a half-life (again as described herein, and preferably expressed as t1/2-beta) that is at least 1.5 times , preferably at least 2-fold, such as at least 5-fold, such as at least 10-fold or greater than 20-fold, than the half-life of the corresponding therapeutic moiety as such (as measured in a human or suitable animal such as a mouse or cynomolgus macaque).

Also preferably, any such compound, polypeptide, fusion protein, or construct has a half-life (again, as described herein and preferably expressed as t1/2-beta) in a human that is elevated by at least 1 hour, preferably more than 2 hours, more preferably more than 6 hours, eg more than 12 hours, compared to the half-life of the corresponding therapeutic component as such.

Also preferably, the compound or polypeptide of the invention has a half-life (again as described herein, and preferably expressed as t1/2-beta) in humans that is greater than 1 hour, preferably greater than 2 hours, more preferably greater than 6 hours eg, more than 12 hours and, eg, about one day, two days, one week, two weeks, and up to the human serum albumin half-life (estimated to be about 19 days).

As mentioned, in one aspect, the serum albumin binding agent of the present invention is used to increase the half-life of (one or more) immunoglobulin single variable domains such as domain antibodies, single domain antibodies, dAb, VHH or nanobodies (such as VHH, humanized VHH or camelized VHs such as camelized human VHs).

Thus, one embodiment of the invention provides a polypeptide, construct, or fusion protein that comprises a serum albumin binding agent of the invention and one or more (e.g., one or two) immunoglobulin single variable domain sequences that are suitably linked to each other, either directly or optionally via one or more suitable linkers or spacers. As mentioned herein, each such single immunoglobulin 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 VHH, humanized VHH, or camelized VH, such as a camelized human VH); and in accordance with one specific, but non-limiting aspect, at least one (and up to all) of these immunoglobulin variable domains contains two or three disulfide bridges. Preferably, all ISVDs present in such a compound of the invention are nanobodies.

When a compound of the invention has an ISVD at its C-terminus (such as a serum albumin binding agent of the invention or an ISVD that is directed against a therapeutic target), then said C-terminal ISVD (and thus, by extension, the entire compound of the invention ) preferably has a C-terminal extension at its C-terminus. This C-terminal extension will be directly linked to the last C-terminal amino acid residue of the ISVD, which will typically be the amino acid residue at Kabat position 113 (unless the ISVD contains one or more amino acid deletions so that the ISVD sequence ends before position 113). Thus, in general, the C-terminal extension will be directly linked to the C-terminal VTVSS sequence (SEQ ID NO: 15) from the C-terminal ISV (and thus, by extension, to the C-terminal TVTSS sequence from the compound of the invention ) or a C-terminal sequence from a C-terminal ISVD that corresponds to a C-terminal ISVD sequence (e.g., wherein said C-terminal sequence of the C-terminal ISVD contains one or more substitutions or deletions compared to a normal VTVSS sequence such as T110K, T110Q, S112K or S112K).

One skilled in the art will also appreciate that in the case where a compound of the invention contains a serum albumin binding agent of the present invention at its C-terminus, said serum albumin binding agent of the present invention will carry said C-terminal extension.

Generally, any C-terminal extension that is used herein (i.e., at the C-terminus of a compound of the invention and/or at the C-terminus of a serum albumin binding agent of the invention) can generally be as described in WO 2012/174741 or WO 2015/173325 (also referred to, for example, WO 2103/024059 and WO2016/118733). In particular, the C-terminal extension may have the formula (X)n, in which n is from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1) ; and each X is a (preferably naturally occurring) amino acid residue which is independently selected from naturally occurring amino acid residues (although in a preferred one aspect it does not contain any cysteine residues) and preferably independently selected from the group consisting of alanine (A), glycine (G ), valine (V), leucine (L), or isoleucine (I).

According to some preferred but non-limiting aspects of such C-terminal extensions, X(n), X and n may be as follows:

(a) n = 1 and X = Ala;

(b) n = 2 and each X = Ala;

(c) n = 3 and each X = Ala;

(d) n=2 and at least one X=Ala (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile);

(e) n=3 and at least one X=Ala (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile);

(f) n=3 and at least two X=Ala (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile);

(g) n = 1 and X = Gly;

(h) n = 2 and each X = Gly;

(i) n = 3 and each X = Gly;

(j) n=2 and at least one X=Gly (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile);

(k) n=3 and at least one X=Gly (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile);

(l) n=3 and at least two X=Gly (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile);

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

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

(o) n=3 and at least one X=Ala or Gly (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile); or

(p) n=3 and at least two X=Ala or Gly (with the remaining amino acid residue(s) X being independently selected from any naturally occurring amino acid, but preferably independently selected from Val, Leu and/or Ile);

wherein aspects (a), (b), (c), (g), (h), (i), (m) and (n) are particularly preferred, with aspects in which n = 1 or 2 are preferred , and aspects in which n = 1 is particularly preferred.

It should also be noted that preferably any C-terminal extension present in the serum albumin binding agent of the invention does not contain a (free) cysteine residue (unless said cysteine residue is used or intended for further functionalization, for example, for PEGylation).

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

Also preferably, when a compound of the invention has an ISVD at its C-terminus (such as a serum albumin binding agent of the invention or an ISVD that is directed against a therapeutic target), then (at least) said C-terminal ISVD preferably contains, even more preferably, in addition to the C-terminal extension as described herein, one or more mutations that reduce binding by preexisting antibodies (i.e., as described herein for the serum albumin binding agents of the invention, and as more broadly described in WO 2012 /175741 and WO 2015/173325, as well as, for example, WO 2013/024059 and WO 2016/118733). In this regard, one skilled in the art will appreciate that in the case where a compound of the present invention has a serum albumin binding agent of the present invention at its C-terminus, then (at least) said serum albumin binding agent of the present invention will preferably contain such mutations ( i.e. preferably in addition to the C-terminal extension).

More generally, according to a particular aspect of the invention, when a compound of the invention contains two or more ISVDs (e.g., a serum albumin binding agent of the invention and one or more ISVDs against a therapeutic target), then preferably all of these ISVDs contain mutations that reduce binding with preexisting antibodies (again, preferably in addition to the C-terminal extension that is linked to the C-terminal ISVD if the compound of the invention has an ISVD at its C-terminus).

When a compound of the invention has an ISVD at its N-terminal portion (such as a serum albumin binding agent of the invention or an ISVD that is directed against a therapeutic target), then said N-terminal ISVD (and thus, by extension, the entire compound along of the invention) preferably contain D at position 1. In this regard, it will again be clear to one skilled in the art that in the case where a compound of the invention has a serum albumin binding agent of the invention at the N-terminus, said serum albumin binding agent of the invention is preferably will have a D at position 1 (eg an E1D mutation compared to, for example, a sequence of SEQ ID NOs: 18 and/or 19, such as the amino acid sequences of the invention SEQ ID NOs: 46-59).

In some additional aspects, the invention relates to a protein, polypeptide or other compound or construct that contains or essentially consists of at least one (and preferably only one) serum albumin binding agent according to the invention, and at least one (for example, one , two or three) therapeutic moieties or substances (in which said serum albumin binding agent and one or more therapeutic moieties or substances are suitably linked, optionally via one or more suitable linkers), wherein the protein, polypeptide, compound, construct are , What:

- when they have an ISVD at their C-terminus, then said protein, polypeptide, compound, construct (their C-terminal ISVD) has a C-terminal extension (X)n (as further described herein) at its C-terminus; and/or

- when they have an ISVD at their C-terminus, then at least said C-terminal ISVD contains one or more mutations that reduce the binding of pre-existing antibodies (as further described herein);

- when they have an ISVD at their N-terminus, then said protein, polypeptide, compound, construct (their N-terminal ISVD) preferably contains a D at position 1; and/or

- in which the protein, polypeptide or other compound may also have an ISVD at its N-terminus, in which case said N-terminal ISVD preferably has a D or E1D at position 1;

Preferably, substantially all ISVDs present in said protein, polypeptide, compound, construct contain one or more mutations that reduce the binding of preexisting antibodies (as further described herein).

According to one specific aspect of the invention, all therapeutic moieties present in a compound of the invention are ISVDs (i.e., ISVDs against a therapeutic target), and in particular heavy chain ISVDs, and more specifically, nanobodies (i.e., nanobodies against a therapeutic target). ).

For example, and without limitation, such compounds of the invention may contain:

one copy of the serum albumin binding agent of the invention and one ISVD (and preferably nanobody) against the therapeutic target; or

- one copy of the serum albumin binding agent of the invention and two ISVDs (and preferably two nanobodies) against a therapeutic target (where the ISVDs may be the same or different, and when they are different, they may be directed against the same target, against different epitopes on the same target or against different therapeutic targets); or

- one copy of the serum albumin binding agent of the invention and three ISVDs (and preferably three nanobodies) against a therapeutic target (where the ISVDs may be the same or different, and when they are different, they may be directed against the same target, against different epitopes on the same target or against different therapeutic targets).

Some non-limiting examples of constructs, fusion proteins or polypeptides of the invention may be schematically represented as follows, where "[Alb]" is the serum albumin binding agent of the invention, "[therapeutic fragment 1]" and "[therapeutic fragment" 2]" represent therapeutic fragments (which, as mentioned, can independently represent a single immunoglobulin variable domain), "-" represents an appropriate linker (which is optional; linkers 9GS and 35GS are suitable examples), and the N-terminus is on the left side, and the C- end - on the right side:

[Alb] - [therapeutic fragment 1]

[therapeutic fragment 1] - [Alb] -X(n)

[alb] - [therapeutic fragment 1] - [therapeutic fragment 1]

[therapeutic fragment 1] - [therapeutic fragment 1] - [Alb] -X(n)

[therapeutic fragment 1] - [Alb] - [therapeutic fragment 1]

[Alyu] - [therapeutic fragment 1] - [therapeutic fragment 2]

[therapeutic fragment 1] - [therapeutic fragment 2] - [Alb] -X(n)

[therapeutic fragment 1] - [Alb] - [therapeutic fragment 2]

When the therapeutic moieties are ISVDs (and preferably nanobodies) against a therapeutic target, preferred, but not limiting, fusion proteins or polypeptides of the invention may be schematically represented as follows, wherein "[Alb]" is the serum albumin binding agent of the invention, "[therapeutic ISVD 1]" and "[therapeutic ISVD 2]" are ISVDs against a therapeutic target (where the ISVDs may be the same or different, and when they are different, they may be directed against the same target, against different epitopes on the same target or against different therapeutic targets), "-" represents an appropriate linker (which is optional), X(n) represents a C-terminal extension as described herein, and the N-terminus is on the left side, and C- end is on the right side:

[Alb] - [therapeutic ISVD 1] -X(n)

[therapeutic ISVD 1] - [Alb] -X(n)

[Alb] - [therapeutic ISVD 1] - [therapeutic ISVD 1] -X(n)

[therapeutic ISVD 1] - [therapeutic ISVD 1] - [Alb] -X(n)

[therapeutic ISVD 1] - [Alb] - [therapeutic ISVD 1] -X(n)

[Alb] - [therapeutic ISVD 1] - [therapeutic ISVD 2] -X(n)

[therapeutic ISVD 1] - [therapeutic ISVD 2] - [Alb] -X(n)

[therapeutic ISVD 1] - [Alb] - [therapeutic ISVD 2] -X(n).

Thus, in another aspect, the invention relates to a polyspecific (and in particular, bispecific) nanobody construct that contains a serum albumin binding agent of the invention and at least one other nanobody (such as one or two other nanobodies, which may be the same or different), wherein said at least one other nanobody is preferably directed against a desired target (which is preferably a therapeutic target) and/or another nanobody that is useful or suitable for therapeutic, prophylactic and/or diagnostic purposes. Again, the serum albumin binding agent of the invention and other nanobodies can be suitably linked to each other, either directly or, if necessary, through one or more suitable linkers or spacers.

For a general description of polyvalent and polyspecific polypeptides containing one or more nanobodies, and their production is also referred to Conrath et al., J. Biol. Chem., Vol. 276, 10.7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; and also, for example, WO 96/34103, WO 99/23221, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

By way of illustration, some examples of the compounds of the present invention are given in SEQ ID NO: 61-67, using the anti-HER2 nanobody of SEQ ID NO: 60 as a typical example of an anti-target nanobody, and with nanobody constituents at different positions in the compound according to the invention. The compounds of SEQ ID NO: 61-64 are examples illustrating the divalent bispecific compounds of the invention, and the compounds of SEQ ID NO: 65-67 are examples illustrating the trivalent bispecific compounds of the invention. In each case, the compounds contain the E1D mutation and a C-terminal alanine residue and contain representative, but non-limiting, examples of the use of suitable linkers (i.e., the 15GS linker in SEQ ID NOs: 62 and 63 and or the 35GS linkers in SEQ ID NOs: 61 and 64 - 67).

Some other examples of certain specific multispecific and/or multivalent polypeptides of the invention can be found in the Ablynx N.V. applications referenced herein. In particular, for a general description of polyvalent and multispecific constructs comprising at least one anti-serum protein nanobody to increase half-life, nucleic acids encoding it, compositions containing them, the preparation of the aforementioned, and the use of the aforementioned, reference is made to the above international applications WO 04/041865 and WO 06/122787 (the serum albumin binding agents described herein can generally be used similarly to extended half-life nanobodies such as Alb-8 described therein), as well as a general description and specific examples of such constructs. given, for example, in WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

The invention also relates to nucleotide sequences or nucleic acids that encode albumin binding agents, compounds or polypeptides of the invention. The invention further includes genetic constructs which include the aforementioned nucleotide sequences or nucleic acids and one or more genetic construct elements known per se. The genetic construct may be in the form of a plasmid or a vector. Again, such constructs may generally be as described in Ablynx N.V. published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

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 albumin binding agents, compounds or polypeptides of the invention. Again, such host cells may be generally as described in Ablynx N.V. published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/ 068627.

The invention also relates to a method for producing an albumin-binding agent, compound, or polypeptide of the invention, which comprises culturing or maintaining a host cell, as described herein, under conditions under which said host cell produces or expresses an albumin-binding agent, compound, or polypeptide. according to the invention, and optionally further comprising isolating the albumin-binding agent, compound or polypeptide of the invention thus obtained. Again, such methods can be carried out as generally described in Ablynx N.V. published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

The invention also relates to a pharmaceutical composition that contains at least one compound or polypeptide according to the invention, and optionally at least one pharmaceutically acceptable carrier, diluent or excipient. Such preparations, carriers, excipients and diluents may generally be as described in Ablynx NV published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/ 068627.

However, since the compounds or polypeptides of the invention have an extended half-life, they are preferably administered into the bloodstream. As such, they can be administered by any suitable route that allows the compound or polypeptide of the invention to enter the bloodstream, such as intravenously, by injection or infusion, or by any other suitable route (including oral, subcutaneous, intramuscular, transdermal, intranasal administration, administration through the lungs, etc.). Suitable modes and routes of administration will be apparent to those skilled in the art, again, for example, also from the teachings of Ablynx NV published patent applications such as, for example, WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO 2009/068627.

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 with a compound or polypeptide of the invention, which method comprises administering to a subject in need thereof a pharmaceutically active amount of the compound or polypeptide according to the invention and/or a pharmaceutical composition containing it. Diseases and disorders that can be prevented or treated using a compound or polypeptide of the invention as described herein will generally be the same as diseases and disorders that can be prevented or treated using a therapeutic moiety or moieties that are present in compound or polypeptide of the invention.

In the context of the present invention, the term "prevention and/or treatment" includes not only the prevention and/or treatment of a disease, but also usually includes preventing the occurrence of a disease, slowing or reversing the progression of a disease, preventing or slowing the development of one or more symptoms associated with a disease, reducing and/or amelioration of one or more symptoms associated with the disease, reduction of the severity and/or duration of the disease and/or any associated symptoms, and/or prevention of further increase in the severity of the disease and/or any associated symptoms, prevention, reduction or reversal of any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

The subject to be treated may be any warm-blooded animal, but in particular a mammal and in particular a human. As will be appreciated by one of skill in the art, the subject to be treated will in particular be a human suffering from or at risk of the diseases and disorders mentioned herein.

In another embodiment, the invention relates to a method of immunotherapy, in particular passive immunotherapy, which comprises administering to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of a compound or polypeptide of the invention and/or a pharmaceutical composition containing them. .

A compound or polypeptide of the invention and/or compositions containing them are administered in accordance with a treatment regimen that is suitable for the prevention and/or treatment of the disease or disorder to be prevented or treated. The clinician will generally be able to determine an appropriate 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 its symptoms, the specific polypeptide of the invention to be used, the specific route of administration and the pharmaceutical formulation or composition to be used, age, sex, body weight, diet, general condition of the patient, and the like, well known to the physician.

Typically, the treatment regimen will include the introduction of one or more compounds or polypeptides according to the invention, or one or more compositions containing them, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to be administered may be determined by the physician, again based on the factors noted above.

Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein, and depending on the particular disease or disorder being treated, the potency and/or half-life of the compounds or polypeptides of the invention to be used, the particular route of administration, and of the particular pharmaceutical composition or formulation employed, the compounds or polypeptides of the invention will generally be administered in an amount of 1 gram to 0.01 microgram per kg of body weight per day, preferably 0.1 gram to 0.1 microgram per kg of body weight per day, such as about 1, 10, 100, or 1000 micrograms per kg of body weight per day, either continuously (eg, by infusion), as a single daily dose, or as several divided doses throughout the day. The clinician will generally be able to determine an appropriate daily dose depending on the factors mentioned herein. It will also be clear that in certain cases the doctor may choose to deviate from these amounts, for example, based on the factors mentioned above and his expert judgment. As a general rule, some indication of administration amounts can be obtained from the amounts normally used for comparable conventional antibodies or antibody fragments against the same target administered in essentially the same route, taking into account, however, differences in affinity/avidity, efficacy, biodistribution, half-life, and similar factors well known to those skilled in the art.

In addition, since the compounds of the invention contain a serum albumin binding agent that increases the half-life of the invention, they do not need to be administered substantially continuously (for example, by infusion), but they can be administered at suitable intervals (as determined by a person skilled in the art). For example, they can be administered (at a suitable dose) once every two days, once every four days, once a week, once every two weeks, and in some cases once every four weeks or even less often, for example, by injection or infusion.

One aspect of the invention relates to a pharmaceutical composition comprising at least one compound or polypeptide of the invention, wherein said composition is intended to be administered at an interval of from once a week to once every 4 weeks, and in particular from once every 7 days to once every 21 days, for example, once every 7 days or 14 days.

Generally, a single polypeptide of the invention will be used in the above method. However, it is within the scope of the invention to use two or more polypeptides of the invention in combination.

The polypeptides of the invention may also be used in combination with one or more other pharmaceutically active compounds or substances, ie as a combination treatment regimen, which may or may not result in a synergistic effect. Again, the clinician will be able to select such additional compounds or substances, as well as an appropriate combination treatment regimen, based on the above factors and their expert judgment.

In particular, the polypeptides of the invention may be used in combination with other pharmaceutically active compounds or substances that are or may be used in the prevention and/or treatment of diseases and disorders that can be prevented or treated with the fusion proteins or constructs of the invention, resulting in a synergistic effect may or may not be achieved.

The efficacy of a treatment regimen used in accordance with the invention may be determined and/or monitored by any method known per se for a given disease or disorder, as will be appreciated by the physician. The physician will also be able, if necessary and on a case-by-case basis, to change or modify a particular treatment regimen in order to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side effects, and / or to ensure an appropriate balance between achieving the desired therapeutic effect, with on the one hand, and preventing, limiting or reducing unwanted side effects on the other hand.

Typically, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or until the desired therapeutic effect is to be maintained. Again, this can be determined by a doctor.

Other aspects, embodiments, advantages and applications of the invention will become apparent from the further description provided herein.

The invention will now be further described by means of the following non-limiting preferred aspects, examples and figures, in which:

fig. 1 is a table listing some of the amino acid positions that will be specifically referred to in this document and their numbering according to some alternative numbering systems (such as Aho and IMGT);

in fig. 2 lists the amino acid sequences mentioned in this document;

fig. 3A and 3B show the alignment of the sequences of SEQ ID NOS: 18 and 19 (according to the invention) with the prior art sequences of SEQ ID NOS: 1 and 2;

fig. 4 shows the alignment of SEQ ID NOs: 18-59;

fig. 5 shows alignment of serum albumin from different mammalian species;

fig. 6 is a graph showing the binding of SEQ ID NO: 1 (reference)-cMycHis6 coated with human serum albumin in the presence of a serum albumin binding agent from SEQ ID NO: 20 (according to the invention). 1.5 nM SEQ ID NO: 1-cMycHis6 and a concentration series of His6Flag3-SEQ ID NO: 1 (reference), Flag3His6-SEQ ID NO: 20 (according to the invention) or cAblys3-Flag3His6 (reference) were incubated on the HSA coating. Linked SEQ ID NO: 1-cMycHis6 was developed using goat anti-cMyc and horseradish peroxidase labeled rabbit anti-goat antibodies;

fig. 7 is a graph showing the binding of HSA to FcRn in the presence of the serum albumin binding agent from SEQ ID NO: 20 (of the invention). The human FcRn-human β2-microglobulin heterodimer was immobilized on a CM5 chip. Binding of 1 μM HSA in the absence or presence of 2 μM nanobody in 50 mM NaPO ₄ + 150 mM NaCl + 0.05% Tween-20 pH 6.0 was monitored on a Biacore T100 instrument.

The entire contents of all references (including literature references, published patents, published patent applications, and pending patent applications) cited herein are hereby incorporated by reference, in particular for those disclosures referenced above.

experimental part

Example 1 Serum Albumin Affinity

Affinity of serum albumin binding agent SEQ ID NO: 19 (according to the invention) for human serum albumin (Sigma-Aldrich A3782), cynomolgus macaque (self-produced), mouse (Albumin Bioscience 2601), rat (Sigma-Aldrich A4538), rabbit (Sigma -Aldrich A0764), guinea pig (Gentaur GPSA62), pig (Sigma-Aldrich A4414), sheep (Sigma-Aldrich A3264), dog (Abcam 119814) and bull (Sigma-Aldrich A3059) were evaluated by surface plasmon resonance (SPR) for instrument ProteOn XPR36 (BioRad). Serum albumin was immobilized by amine binding on a ProteOn GLC chip using the ProteOn amine immobilization kit (BioRad). Various concentrations (300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM and 1.23 nM) of the serum albumin binding agent SEQ ID NO: 19 (according to the invention) were administered in HBS-P + pH buffer 7.4 (GE Healthcare) at 45 µl/min for 120 sec, followed by dissociation for 900 sec. There was no or very low binding for SEQ ID NO: 19 to sheep and bovine CA. The affinity of SEQ ID NO: 19 for human, cynomolgus, rat, mouse and guinea pig CA was in the picomolar range (Table 1). In addition, there was a cross-reaction of SEQ ID NO: 19 with dog CA, as well as low affinity binding with porcine and rabbit CA.

Table 1. Binding kinetic parameters for the serum albumin binding agent SEQ ID NO: 19 (according to the invention) with CA of different species.

° SEQ ID NO:19 (according to the invention) SEQ ID NO:1 (reference) SA ka
(s-1M-1) kd
(s-1) KD
(M) ka
(s-1M-1) kd
(s-1) KD
(M) Human 7.9E+05 3.7E-05 4.6E-11 4.9E+05 1.6E-03 3.3E-09 Javanese macaque 8.3E+05 1.4E-05 1.6E-11 4.6E+05 1.4E-03 3.1E-09 Rat 1.2E+06 1.5E-05 1.2E-11 3.9E+05 2.6E-01 6.7E-07 mouse 9.4E+05 1.5E-05 1.6E-11 6.6E+05 3.0E-02 3.9E-08 guinea pig 9.4E+05 5.6E-05 5.9E-11 9.4E+05 1.9E-02 2.0E-08 Canine 9.0E+05 2.5E-03 2.8E-09 No binding Pork 1.2E+05 3.6E-02 2.9E-07 No binding The serum albumin binding agent SEQ ID NO:19 (according to the invention) was injected at various concentrations onto the immobilized CA on a ProteOn instrument. The kinetics of binding and dissociation were analyzed at pH 7.4. The kinetic parameters for SEQ ID NO:1 are listed as a reference and were determined in separate experiments.

The long half-life of albumin in the blood is due mainly to two characteristics: (i) the large size (65 kDa) of albumin limits its glomerular filtration; and (ii) albumin binds to FcRn at low pH (pH 6), which protects albumin from degradation in lysosomes after passive endocytosis in endothelial and epithelial cells by recycling from the early endosome back to the extracellular environment. In order for albumin-binding nanobodies to lead to a long serum half-life through albumin binding and subsequent recycling, they must remain bound to albumin in the pH range of 5.0 to 7.4. The rate of dissociation of the serum albumin binding agent SEQ ID NO: 19 (according to the invention) with HSA at pH 5, pH 6 and pH 7.4 was measured on the ProteOn instrument as described above, including SEQ ID NO: 1 as reference. Serum albumin binding agents SEQ ID NO: 19 (according to the invention) and SEQ ID NO: 1 were administered at 500 nM and 300 nM, respectively, in HBS-P+ pH 7.4 buffer. Dissociation buffers were 50 mM NaOAc/HOAc + 150 mM NaCl + 0.05% Tween-20, pH 5.0; 50 mM NaOAc/HOAc + 150 mM NaCl + 0.05% Tween-20, pH 6.0; and HBS-P+ pH 7.4, respectively. Dissociation was analyzed for 2700 s. The dissociation rates for SEQ ID NO: 19 do not differ significantly over the pH range of 5.0 to 7.4 (Table 2).

Table 2 Dissociation rate of serum albumin binding agent SEQ ID NO: 19 (according to the invention) with HSA at various pH values.

° Nd (s-1) SEQ ID NO:19 (according to the invention) or SEQ ID NO:1 (reference) was injected onto the immobilized HSA.
Dissociation was monitored at pH 5.0; 6.0 and 7.4 on the ProteOn instrument. pH 7.4 6.0E-05 1.3E-03 pH 6.0 6.7E-05 9.2E-04 pH 5.0 7.5E-05 1.1E-03 °pH SEQ ID NO:19 (according to the invention) SEQ ID NO:1 (reference)

Example 2 Epitope

Epitope binding was analyzed in a competitive ELISA. Human serum albumin was applied at a concentration of 125 ng/ml in PBS at 4° C. overnight. After blocking with PBS + 1% casein, 1.5 nM SEQ ID NO: 1-cMycHis6 and a series of competitor concentrations (His6Flag3-SEQ ID NO: 20, His6Flag3-SEQ ID NO: 1) were added as a positive control or hen egg lysozyme binding cAblys3-Flag3His6 single domain antibody as a negative control). Linked SEQ ID NO: 1-cMycHis6 was developed using goat anti-cMyc (Abcam ab19234) and HRP-labeled rabbit anti-goat (Genway 18-511-244226) antibodies. The albumin binding agent of SEQ ID NO: 20 and SEQ ID NO: 1 did not bind identical epitopes on HSA (FIG. 6).

Example 3 Interference with interaction between SA and FcRn

For a serum albumin binding agent of SEQ ID NO: 19 (according to the invention), in order to ensure a long half-life through albumin binding and subsequent recycling, there should be no interference with albumin binding to FcRn. This was analyzed in SPR on the Biacore T100 instrument (GE Healthcare). A human FcRn-human β2-microglobulin heterodimer (Sino Biological CT009-H08H) was immobilized on a CM5 chip using a standard amino group immobilization (Biacore amino group immobilization kit). Mixture of 1 μM HSA and 2 μM nanobodies (His6Flag3-SEQ ID NO: 20, His6Flag3-SEQ ID NO: 1 or cAblys3-Flag3His6) in 50 mM NaPO ₄ + 150 mM NaCl + 0.05% Tween-20, pH 6, 0 was injected at a rate of 10 μl/min for 120 seconds followed by dissociation for 600 seconds. The binding curves were qualitatively compared with the binding curve of 1 μM HSA in the absence of the nanobody. The albumin binding agent of SEQ ID NO: 20 (according to the invention) did not affect the binding of HSA to FcRn (FIG. 7).

Example 4: Pharmacokinetic profile in a dog.

The pharmacokinetics of the trivalent nanobody construct (SEQ ID NO: 68) after a single intravenous administration was studied in dogs. A trivalent construct was generated in Pichia pastoris and contained two anti-respiratory syncytial virus (RSV) nanobodies with an albumin-binding nanobody of SEQ ID NO: 19 at the C-terminus (where the three nanobodies are linked to each other by (Gly4Ser )7 linkers).

The pharmacokinetic profile of the construct was evaluated in a pharmacokinetic study (non-crossover, single dose) in twelve healthy fasting beagle dogs (healthy as defined by the preliminary health study). The construct was administered intravenously.

Animals did not receive food (without restriction of access to water) during the night before the administration of the dose and before the collection of blood samples before the administration of the drug, 24, 168 and 336 hours after the administration of the drug (total fasting time no more than 24 hours). Food was returned 4 hours after drug administration or immediately after blood sampling at 24, 168 and 336 hours. Body weight was also recorded and monitored during the study.

1 ml of whole blood (or 2-3 ml before the administration of the drug and after 24, 168 and 336 hours) to obtain plasma was taken from the peripheral vessel before the administration of the drug and after 5, 15, 30 minutes and after 1, 4, 8, 12, 24, 72, 168, 240, 336, 504 and 672 hours after initial drug administration. The collected serum samples were stored at room temperature and allowed to clot. Blood was centrifuged at 10,000 rpm for 2 minutes using a refrigerated centrifuge (set to maintain 4°C). Samples were frozen within 30 minutes of collection and stored until submitted for analysis (including measurement of the concentration of the respective nanobody construct).

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SEQUENCE LIST

<110> ABLINX NV

<120> ADVANCED SERUM ALBUMIN BINDING AGENTS

<130> 202811

<150> US62/446,992

<151> 2017-01-17

<160> 78

<170>PatentIn version 3.5

<210> 1

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Reference

<400> 1

Glu 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

<210> 2

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> Reference

<400> 2

Glu 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

<210> 3

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 (Kabat)

<400> 3

Ser Phe Gly Met Ser

15

<210> 4

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 (Kabat)

<400> 4

Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 5

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 (Kabat/Abm)

<400> 5

Gly Gly Ser Leu Ser Arg

15

<210> 6

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 (Abm)

<400> 6

Gly Phe Thr Phe Arg Ser Phe Gly Met Ser

1 5 10

<210> 7

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 (Abm)

<400> 7

Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu

1 5 10

<210> 8

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 (Kabat/Abm)

<400> 8

Gly Gly Ser Leu Ser Arg

15

<210> 9

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 (Kabat)

<400> 9

Ser Tyr Val Met Gly

15

<210> 10

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 (Kabat)

<400> 10

Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 11

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 (Abm)

<400> 11

Gly Ser Asn Ile Ser Ser Tyr Val Met Gly

1 5 10

<210> 12

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR1 (Abm)

<400> 12

Gly Ser Thr Ile Ser Ser Tyr Val Met Gly

1 5 10

<210> 13

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 (Abm)

<400> 13

Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr

1 5 10

<210> 14

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 (Abm)

<400> 14

Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp Tyr

1 5 10 15

<210> 15

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> C-terminal sequence

<400> 15

Val Thr Val Ser Ser

15

<210> 16

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> 15 GS linker

<400> 16

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 17

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> 35 GS linker

<400> 17

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser

35

<210> 18

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 19

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 20

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 21

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 23

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 25

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 26

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 26

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 27

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 29

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 30

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 30

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 31

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 31

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 32

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 33

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 33

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 34

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 35

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 36

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 37

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 37

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 38

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 38

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 39

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 39

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 40

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 41

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 42

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 43

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 44

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 44

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 45

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

<210> 46

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 46

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 47

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 47

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 48

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Asn Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 49

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 49

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 50

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 51

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 51

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 52

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 52

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 53

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 53

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 54

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 55

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 55

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 56

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 56

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 57

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 57

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 58

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 58

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 59

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> ISVD

<400> 59

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr 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 Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 60

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-HER2-Nanobody

<400> 60

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 61

<211> 278

<212> PRT

<213> Artificial Sequence

<220>

<223> bivalent bispecific compound

<400> 61

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

145 150 155 160

Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

165 170 175

Ala Ser Gly Ser Thr Ile Ser Ser Tyr Val Met Gly Trp Phe Arg Arg

180 185 190

Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly

195 200 205

Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

210 215 220

Arg Asp Asn Ser Lys Lys Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg

225 230 235 240

Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala Gly Arg Tyr Ser Ala

245 250 255

Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Ala

275

<210> 62

<211> 258

<212> PRT

<213> Artificial Sequence

<220>

<223> bivalent bispecific compound

<400> 62

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val

130 135 140

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser

145 150 155 160

Thr Ile Ser Ser Tyr Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys

165 170 175

Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr

180 185 190

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

195 200 205

Lys Lys Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr

210 215 220

Ala Leu Tyr Tyr Cys Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln

225 230 235 240

Ser Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250 255

Ser Ala

<210> 63

<211> 258

<212> PRT

<213> Artificial Sequence

<220>

<223> bivalent bispecific compound

<400> 63

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Glyn 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 Glu Val Gln Leu Val

130 135 140

Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

145 150 155 160

Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn Thr Met Gly Trp Tyr

165 170 175

Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Leu Ile Ser Ser

180 185 190

Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

195 200 205

Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu

210 215 220

Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Arg Phe Arg Thr Ala

225 230 235 240

Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

245 250 255

Ser Ala

<210> 64

<211> 278

<212> PRT

<213> Artificial Sequence

<220>

<223> bivalent bispecific compound

<400> 64

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Glyn 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 Ala Gly Gly Ser

165 170 175

Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn Thr

180 185 190

Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

195 200 205

Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly

210 215 220

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

225 230 235 240

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Arg

245 250 255

Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Ala

275

<210> 65

<211> 431

<212> PRT

<213> Artificial Sequence

<220>

<223> trivalent bispecific compound

<400> 65

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

145 150 155 160

Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala

165 170 175

Ala Ser Gly Ile Thr Phe Ser Ile Asn Thr Met Gly Trp Tyr Arg Gln

180 185 190

Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Leu Ile Ser Ser Ile Gly

195 200 205

Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

210 215 220

Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro

225 230 235 240

Glu Asp Thr Ala Val Tyr Tyr Cys Lys Arg Phe Arg Thr Ala Ala Gln

245 250 255

Gly Thr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

260 265 270

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

290 295 300

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

305 310 315 320

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser

325 330 335

Ser Tyr Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu

340 345 350

Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp

355 360 365

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr

370 375 380

Ala Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr

385 390 395 400

Tyr Cys Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu

405 410 415

Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

420 425 430

<210> 66

<211> 431

<212> PRT

<213> Artificial Sequence

<220>

<223> trivalent bispecific compound

<400> 66

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn

20 25 30

Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys

85 90 95

Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

145 150 155 160

Gly Gly Gly Val Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

165 170 175

Ala Ser Gly Ser Thr Ile Ser Ser Tyr Val Met Gly Trp Phe Arg Arg

180 185 190

Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly

195 200 205

Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser

210 215 220

Arg Asp Asn Ser Lys Lys Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg

225 230 235 240

Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala Gly Arg Tyr Ser Ala

245 250 255

Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Ile Thr Phe Ser Ile Asn Thr Met Gly Trp Tyr Arg Gln Ala

340 345 350

Pro Gly Lys Gln Arg Glu Leu Val Ala Leu Ile Ser Ser Ile Gly Asp

355 360 365

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

370 375 380

Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

385 390 395 400

Asp Thr Ala Val Tyr Tyr Cys Lys Arg Phe Arg Thr Ala Ala Gln Gly

405 410 415

Thr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

420 425 430

<210> 67

<211> 431

<212> PRT

<213> Artificial Sequence

<220>

<223> trivalent bispecific compound

<400> 67

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Ser Ser Tyr

20 25 30

Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser Tyr Glu Tyr Asp

100 105 110

Tyr Trp Gly Glyn 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 Ala Gly Gly Ser

165 170 175

Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn Thr

180 185 190

Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala

195 200 205

Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys Gly

210 215 220

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln

225 230 235 240

Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys Arg

245 250 255

Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr Leu

260 265 270

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly

305 310 315 320

Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

325 330 335

Ser Gly Ile Thr Phe Ser Ile Asn Thr Met Gly Trp Tyr Arg Gln Ala

340 345 350

Pro Gly Lys Gln Arg Glu Leu Val Ala Leu Ile Ser Ser Ile Gly Asp

355 360 365

Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

370 375 380

Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu

385 390 395 400

Asp Thr Ala Val Tyr Tyr Cys Lys Arg Phe Arg Thr Ala Ala Gln Gly

405 410 415

Thr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala

420 425 430

<210> 68

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> trivalent compound

<400> 68

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Ser Trp Ser Asp Gly Ser 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 Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr Ile Tyr Ile Trp

100 105 110

Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro

165 170 175

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser

180 185 190

Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

195 200 205

Phe Val Ala Ala Ile Ser Trp Ser Asp Gly Ser Thr Tyr Tyr Ala Asp

210 215 220

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

225 230 235 240

Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr

245 250 255

Tyr Cys Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr Ile Tyr

260 265 270

Ile Trp Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

275 280 285

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

290 295 300

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

305 310 315 320

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

325 330 335

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr

340 345 350

Ile Ser Ser Tyr Val Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu

355 360 365

Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Tyr Thr Tyr Tyr

370 375 380

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys

385 390 395 400

Lys Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala

405 410 415

Leu Tyr Tyr Cys Ala Ala Gly Arg Tyr Ser Ala Trp Tyr Ser Gln Ser

420 425 430

Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

435 440 445

<210> 69

<211> 609

<212> PRT

<213> Homo sapiens

<400> 69

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu

35 40 45

Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala

100 105 110

Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val

130 135 140

Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys

145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys

180 185 190

Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu

195 200 205

Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys

210 215 220

Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

225 230 235 240

Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser

245 250 255

Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

275 280 285

Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu

290 295 300

Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp

305 310 315 320

Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser

325 330 335

Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val

355 360 365

Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys

370 375 380

Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu

385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys

405 410 415

Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

450 455 460

Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val

465 470 475 480

Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg

485 490 495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu

530 535 540

Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys

545 550 555 560

Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala

565 570 575

Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe

580 585 590

Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly

595 600 605

Leu

<210> 70

<211> 607

<212> PRT

<213> Mus musculus

<400> 70

Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala

1 5 10 15

Phe Ser Arg Gly Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala His

20 25 30

Arg Tyr Asn Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu Ile

35 40 45

Ala Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala Lys

50 55 60

Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp Glu

65 70 75 80

Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys

85 90 95

Leu Cys Ala Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala Asp

100 105 110

Cys Cys Thr Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His

115 120 125

Lys Asp Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala Glu

130 135 140

Ala Met Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly His

145 150 155 160

Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu

165 170 175

Leu Leu Tyr Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys

180 185 190

Ala Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly Val

195 200 205

Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys Ser

210 215 220

Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala

225 230 235 240

Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr Lys

245 250 255

Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly Asp

260 265 270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met Cys

275 280 285

Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys Cys Asp Lys

290 295 300

Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val Glu His Asp Thr

305 310 315 320

Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp Phe Val Glu Asp Gln

325 330 335

Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Thr

340 345 350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu

355 360 365

Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys Cys

370 375 380

Ala Glu Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu Phe

385 390 395 400

Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys Asp

405 410 415

Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val

420 425 430

Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu

435 440 445

Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu Pro

450 455 460

Glu Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu

465 470 475 480

Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val

485 490 495

Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe Ser

500 505 510

Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala Glu

515 520 525

Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu Lys Glu Lys

530 535 540

Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys Pro

545 550 555 560

Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met Asp Asp Phe Ala Gln

565 570 575

Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe Ser

580 585 590

Thr Glu Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala

595 600 605

<210> 71

<211> 608

<212> PRT

<213> Rattus norvegicus

<400> 71

Met Lys Trp Val Thr Phe Leu Leu Leu Leu Phe Ile Ser Gly Ser Ala

1 5 10 15

Phe Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu

35 40 45

Ile Ala Phe Ser Gln Tyr Leu Gln Lys Cys Pro Tyr Glu Glu His Ile

50 55 60

Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Asn Ala Glu Asn Cys Asp Lys Ser Ile His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Ala Ile Pro Lys Leu Arg Asp Asn Tyr Gly Glu Leu Ala

100 105 110

Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asp Asp Asn Pro Asn Leu Pro Pro Phe Gln Arg Pro Glu Ala

130 135 140

Glu Ala Met Cys Thr Ser Phe Gln Glu Asn Pro Thr Ser Phe Leu Gly

145 150 155 160

His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Tyr Tyr Ala Glu Lys Tyr Asn Glu Val Leu Thr Gln Cys

180 185 190

Cys Thr Glu Ser Asp Lys Ala Ala Cys Leu Thr Pro Lys Leu Asp Ala

195 200 205

Val Lys Glu Lys Ala Leu Val Ala Ala Val Arg Gln Arg Met Lys Cys

210 215 220

Ser Ser Met Gln Arg Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

225 230 235 240

Ala Arg Met Ser Gln Arg Phe Pro Asn Ala Glu Phe Ala Glu Ile Thr

245 250 255

Lys Leu Ala Thr Asp Leu Thr Lys Ile Asn Lys Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met

275 280 285

Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Ala Cys Cys Asp

290 295 300

Lys Pro Val Leu Gln Lys Ser Gln Cys Leu Ala Glu Ile Glu His Asp

305 310 315 320

Asn Ile Pro Ala Asp Leu Pro Ser Ile Ala Ala Asp Phe Val Glu Asp

325 330 335

Lys Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser

355 360 365

Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Glu Lys Cys

370 375 380

Cys Ala Glu Gly Asp Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu

385 390 395 400

Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys

405 410 415

Glu Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu

420 425 430

Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu

450 455 460

Pro Glu Ala Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile

465 470 475 480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys

485 490 495

Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala

515 520 525

Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Asp Lys Glu

530 535 540

Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys

545 550 555 560

Pro Lys Ala Thr Glu Asp Gln Leu Lys Thr Val Met Gly Asp Phe Ala

565 570 575

Gln Phe Val Asp Lys Cys Cys Lys Ala Ala Asp Lys Asp Asn Cys Phe

580 585 590

Ala Thr Glu Gly Pro Asn Leu Val Ala Arg Ser Lys Glu Ala Leu Ala

595 600 605

<210> 72

<211> 607

<212> PRT

<213> Bos taurus

<400> 72

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Ile Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Glu His Phe Lys Gly Leu Val Leu

35 40 45

Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His Val

50 55 60

Lys Leu Val Asn Glu Leu Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser His Ala Gly Cys Glu Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Glu Leu Cys Lys Val Ala Ser Leu Arg Glu Thr Tyr Gly Asp Met Ala

100 105 110

Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Ser

115 120 125

His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Asp Pro Asn

130 135 140

Thr Leu Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys

145 150 155 160

Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu

165 170 175

Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Cys

180 185 190

Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Glu Thr Met

195 200 205

Arg Glu Lys Val Leu Thr Ser Ser Ala Arg Gln Arg Leu Arg Cys Ala

210 215 220

Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala

225 230 235 240

Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Val Glu Val Thr Lys

245 250 255

Leu Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp

260 265 270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys

275 280 285

Asp Asn Gln Asp Thr Ile Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys

290 295 300

Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Lys Asp Ala

305 310 315 320

Ile Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys

325 330 335

Asp Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Ala Phe Leu Gly Ser

340 345 350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val

355 360 365

Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Glu Cys Cys

370 375 380

Ala Lys Asp Asp Pro His Ala Cys Tyr Ser Thr Val Phe Asp Lys Leu

385 390 395 400

Lys His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Asp

405 410 415

Gln Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val

420 425 430

Arg Tyr Thr Arg Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu

435 440 445

Val Ser Arg Ser Leu Gly Lys Val Gly Thr Arg Cys Cys Thr Lys Pro

450 455 460

Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu

465 470 475 480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val

485 490 495

Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser

500 505 510

Ala Leu Thr Pro Asp Glu Thr Tyr Val Pro Lys Ala Phe Asp Glu Lys

515 520 525

Leu Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys

530 535 540

Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro

545 550 555 560

Lys Ala Thr Glu Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala

565 570 575

Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Ala Cys Phe Ala

580 585 590

Val Glu Gly Pro Lys Leu Val Val Ser Thr Gln Thr Ala Leu Ala

595 600 605

<210> 73

<211> 607

<212> PRT

<213> Sus scrofa

<400> 73

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr Tyr Lys Ser Glu Ile Ala

20 25 30

His Arg Phe Lys Asp Leu Gly Glu Gln Tyr Phe Lys Gly Leu Val Leu

35 40 45

Ile Ala Phe Ser Gln His Leu Gln Gln Cys Pro Tyr Glu Glu His Val

50 55 60

Lys Leu Val Arg Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Ile His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Ala Ile Pro Ser Leu Arg Glu His Tyr Gly Asp Leu Ala

100 105 110

Asp Cys Cys Glu Lys Glu Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asn Asp Asn Pro Asp Ile Pro Lys Leu Lys Pro Asp Pro Val

130 135 140

Ala Leu Cys Ala Asp Phe Gln Glu Asp Glu Gln Lys Phe Trp Gly Lys

145 150 155 160

Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu

165 170 175

Leu Leu Tyr Tyr Ala Ile Ile Tyr Lys Asp Val Phe Ser Glu Cys Cys

180 185 190

Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Ile Glu His Leu

195 200 205

Arg Glu Lys Val Leu Thr Ser Ala Ala Lys Gln Arg Leu Lys Cys Ala

210 215 220

Ser Ile Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Leu Ala

225 230 235 240

Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Thr Glu Ile Ser Lys

245 250 255

Ile Val Thr Asp Leu Ala Lys Val His Lys Glu Cys Cys His Gly Asp

260 265 270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys

275 280 285

Glu Asn Gln Asp Thr Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp Lys

290 295 300

Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Ala Lys Arg Asp Glu

305 310 315 320

Leu Pro Ala Asp Leu Asn Pro Leu Glu His Asp Phe Val Glu Asp Lys

325 330 335

Glu Val Cys Lys Asn Tyr Lys Glu Ala Lys His Val Phe Leu Gly Thr

340 345 350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu

355 360 365

Leu Leu Arg Ile Ala Lys Ile Tyr Glu Ala Thr Leu Glu Asp Cys Cys

370 375 380

Ala Lys Glu Asp Pro Pro Ala Cys Tyr Ala Thr Val Phe Asp Lys Phe

385 390 395 400

Gln Pro Leu Val Asp Glu Pro Lys Asn Leu Ile Lys Gln Asn Cys Glu

405 410 415

Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val

420 425 430

Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu

435 440 445

Val Ala Arg Lys Leu Gly Leu Val Gly Ser Arg Cys Cys Lys Arg Pro

450 455 460

Glu Glu Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Leu Val Leu

465 470 475 480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val

485 490 495

Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser

500 505 510

Ala Leu Thr Pro Asp Glu Thr Tyr Lys Pro Lys Glu Phe Val Glu Gly

515 520 525

Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Asp Glu Lys

530 535 540

Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro

545 550 555 560

His Ala Thr Glu Glu Gln Leu Arg Thr Val Leu Gly Asn Phe Ala Ala

565 570 575

Phe Val Gln Lys Cys Cys Ala Ala Pro Asp His Glu Ala Cys Phe Ala

580 585 590

Val Glu Gly Pro Lys Phe Val Ile Glu Ile Arg Gly Ile Leu Ala

595 600 605

<210> 74

<211> 608

<212> PRT

<213> Canis lupus

<400> 74

Met Lys Trp Val Thr Phe Ile Ser Leu Phe Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Leu Val Arg Arg Glu Ala Tyr Lys Ser Glu Ile Ala

20 25 30

His Arg Tyr Asn Asp Leu Gly Glu Glu His Phe Arg Gly Leu Val Leu

35 40 45

Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Ala Lys Glu Val Thr Glu Phe Ala Lys Ala Cys Ala Ala Glu

65 70 75 80

Glu Ser Gly Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Ser Leu Arg Asp Lys Tyr Gly Asp Met Ala

100 105 110

Asp Cys Cys Glu Lys Gln Glu Pro Asp Arg Asn Glu Cys Phe Leu Ala

115 120 125

His Lys Asp Asp Asn Pro Gly Phe Pro Pro Leu Val Ala Pro Glu Pro

130 135 140

Asp Ala Leu Cys Ala Ala Phe Gln Asp Asn Glu Gln Leu Phe Leu Gly

145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Tyr Tyr Ala Gln Gln Tyr Lys Gly Val Phe Ala Glu Cys

180 185 190

Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Gly Pro Lys Ile Glu Ala

195 200 205

Leu Arg Glu Lys Val Leu Leu Ser Ser Ala Lys Glu Arg Phe Lys Cys

210 215 220

Ala Ser Leu Gln Lys Phe Gly Asp Arg Ala Phe Lys Ala Trp Ser Val

225 230 235 240

Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Ala Glu Ile Ser

245 250 255

Lys Val Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Met

275 280 285

Cys Glu Asn Gln Asp Ser Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp

290 295 300

Lys Pro Val Leu Glu Lys Ser Gln Cys Leu Ala Glu Val Glu Arg Asp

305 310 315 320

Glu Leu Pro Gly Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Asp

325 330 335

Lys Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Thr Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Glu Tyr Ser Val Ser

355 360 365

Leu Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Lys Cys

370 375 380

Cys Ala Thr Asp Asp Pro Pro Thr Cys Tyr Ala Lys Val Leu Asp Glu

385 390 395 400

Phe Lys Pro Leu Val Asp Glu Pro Gln Asn Leu Val Lys Thr Asn Cys

405 410 415

Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Val Ser Arg Lys Leu Gly Lys Val Gly Thr Lys Cys Cys Lys Lys

450 455 460

Pro Glu Ser Glu Arg Met Ser Cys Ala Glu Asp Phe Leu Ser Val Val

465 470 475 480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Arg

485 490 495

Val Thr Lys Cys Cys Ser Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Gly Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Ala Glu

530 535 540

Lys Gln Val Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys

545 550 555 560

Pro Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Gly Asp Phe Gly

565 570 575

Ala Phe Val Glu Lys Cys Cys Ala Ala Glu Asn Lys Glu Gly Cys Phe

580 585 590

Ser Glu Glu Gly Pro Lys Leu Val Ala Ala Ala Gln Ala Ala Leu Val

595 600 605

<210> 75

<211> 608

<212> PRT

<213> Felis catus

<400> 75

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Thr Arg Arg Glu Ala His Gln Ser Glu Ile Ala

20 25 30

His Arg Phe Asn Asp Leu Gly Glu Glu His Phe Arg Gly Leu Val Leu

35 40 45

Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Gly Cys Val Ala Asp

65 70 75 80

Gln Ser Ala Ala Asn Cys Glu Lys Ser Leu His Glu Leu Leu Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Ser Leu Arg Asp Lys Tyr Gly Glu Met Ala

100 105 110

Asp Cys Cys Glu Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

115 120 125

His Lys Asp Asp Asn Pro Gly Phe Gly Gln Leu Val Thr Pro Glu Ala

130 135 140

Asp Ala Met Cys Thr Ala Phe His Glu Asn Glu Gln Arg Phe Leu Gly

145 150 155 160

Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Tyr Tyr Ala Glu Glu Tyr Lys Gly Val Phe Thr Glu Cys

180 185 190

Cys Glu Ala Ala Asp Lys Ala Ala Cys Leu Thr Pro Lys Val Asp Ala

195 200 205

Leu Arg Glu Lys Val Leu Ala Ser Ser Ala Lys Glu Arg Leu Lys Cys

210 215 220

Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Val

225 230 235 240

Ala Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Ala Glu Ile Ser

245 250 255

Lys Leu Val Thr Asp Leu Ala Lys Ile His Lys Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile

275 280 285

Cys Glu Asn Gln Asp Ser Ile Ser Thr Lys Leu Lys Glu Cys Cys Gly

290 295 300

Lys Pro Val Leu Glu Lys Ser His Cys Ile Ser Glu Val Glu Arg Asp

305 310 315 320

Glu Leu Pro Ala Asp Leu Pro Pro Leu Ala Val Asp Phe Val Glu Asp

325 330 335

Lys Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ser Val Ser

355 360 365

Leu Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Lys Cys

370 375 380

Cys Ala Thr Asp Asp Pro Pro Ala Cys Tyr Ala His Val Phe Asp Glu

385 390 395 400

Phe Lys Pro Leu Val Glu Glu Pro His Asn Leu Val Lys Thr Asn Cys

405 410 415

Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Val Ser Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Thr His

450 455 460

Pro Glu Ala Glu Arg Leu Ser Cys Ala Glu Asp Tyr Leu Ser Val Val

465 470 475 480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Arg

485 490 495

Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Gln Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Ser Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Ala Glu

530 535 540

Lys Gln Ile Lys Lys Gln Ser Ala Leu Val Glu Leu Leu Lys His Lys

545 550 555 560

Pro Lys Ala Thr Glu Glu Gln Leu Lys Thr Val Met Gly Asp Phe Gly

565 570 575

Ser Phe Val Asp Lys Cys Cys Ala Ala Glu Asp Lys Glu Ala Cys Phe

580 585 590

Ala Glu Glu Gly Pro Lys Leu Val Ala Ala Ala Gln Ala Ala Leu Ala

595 600 605

<210> 76

<211> 607

<212> PRT

<213> Ovis aries

<400> 76

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Ile Ala

20 25 30

His Arg Phe Asn Asp Leu Gly Glu Glu Asn Phe Gln Gly Leu Val Leu

35 40 45

Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His Val

50 55 60

Lys Leu Val Lys Glu Leu Thr Glu Phe Ala Lys Thr Cys Val Ala Asp

65 70 75 80

Glu Ser His Ala Gly Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Glu Leu Cys Lys Val Ala Thr Leu Arg Glu Thr Tyr Gly Asp Met Ala

100 105 110

Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Asn

115 120 125

His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Glu Pro Asp

130 135 140

Thr Leu Cys Ala Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys

145 150 155 160

Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu

165 170 175

Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Cys

180 185 190

Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Asp Ala Met

195 200 205

Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gln Arg Leu Arg Cys Ala

210 215 220

Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala

225 230 235 240

Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Thr Asp Val Thr Lys

245 250 255

Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp

260 265 270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys

275 280 285

Asp His Gln Asp Ala Leu Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys

290 295 300

Pro Val Leu Glu Lys Ser His Cys Ile Ala Glu Val Asp Lys Asp Ala

305 310 315 320

Val Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys

325 330 335

Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly Ser

340 345 350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val

355 360 365

Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Asp Cys Cys

370 375 380

Ala Lys Glu Asp Pro His Ala Cys Tyr Ala Thr Val Phe Asp Lys Leu

385 390 395 400

Lys His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Lys Asn Cys Glu

405 410 415

Leu Phe Glu Lys His Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val

420 425 430

Arg Tyr Thr Arg Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu

435 440 445

Ile Ser Arg Ser Leu Gly Lys Val Gly Thr Lys Cys Cys Ala Lys Pro

450 455 460

Glu Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu

465 470 475 480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val

485 490 495

Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser

500 505 510

Asp Leu Thr Leu Asp Glu Thr Tyr Val Pro Lys Pro Phe Asp Glu Lys

515 520 525

Phe Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys

530 535 540

Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys Pro

545 550 555 560

Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala

565 570 575

Phe Val Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Gly Cys Phe Val

580 585 590

Leu Glu Gly Pro Lys Leu Val Ala Ser Thr Gln Ala Ala Leu Ala

595 600 605

<210> 77

<211> 607

<212> PRT

<213> Equus caballus

<400> 77

Met Lys Trp Val Thr Phe Val Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Leu Arg Arg Asp Thr His Lys Ser Glu Ile Ala

20 25 30

His Arg Phe Asn Asp Leu Gly Glu Lys His Phe Lys Gly Leu Val Leu

35 40 45

Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val

50 55 60

Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Lys Cys Ala Ala Asp

65 70 75 80

Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

85 90 95

Lys Leu Cys Thr Val Ala Thr Leu Arg Ala Thr Tyr Gly Glu Leu Ala

100 105 110

Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Thr

115 120 125

His Lys Asp Asp His Pro Asn Leu Pro Lys Leu Lys Pro Glu Pro Asp

130 135 140

Ala Gln Cys Ala Ala Phe Gln Glu Asp Pro Asp Lys Phe Leu Gly Lys

145 150 155 160

Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Gly Pro Glu

165 170 175

Leu Leu Phe His Ala Glu Glu Tyr Lys Ala Asp Phe Thr Glu Cys Cys

180 185 190

Pro Ala Asp Asp Lys Leu Ala Cys Leu Ile Pro Lys Leu Asp Ala Leu

195 200 205

Lys Glu Arg Ile Leu Leu Ser Ser Ala Lys Glu Arg Leu Lys Cys Ser

210 215 220

Ser Phe Gln Asn Phe Gly Glu Arg Ala Val Lys Ala Trp Ser Val Ala

225 230 235 240

Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Glu Val Ser Lys

245 250 255

Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly Asp

260 265 270

Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys

275 280 285

Glu His Gln Asp Ser Ile Ser Gly Lys Leu Lys Ala Cys Cys Asp Lys

290 295 300

Pro Leu Leu Gln Lys Ser His Cys Ile Ala Glu Val Lys Glu Asp Asp

305 310 315 320

Leu Pro Ser Asp Leu Pro Ala Leu Ala Ala Asp Phe Ala Glu Asp Lys

325 330 335

Glu Ile Cys Lys His Tyr Lys Asp Ala Lys Asp Val Phe Leu Gly Thr

340 345 350

Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu

355 360 365

Leu Leu Arg Ile Ala Lys Thr Tyr Glu Ala Thr Leu Glu Lys Cys Cys

370 375 380

Ala Glu Ala Asp Pro Pro Ala Cys Tyr Arg Thr Val Phe Asp Gln Phe

385 390 395 400

Thr Pro Leu Val Glu Glu Pro Lys Ser Leu Val Lys Lys Asn Cys Asp

405 410 415

Leu Phe Glu Glu Val Gly Glu Tyr Asp Phe Gln Asn Ala Leu Ile Val

420 425 430

Arg Tyr Thr Lys Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu

435 440 445

Ile Gly Arg Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro

450 455 460

Glu Ser Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu Ala Leu

465 470 475 480

Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Ile

485 490 495

Thr Lys Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro Cys Phe Ser

500 505 510

Ala Leu Glu Leu Asp Glu Gly Tyr Val Pro Lys Glu Phe Lys Ala Glu

515 520 525

Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Asp Glu Lys

530 535 540

Gln Ile Lys Lys Gln Ser Ala Leu Ala Glu Leu Val Lys His Lys Pro

545 550 555 560

Lys Ala Thr Lys Glu Gln Leu Lys Thr Val Leu Gly Asn Phe Ser Ala

565 570 575

Phe Val Ala Lys Cys Cys Gly Arg Glu Asp Lys Glu Ala Cys Phe Ala

580 585 590

Glu Glu Gly Pro Lys Leu Val Ala Ser Ser Gln Leu Ala Leu Ala

595 600 605

<210> 78

<211> 608

<212> PRT

<213> Oryctolagus cuniculus

<400> 78

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala

20 25 30

His Arg Phe Asn Asp Val Gly Glu Glu His Phe Ile Gly Leu Val Leu

35 40 45

Ile Thr Phe Ser Gln Tyr Leu Gln Lys Cys Pro Tyr Glu Glu His Ala

50 55 60

Lys Leu Val Lys Glu Val Thr Asp Leu Ala Lys Ala Cys Val Ala Asp

65 70 75 80

Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Asp Ile Phe Gly Asp

85 90 95

Lys Ile Cys Ala Leu Pro Ser Leu Arg Asp Thr Tyr Gly Asp Val Ala

100 105 110

Asp Cys Cys Glu Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe Leu His

115 120 125

His Lys Asp Asp Lys Pro Asp Leu Pro Pro Phe Ala Arg Pro Glu Ala

130 135 140

Asp Val Leu Cys Lys Ala Phe His Asp Asp Glu Lys Ala Phe Phe Gly

145 150 155 160

His Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

165 170 175

Glu Leu Leu Tyr Tyr Ala Gln Lys Tyr Lys Ala Ile Leu Thr Glu Cys

180 185 190

Cys Glu Ala Ala Asp Lys Gly Ala Cys Leu Thr Pro Lys Leu Asp Ala

195 200 205

Leu Glu Gly Lys Ser Leu Ile Ser Ala Ala Gln Glu Arg Leu Arg Cys

210 215 220

Ala Ser Ile Gln Lys Phe Gly Asp Arg Ala Tyr Lys Ala Trp Ala Leu

225 230 235 240

Val Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Thr Asp Ile Ser

245 250 255

Lys Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Met

275 280 285

Cys Glu His Gln Glu Thr Ile Ser Ser His Leu Lys Glu Cys Cys Asp

290 295 300

Lys Pro Ile Leu Glu Lys Ala His Cys Ile Tyr Gly Leu His Asn Asp

305 310 315 320

Glu Thr Pro Ala Gly Leu Pro Ala Val Ala Glu Glu Phe Val Glu Asp

325 330 335

Lys Asp Val Cys Lys Asn Tyr Glu Glu Ala Lys Asp Leu Phe Leu Gly

340 345 350

Lys Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Val

355 360 365

Leu Leu Leu Arg Leu Gly Lys Ala Tyr Glu Ala Thr Leu Lys Lys Cys

370 375 380

Cys Ala Thr Asp Asp Pro His Ala Cys Tyr Ala Lys Val Leu Asp Glu

385 390 395 400

Phe Gln Pro Leu Val Asp Glu Pro Lys Asn Leu Val Lys Gln Asn Cys

405 410 415

Glu Leu Tyr Glu Gln Leu Gly Asp Tyr Asn Phe Gln Asn Ala Leu Leu

420 425 430

Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Ile Ser Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His

450 455 460

Pro Glu Ala Glu Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Val Val

465 470 475 480

Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys

485 490 495

Val Thr Lys Cys Cys Ser Glu Ser Leu Val Asp Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Gly Pro Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala

515 520 525

Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Glu Thr Glu

530 535 540

Arg Lys Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys

545 550 555 560

Pro His Ala Thr Asn Asp Gln Leu Lys Thr Val Val Gly Glu Phe Thr

565 570 575

Ala Leu Leu Asp Lys Cys Cys Ser Ala Glu Asp Lys Glu Ala Cys Phe

580 585 590

Ala Val Glu Gly Pro Lys Leu Val Glu Ser Ser Lys Ala Thr Leu Gly

595 600 605

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Claims (43)

1. An immunoglobulin single variable domain (ISVD) capable of specifically binding to serum albumin, which has: CDR1 according to Abm, which is the amino acid sequence GSNISSYVMG shown in SEQ ID NO: 11 or GSTISSYVMG shown in SEQ ID NO: 12; And CDR2 according to Abm, which is the amino acid sequence AISRSGGYTY shown in SEQ ID NO: 13; And CDR3 according to Abm, which is the GRYSAWYSQSYEYDY amino acid sequence shown in SEQ ID NO: 14. 2. An ISVD according to claim 1 that can bind to human serum albumin with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM, as determined using ProteOn. 3. ISVD according to any one of paragraphs. 1, 2, which is a single variable domain of the immunoglobulin heavy chain. 4. ISVD according to any one of paragraphs. 1-3, which is VHH, humanized VHH, or camelized VH, and in particular camelized human VH. 5. ISVD according to any one of paragraphs. 1-4, which is such that: has a human serum half-life, expressed as t1/2 beta, that is greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours; and/or such that: when linked to a therapeutic moiety or substance, it provides the resulting polypeptide of the invention with a human serum half-life, expressed as t1/2 beta, that is greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably over 72 hours. 6. ISVD according to any one of paragraphs. 1-5 which has: degree of sequence identity with SEQ ID NO: 18 and/or 19 of at least 85%, preferably at least 90%, more preferably at least 95%, where any C-terminal extension that may be present and CDRs are ignored to determine the degree of sequence identity; and/or which has: no more than 7, preferably no more than 5, eg only 3, 2 or 1 "amino acid differences" of SEQ ID NO: 18 and/or 19, ignoring any C-terminal extension that may be present and CDRs. 7. ISVD according to any one of paragraphs. 1-6 which has: a degree of sequence identity with SEQ ID NO: 18 of at least 85%, preferably at least 90%, more preferably at least 95%, where any C-terminal extension that may be present and CDRs are not taken into account to determine the degree of identity sequences; and/or which has: no more than 7, preferably no more than 5, eg only 3, 2 or 1 "amino acid differences" of SEQ ID NO: 18, ignoring any C-terminal extension that may be present and CDRs. 8. ISVD according to any one of paragraphs. 1-6 which has: a degree of sequence identity with the sequence of SEQ ID NO: 19 of at least 85%, preferably at least 90%, more preferably at least 95%, where any C-terminal extension that may be present and CDRs are not taken into account to determine the degree of identity sequences; and/or which has: no more than 7, preferably no more than 5, eg only 3, 2 or 1 "amino acid differences" of SEQ ID NO: 19, ignoring any C-terminal extension that may be present and CDRs. 9. ISVD according to any one of paragraphs. 1-8, which contains, compared to the sequence of SEQ ID NO: 18, one or more mutations that reduce the binding of preexisting antibodies. 10. ISVD according to any one of paragraphs. 1-9, which is VHH and which contains, compared to the sequence of SEQ ID NO: 18, one or more humanizing substitutions. 11. An immunoglobulin single variable domain capable of specifically binding to human serum albumin and selected from SEQ ID NOs: 18-59. 12. A protein construct that specifically binds to human serum albumin and contains a protein, polypeptide or other construct and at least one ISVD against human serum albumin, characterized in that said ISVD is an ISVD according to any one of paragraphs. 1-11. 13. Protein construct according to claim 12, which also contains at least one therapeutic fragment or substance. 14. Protein construct according to claim 12 or 13, which is a fusion protein. 15. Protein structure according to any one of paragraphs. 12-14 which has a human serum half-life, expressed as t1/2 beta, which is greater than 6 hours, preferably greater than 12 hours, more preferably greater than 24 hours, even more preferably greater than 72 hours. 16. Protein structure according to any one of paragraphs. 12-15, being such that: when it has an ISVD at its C-terminus, then the C-terminal ISVD of said protein construct has a C-terminal extension (X)n at its C-terminus, where n is 1 to 10 and each X is independently selected from natural amino acid residues; and/or when it has an ISVD on its C-terminal portion, then at least said C-terminal ISVD contains one or more mutations that reduce the binding of preexisting antibodies; and/or when it has an ISVD at its N-terminal portion, then the N-terminal ISVD of said protein construct preferably contains a D at position 1; and/or preferably, substantially all ISVDs present in said protein construct contain one or more mutations that reduce the binding of preexisting antibodies. 17. Pharmaceutical composition containing a protein structure according to any one of paragraphs. 12-16 and at least one pharmaceutically acceptable carrier, diluent or excipient. 18. Nucleic acid encoding ISVD according to any one of paragraphs. 1-11, optionally included in the genetic construct. 19. Nucleic acid encoding a protein construct according to any one of paragraphs. 12-16, optionally included in the genetic construct 20. The host cell for the expression of ISVD according to any one of paragraphs. 1-11 containing the nucleic acid according to claim 18. 21. A host cell for expression of a protein construct according to any one of paragraphs. 12-16 containing the nucleic acid according to claim 19. 22. The method of obtaining ISVD according to any one of paragraphs. 1-11, including cultivating or maintaining a host cell according to claim 20 under such conditions that said host cell produces or expresses an ISVD according to any one of paragraphs. 1-11, and optionally further including the allocation of ISVD according to any one of paragraphs. 1-11 obtained in this way. 23. The method of obtaining a protein construct according to any one of paragraphs. 12-16, including cultivating or maintaining a host cell according to claim 21 under such conditions that said host cell produces or expresses a protein construct according to any one of claims. 12-16, and optionally further comprising isolating the protein construct according to any one of paragraphs. 12-16 obtained in this way. 24. Protein structure according to any one of paragraphs. 13-16 for use in 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 therapeutic fragment(s) or substance(s) that are present in the protein construct according to any one of paragraphs. 13-16.

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